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Objectives Changes in protein glycosylation are a hallmark of immune-mediated diseases. Glycans are master regulators of the inflammatory response being important molecules for the discrimination between "self"/"non-self". We here explored whether lupus nephritis (LN) exhibit an altered cellular glycosylation aiming to identify a unique glycosignature that characterizes LN pathogenesis. Methods A comprehensive tissue glycomics characterization was performed in a cohort of human biopsy-proven LN clinical samples from SLE patients. A combination of advanced tissue mass spectrometry imaging (MSI); in situ glyco-characterization and ex vivo glycophenotyping of human kidney biopsies were performed to structurally map the N-glycans repertoire in LN samples. Results LN revealed a unique glycan signature characterized by an increased abundance and spatial distribution of unusual mannose-enriched glycans that are typically found in lower microorganisms; this glycosignature was specific of LN as it was not observed in other kidney diseases. Exposure of mannosylated glycans in LN was found to occur at the cell surface of kidney cells promoting an increased recognition by specific glycans-recognizing receptors, expressed by immune cells. This abnormal glyco-signature of LN was demonstrated to be due to a deficient complex N-glycosylation and a proficient O-mannosylation pathway. Moreover, levels of mannosylation detected in kidney biopsies from LN patients at diagnosis were demonstrated to predict the development of chronic kidney disease (CKD) with 93% of specificity. Conclusions Cellular mannosylation is a marker of LN, predicting the development of CKD, and thus constituting a potential glycobiomarker to be included in the diagnostic and prognostic algorithm of LN.

Simple Summary Comparative oncology is centered around the study of naturally occurring tumors in animals as a parallel and complementary model for human cancer research. Canine mammary tumors pose as excellent models since they share similarities in their spontaneous nature, histological subtypes, genetic background, and clinical course, which would be impossible to reproduce in murine models. Our study aimed to investigate cancer heterogeneity in primary tumors and metastasis, by applying bottom-up proteomics and mass spectrometry imaging to identify potential disease-state markers. We have demonstrated that the malignant phenotype may have arisen as a consequence of alterations in the expression of proteins involved in immune evasion facilitating metastatic events. To our knowledge, this is the first study to use mass spectrometry imaging in a dog model of breast cancer, that have demonstrated that poorly described proteins might play important roles in cancer spreading and should be further validated as potential early-stage tumor biomarkers. Abstract New insights into the underlying biological processes of breast cancer are needed for the development of improved markers and treatments. The complex nature of mammary cancer in dogs makes it a great model to study cancer biology since they present a high degree of tumor heterogeneity. In search of disease-state biomarkers candidates, we applied proteomic mass spectrometry imaging in order to simultaneously detect histopathological and molecular alterations whilst preserving morphological integrity, comparing peptide expression between intratumor populations in distinct levels of differentiation. Peptides assigned to FNDC1, A1BG, and double-matching keratins 18 and 19 presented a higher intensity in poorly differentiated regions. In contrast, we observed a lower intensity of peptides matching calnexin, PDIA3, and HSPA5 in poorly differentiated cells, which enriched for protein folding in the endoplasmic reticulum and antigen processing, assembly, and loading of class I MHC. Over-representation of collagen metabolism, coagulation cascade, extracellular matrix components, cadherin-binding and cell adhesion pathways also distinguished cell populations. Finally, an independent validation showed FNDC1, A1BG, PDIA3, HSPA5, and calnexin as significant prognostic markers for human breast cancer patients. Thus, through a spatially correlated characterization of spontaneous carcinomas, we described key proteins which can be further validated as potential prognostic biomarkers.

Myxoid liposarcoma (MLS) is the second most common subtype of liposarcoma, accounting for 6% of all sarcomas. MLS is characterized by a pathognomonic FUS-DDIT3, or rarely EWSR1-DDIT3, gene fusion. The presence of >= 5% hypercellular round cell areas is associated with a worse prognosis for the patient and is considered high grade. The prognostic significance of areas with moderately increased cellularity (intermediate) is currently unknown. Here we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging to analyze the spatial distribution of N-linked glycans on an MLS microarray in order to identify molecular markers for tumor progression. Comparison of the N-glycan profiles revealed that increased relative abundances of high-mannose type glycans were associated with tumor progression. Concomitantly, an increase of the average number of mannoses on high-mannose glycans was observed. Although overall levels of complex-type glycans decreased, an increase of tri- and tetra-antennary N-glycans was observed with morphological tumor progression and increased tumor histological grade. The high abundance of tri-antennary N-glycan species was also associated with poor disease-specific survival. These findings mirror recent observations in colorectal cancer, breast cancer, ovarian cancer, and cholangiocarcinoma, and are in line with a general role of high-mannose glycans and higher-antennary complex-type glycans in cancer progression.

Carotid atherosclerosis is one of the main causes of stroke, mortality and disability worldwide. The disease is characterized by plaques, heterogeneous depositions of lipids and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 lipids in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-driven multivariate analysis. These data allowed us to compare the spatial lipid patterns with morphological plaque features that have been associated with enhanced risk of symptoms. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, while diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. This is the first study to systematically investigate spatial lipid patterns in atherosclerosis, analyze their relation to histological tissue type, and to demonstrate a clear co-localization between plaque features and specific lipid classes and individual lipid molecules in high-risk atherosclerotic plaques.

Mass spectrometry imaging (MSI) has provided many results with translational character, which still have to be proven robust in large patient cohorts and across different centers. Although formalin-fixed paraffin-embedded (FFPE) specimens are most common in clinical practice, no MSI multicenter study has been reported for FFPE samples. Here, we report the results of the first round robin MSI study on FFPE tissues with the goal to investigate the consequences of inter- and intracenter technical variation on masking biological effects. A total of four centers were involved with similar MSI instrumentation and sample preparation equipment. A FFPE multi-organ tissue microarray containing eight different types of tissue was analyzed on a peptide and metabolite level, which enabled investigating different molecular and biological differences. Statistical analyses revealed that peptide intercenter variation was significantly lower and metabolite intercenter variation was significantly higher than the respective intracenter variations. When looking at relative univariate effects of mass signals with statistical discriminatory power, the metabolite data was more reproducible across centers compared to the peptide data. With respect to absolute effects (cross-center common intensity scale), multivariate classifiers were able to reach on average > 90% accuracy for peptides and > 80% for metabolites if trained with sufficient amount of cross-center data. Overall, our study showed that MSI data from FFPE samples could be reproduced to a high degree across centers. While metabolite data exhibited more reproducibility with respect to relative effects, peptide data-based classifiers were more directly transferable between centers and therefore more robust than expected. Graphical abstractᅟ Electronic supplementary material The online version of this article (10.1007/s00216-018-1216-2) contains supplementary material, which is available to authorized users.

Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.

Background Donors for allogeneic stem cell transplantation are preferentially matched with patients for HLA-A, -B, -C, and -DRB1. Mismatches between donor and patient in these alleles are associated with an increased risk of graft-versus-host disease (GVHD). In contrast, HLA-DRB3, 4 and 5, HLA-DQ and HLA-DP are usually assumed to be low expression loci with limited relevance, although mismatches in HLA-DQ and HLA-DP can result in alloimmune responses. Mismatches in HLA-DRB3, 4, and 5 are usually not taken into account in donor selection. Methods Conversion of chimerism in the presence of GVHD after CD4 donor lymphocyte infusion was observed in a patient, HLA 10/10 matched, but mismatched for HLA-DRB3 and HLA-DPB1 compared with the donor. Alloreactive CD4 T cells were isolated from peripheral blood after CD4 donor lymphocyte infusion and recognition of donor-derived target cells transduced with the mismatched patient variant HLA-DRB3 and HLA-DPB1 molecule was tested. Results A dominant polyclonal CD4 T cell response against patient's mismatched HLA-DRB3 molecule was found in addition to an immune response against patient's mismatched HLA-DPB1 molecule. CD4 T cells specific for these HLA class II molecules recognized both hematopoietic target cells as well as GVHD target cells. Conclusions In contrast to the assumption that mismatches in HLA-DRB3, 4, and 5 are not of immunogenic significance after HLA 10/10 matched allogeneic stem cell transplantation, we show that in this matched setting not only mismatches in HLA-DPB1, but also mismatches in HLA-DRB3 may induce a polyclonal allo-immune response associated with conversion of chimerism and severe GVHD.

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.

Background Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft- versus -leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft- versus -host disease. Design and Methods We analyzed in detail CD8+ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft- versus -host disease of the skin. CD8+ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens. Results Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones. Conclusions Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft- versus -leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT. Cancer Res; 70(22); 9073–83. ©2010 AACR.

Clinical studies have indicated that HLA-DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD), but also a decreased risk of disease relapse. However, specific HLA-DPB1 mismatches were associated with poor clinical outcome. It was suggested that this unfavorable effect was caused by a difference in immunogenicity between HLA-DPB1 alleles. To analyze whether immunogenicity of HLA-DPB1 mismatches could be predicted based on the presence or absence of specific amino acid sequences we developed a model to generate allo-HLA-DPB1 responses in vitro. We tested in total 48 different stimulator/responder combinations by stimulating CD4(+) T cells from 5 HLA-DPB1 homozygous individuals with the same antigen-presenting cells transduced with different allo-HLA-DPB1 molecules. HLA-DPB1 molecules used for stimulation comprised 76% to 99% of HLA-DPB1 molecules present in different ethnic populations. We show that all HLA-DPB1 mismatches as defined by allele typing resulted in high-frequency immune responses. Furthermore, we show that crossrecognition of different HLA-DPB1 molecules is a broadly observed phenomenon. We confirm previously described patterns in crossrecognition, and demonstrate that a high degree in similarity between HLA-DPB1 molecules is predictive for crossrecognition, but not for immunogenicity.

α-Globin gene triplications may exacerbate the α chain and β chain imbalance in β-thalassemia (β-thal) and may compensate for the effect of α-globin gene deletion in α-thal. Identification of an α-globin gene triplication is, therefore, valuable in predicting the clinical phenotype of the thalassemias. To be able to detect α-globin gene triplications, we have modified an existing multiplex polymerase chain reaction (PCR) assay for the seven most prevalent α-globin gene deletions by incorporating two triplication-specific primers and concurrently substituting one of the original primers by a newly designed primer. This modified multiplex PCR assay was evaluated by performing the assay on archival DNA samples and on peripheral blood samples from 163 suspected thalassemia cases. It was found to function properly. Our assay thereby represents the first multiplex PCR assay that can detect both the seven most prevalent α-globin gene deletions and the αααanti 3.7 α-globin gene triplication in a single-tube reaction.

Bacterial and archaeal communities in sediments obtained from three geographically-distant mud volcanoes, a control site and a microbial mat in the Eastern Mediterranean deep-sea were characterized using direct 16S rRNA gene analyses. The data were thus in relation to the chemical characteristics of the (stratified) habitats to infer community structure-habitat relationships. The bacterial sequences in the different habitats were related to those of Actinobacteria, Bacilli, Chloroflexi, Alpha-, Beta-, Gamma-, Delta- and Epsilonproteobacteria and unclassified bacteria, including the JS1 group. The archaeal sequences found were affiliated with those of the Methanosarcinales, Thermoplasmales, Halobacteriales and Crenarchaea belonging to marine benthic group I and B, as well as MCG group archaea. In each sample, the communities were diverse and unique at the phylotype level. However, at higher taxonomic levels, similar groups were found in different sediments, and similar depth layers tended to contain similar communities. The sequences that dominated in all top layers (as well as in the mat) probably represented organisms involved in aerobic heterotrophy, sulfide-based chemoautotrophy and methanotrophy and/or methylotrophy. Sequences of organisms most likely involved in anaerobic methane oxidation, sulfate reduction and anaerobic heterotrophy were predominantly found in deeper layers. The data supported the notion of (1) uniqueness of each habitat at fine taxonomic levels, (2) stratification in depth and (3) conservation of function in the sediments.

Background and Objectives Cytotoxic T lymphocytes (CTL) may use two effector mechanisms to kill their target cells: perforin (PFN) and granzyme B (GrB)-dependent granule-mediated cell death and death receptor-mediated cell death. Controversy exists whether, in addition to PFN/GrB-mediated apoptosis, death receptor-induced apoptosis contributes to the elimination of human tumor cells by CTL.Design and Methods Since the two CTL-mediated effector mechanisms differ in time required to eliminate target cells, lysis of target cells was analyzed using CTL clones with slow and rapid kinetics of killing derived from a patient with chronic myeloid leukemia. To determine the involvement of the death receptor pathway, a retroviral construct encoding the antiapoptotic gene FLICE inhibitory protein (FLIP) was introduced into these target cells.Results A CTL clone capable of killing 50% of the target cells within 2 hours of incubation primarily acted by release of PFN and GrB. In contrast, two CTL clones showing slower target cell killing kinetics partially used the death receptor pathway (~30% inhibition by FLIP).Interpretation and Conclusions We demonstrated that the death receptor pathway contributes to T-cell-mediated cell death if not all target cells are destroyed by release of PFN and GrB.

This paper measures the direct costs that the terrorist attacks of March 11 caused on the economy of the region of Madrid. The evaluation has been made applying a conservative criterion, and the results obtained have to be considered as minimum. The result indicates that the terrorist attacks caused a loss of nearly 212 million euros to the regional economy of Madrid, equivalent to 0,16 per cent of the regional GDP (0,03 of the national GDP). This confirms that the immediate economic dimension of a terrorist attack such as the one of March 11 apart from human catastrophic consequences is relatively low.

In the Eastern Mediterranean Sea, deep hypersaline anoxic basins (DHABs) and deep-sea sediment contain anoxic environments where sulfate reduction is an important microbial metabolic process. The objective of this study was to characterize the sulfate-reducing community in the brine and interface of the DHABs L'Atalante and Urania based on a phylogenetic analysis of the dissimilatory sulfite reductase gene (dsrA). Results demonstrated that the sulfate-reducing community was diverse, except for the sulfidogenic brine of the Urania basin. The similarity of the dsrA sequences between different environments was very low demonstrating that each environment had a unique sulfate-reducing community. Sequences had 67.6-93.3% similarity to dsrA sequences from GenBank database and were mostly related to the delta-proteobacteria. Each environment was dominated by a different family within the delta-proteobacteria except for the Urania interface, which was dominated by sequences related to the Gram-positive Peptococcaceae. We conclude that sulfate-reducing communities inhabiting the L'Atalante and Urania basins are highly diverse with low similarities to each other and contain a sulfate-reducing species composition that is very different from sulfate-reducing species compositions in previously studied ecosystems.

A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-microm filters to coarsely separate unicellular and filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the mat microbial community and the relatively low delta(13)C carbon isotope values of bulk bacterial lipids suggested the occurrence of methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a combination of methane- and sulfide-driven chemotrophy.

In HLA-matched allogeneic hematopoietic stem cell transplantation (SCT), donor T cells can mediate graft-versus-leukemia/lymphoma (GvL) reactivity and graft-versus-host disease (GvHD) by recognition of minor histocompatibility antigens (MiHA).1–4 Only a minority of MiHA shows hematopoiesis-restricted expression, and donor T cells for these MiHA may induce beneficial GvL reactivity without GvHD. The number of well-characterized MiHA with therapeutic relevance based on hematopoiesis-restricted expression remains limited and only 25% and 40% of recipients transplanted with grafts from sibling and unrelated donors, respectively, are eligible for therapies targeting known hematopoietic MiHA.3,4 Thus, in order to increase the efficacy and applicability of cellular therapy for selective GvL induction, more hematopoiesis-restricted MiHA with balanced population frequencies in common HLA molecules must be identified. Here, we investigated the therapeutic significance of a MiHA encoded by ARHGDIB.5 We demonstrated hematopoiesis-restricted gene expression with the exception of intermediate mRNA expression in endothelial cells and showed that T cells recognized LB-ARHGDIB-1R presented by HLA-B*07:02 on primary leukemic cells, but not on [interferon-gamma (IFN-γ)]-treated fibroblasts and keratinocytes. To evaluate potential toxicity against endothelial cells, we tested T cell recognition of LB-ARHGDIB-1R on human umbilical vein endothelial cells (HUVEC) and found only limited reactivity under inflammatory conditions. Furthermore, we demonstrated in vivo targeting of LB-ARHGDIB-1R in eight out of ten patients who were screened for post-transplant specific T-cell responses. In one patient with relapsed lymphoma, high T-cell frequencies were induced after donor lymphocyte infusion (DLI), coinciding with long-lasting anti-lymphoma immunity without GvHD. Our data thus support the relevance of LB-ARHGDIB-1R as a therapeutic target with the potential to induce selective GvL reactivity.

Objective Previously, we observed an increased recognition of malignant cells by cytotoxic T lymphocytes (CTL) when the target cells were cultured in vitro for 24 hours. In this study, we analyzed the expression of costimulatory and adhesion molecules on acute myeloid leukemia (AML) cells and determined whether 24-hour culture of the cells was associated with upregulation of these molecules. We analyzed whether this incubation period improved recognition of AML cells by CTL. Materials and Methods Expression of costimulatory and adhesion molecules on leukemic blasts of 34 patients comprising each AML FAB subclassification were analyzed directly and after 24 hours of culture, and the recognition of these AML cells by an HLA-A2 restricted CTL clone was determined. Blocking studies were performed with antibodies against CD54, CD58, and CD11a. Results Immunophenotyping showed a low expression of CD80 and CD40 and a variable CD86 expression on most AML cells. CD54 expression was generally low, CD58 expression was high, and CD11a expression was variable, with a higher expression in AML M0, M1, M4, and M5. Twenty-four hours of culture resulted in a significant upregulation of CD40, CD54, and CD58. Impaired recognition of AML cells by the HLA-A2 restricted CTL clone was enhanced 100–200% by 24 hours of preincubation of the leukemic cells. Blocking studies showed the importance of multiple adhesion molecules on the AML cells. Conclusion Low expression of multiple costimulatory and adhesion molecules on AML could be upregulated by 24 hours of culture, which was associated with increased recognition of the AML blasts by CTL. Blocking multiple adhesion molecules completely abolished CTL recognition, showing the importance of the combination of these molecules for T-cell interaction with AML.

Microbiological and environmental variables involved in the removal of free sulfide were studied along an eutrophication transect in the Bassin d'Arcachon (France). At four sites, analyses were carried out on reduced sulfur compounds, iron species and total numbers of viable sulfur bacteria (sulfide-producing bacteria, colorless sulfur bacteria and purple sulfur bacteria). In addition, the chemical buffering capacity towards free sulfide and the potential microbiological sulfide oxidation rates were determined. In the ecosystem, no free sulfide occurs in the top layers of the sediment at all four sites, despite a high nutrient load and hence favourable conditions for sulfide-producing bacteria. The explanation of this apparent discrepancy was shown to be the high biological sulfide oxidizing capacity in combination with a high chemical buffering capacity. The data presented illustrate that the buffering capacity of sediments towards free sulfide is the combined result of the chemical and biological processes. The ratio between these were found to depend on the degree of eutrophication. It was shown that the chemical buffering capacity towards sulfide is severely overestimated when based on the pool of chemically reactive iron, a more realistic value is obtained by estimating the total amount of sulfide that can be added before free sulfide can be detected. A clear difference was observed between the numbers of colorless sulfur bacteria and the activity of the entire population. For a proper quantification of the sulfide buffering capacity of sediments, it is essential to estimate the concentration of iron and sulfur compounds that actually can react with sulfide, as well as to analyze the activities of sulfide-oxidizing microbes.

After allogeneic bone marrow transplantation (BMT), the beneficial graft-versus-leukemia (GVL) effect but also the life-threatening graft-versus-host disease (GVHD) are mediated by T cells of the grafted marrow. The identification of leukemia cell–reactive T cells and their ligands are, therefore, crucial for the development of new anti leukemia strategies. Here we describe a leukemia-reactive allo-HLA class II restricted CD4+ T-cell clone, 6.2, isolated from a healthy individual after stimulation with allogeneic leukemic cells. Clone 6.2 recognizes leukemic cells from several AML patients without showing reactivity to unfractioned peripheral blood mononuclear cells, monocytes, B cells, T-cell blasts, and proximal tubulus epithelial cells. Interestingly, clone 6.2 also recognizes BM cells derived from healthy individuals and inhibits the colony formation of myeloid and erythroid cell lineages. In the BM, clone 6.2 recognizes only CD34+ early precursor cells but not CD34−, more differentiated cells. Thus, the target antigen of clone 6.2 is developmentally regulated and expressed only by leukemic cells and CD34+ early progenitor cells in the hematopoietic system. We suggest that targeting the T-cell immune response to leukemia-associated, developmentally regulated antigens of the hematopoietic system can provide a basis for the separation of GVL from GVHD, and may lead to new therapeutic approaches for residual and relapsed leukemia.

Umbilical cord blood (UCB) has been successfully used as an alternative source of hematopoietic stem cells for allogeneic transplantation. A relatively low incidence and severity of graft-versus-host disease (GVHD) following UCB transplants has been reported, and it has been suggested that this may be caused by a low frequency of alloreactive lymphocytes in UCB. Low frequencies of alloreactive T lymphocytes in UCB may allow transplantation across major MHC barriers with an acceptable risk of GVHD. We investigated cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) frequencies in UCB. Normal frequencies of CTLp and HTLp were measured against 1-2 HLA class I and 0-1 HLA-DR mismatched stimulator cells. Since it has been postulated that due to maternal fetal transfusion during pregnancy, fetal blood lymphocytes may become tolerant for noninherited maternal antigens (NIMA), allowing transplantation over certain HLA barriers, reactivity of 24 umbilical cord blood samples was analyzed against both parents. The median frequencies of CTLp against NIMA with 1 class-I mismatch was 79 per 10(6) nucleated cells (range 16-428) and with 2 class I mismatches 121 (range 33-748). CTLp frequencies against noninherited paternal antigens (NIPA) were not statistically different from those against NIMA, with a median of 115 (range 8-336) and 176 (range 50-725) for 1 or 2 HLA class I mismatches, respectively. HTLp frequencies in UCB against parents with 0 or 1 HLA-DR antigen mismatches were similar with respect to NIMA (median 74, range 43-233 and 88, range 16-777, respectively) and NIPA (median 125, range 18-174, and 110, range 28-350, respectively). In four cases, UCB from two HLA-identical siblings was tested against both parents. A correlation between the frequencies of CTLp and HTLp from HLA-identical individuals was found, illustrating that these frequencies are genetically determined. These results illustrate that UCB contains normal frequencies of CTLp and HTLp against MHC alloantigens.

We investigated whether minor histocompatibility (mH) antigen-specific cytotoxic T lymphocytes (CTL) can discriminate between leukemic hematopoietic progenitor cells (leukemic-HPC) from AML or CML patients, the HPC from their remission bone marrow (remission-HPC), and normal HPC from their HLA-identical sibling bone marrow donor (donor-HPC). Specific lysis by CD8+ CTL clones was observed not only of the leukemic-HPC but also of the donor-HPC in 3/4 patient/donor combinations expressing mH antigen HA-1, 3/5 combinations expressing mH antigen HA-2, 2/3 combinations expressing mH antigen HA-3, and 2/2 combinations expressing mH antigen HY-A1. In four patient/donor combinations the recognition of the donor-HPC was clearly less than of the leukemic-HPC, indicating differential susceptibility to lysis by these mH CTL clones. In addition, differential recognition of leukemic-HPC and remission-HPC within seven patients was analyzed. In one patient expressing the HA-2 antigen on the leukemic cells the recognition of the remission-HPC was clearly less than of the leukemic-HPC. One CD4+ CTL clone showed specific lysis of the leukemic-HPC from an AML patient and a CML patient as well as of normal remission-HPC and donor-HPC. These results illustrate that in general CD8+ and CD4+ mH antigen specific CTL clones do not differentially recognize leukemic-HPC and normal-HPC. However, differences in susceptibility to lysis of malignant versus normal cells may contribute to a differential GVL effect.

Human umbilical cord blood (UCB) may be used as an alternative source of bone marrow repopulating cells in allogeneic bone marrow transplantation in children. It has been reported that high numbers of hematopoietic progenitor cells (HPC) from umbilical cord blood may be lost during simple cell-separation techniques. This may seriously hamper the use of UCB as an alternative source of bone marrow repopulating cells. In this study we demonstrate that UCB can be separated into various cell fractions using several cell-separation methods including red blood cell lysis, methylcellulose sedimentation, and density gradients without significant loss of HPC, when cell separations are initiated within 8 h. We demonstrate that UCB contains a high concentration of immature HPC as compared with bone marrow grafts. Using FACS analysis of cells harvested from single colonies derived from single cell- single well-sorted CD34++ CD33- UCB cells, the high frequency of multipotential HPC was illustrated. These results suggest that UCB may contain sufficient HPC for hematopoietic stem cell transplantation in adults.

To the editor: Clinical studies have indicated that human leukocyte antigen (HLA)–DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation (SCT). Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD) but also a

Before and after bone marrow transplantation (BMT) for hematologic malignancies, peripheral blood mononuclear cells from 10 patients were obtained. The relative and absolute numbers of CD3+ T-cell receptor yS+ (TCRyS+) cells, as defined by the reaction of monoclonal antibodies (MoAbs) directed against CD3 and the TCR yS (anti-TCRyS-1). were determined. Before transplantation, eight of nine patients tested had less than 10% CD3+TCRyS+ cells. Consistent increased numbers of yS cells up to eightfold the pretransplant level can be seen in four of nine patients tested within the first 4 months after BMT. The large majority of early posttransplant yS and 4 T cells express the CD45RO antigen, which is usually expressed on "memory" cells only. The V-region usage of the TCRyS' T cells was analyzed using fresh mononuclear cells and MoAbs against known Vy and V6 regions. For more detailed analysis, CD3'TCRyS + cells were WO DISTINCT FORMS of the T-cell receptor (TCR) T have been described, the UP TCR and the y8 TCR.'32 The y8 TCR is expressed by a minority of CD3+ cells (0.5% to 17%) in peripheral blood (PB) and thymus.',3 The y8 cells form a distinct and functionally heterogeneous T-cell lineage and are evenly distributed throughout the lymphoid ~ystem.~ Monoclonal antibodies (MoAbs) specific for the human y8 TCR as well as for V-regions have been described?-" Although the function of TCRy8 cells remains largely unknown, recent reports indicate that these cells are capable of interacting with a heterogeneous set of ligands, among which are alloantigenic determinants, tetanus toxoid, mycobacterial (heat shock) proteins,''~'Z and Staphylococcal enterotoxin A (SEA).I3 Studies in the mouse have indicated that the y8 TCR is expressed before the aP TCR during thymic ontogeny. Populations of y8 TCR' cells are intermittently generated that express characteristic TCRS.'~,'' These waves appear to home specifically to certain epithelia in the skin and in the gut.I6 It has already been shown that rearrangements involving Cy1 precede those involving Cy2, and recent results indicate that, during early fetal ontogeny, rearrangements involving the Cy1 constant region occur before those involving the Cy2 constant region gene ~egment.'~"~ Also, early in human fetal development a wave of TCRy8 cells exists that preferentially uses the V82 gene segment." The kinetics of engraftment after bone marrow transplantation (BMT) may serve as a model system to study the TCRy8 repopulation. After BMT, relative and absolute increased numbers of TCRyG cells have been reported, suggesting differences in repopulation rates between TCRaP and TCRy8 cells after

Minor histocompatibility (mH) antigens appear to play a major role in bone marrow transplantation (BMT) using HLA-identical donors. Previously, we reported the isolation of major histocompatibility complex (MHC)-restricted mH antigen-specific cytotoxic T lymphocytes (CTL) from patients with graft-vs.-host disease or rejection after HLA-identical BMT. We have demonstrated that mH antigens can be recognized on hematopoietic progenitor cells, and residual recipient CTL specific for mH antigens expressed on donor hematopoietic progenitor cells may be responsible for graft rejection in spite of intensive conditioning regimens in HLA-identical BMT. Here, we investigated whether mH antigen-specific CTL directed against the mH antigens HA-1 to HA-5 and the male-specific antigen H-Y were capable of antigen-specific inhibition of in vitro growth of clonogenic leukemic precursor cells. We demonstrate that mH antigen-specific CTL against all mH antigens tested can lyse freshly obtained myeloid leukemic cells, that these mH antigen-specific CTL can inhibit their clonogenic leukemic growth in vitro, and that this recognition is MHC restricted. We illustrate that leukemic (precursor) cells can escape elimination by mH antigen-specific CTL by impaired expression of the relevant MHC restriction molecule. We suggest that mH antigen-specific MHC-restricted CTL may be involved in vivo in the graft-vs.-leukemia reactivity after BMT.

Mismatching for human leukocyte antigen (HLA)-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a decreased risk of disease relapse, indicating that HLA-DP may represent a target for graft-versus-leukemia (GVL) reactivity in HLA class II-expressing hematological malignancies. To investigate whether HLA-DP-specific T cells could mediate GVL reactivity following HLA-DPB1-mismatched URD-SCT and donor lymphocyte infusion (DLI), we analyzed the immune response in a patient with leukemic lymphoplasmacytic lymphoma responding to DLI without graft-versus-host disease. The emergence of leukemia-reactive CD4+ T cells during the clinical immune response was demonstrated by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot(ELISPOT)analysis. Following clonal isolation of these leukemia-reactive CD4+ T cells, blocking studies, panel studies and retroviral transduction experiments of both mismatched HLA-DPB1 alleles identified HLA-DPB1(*)0201 and HLA-DPB1(*)0301 as the targets of this immune response. The HLA-DPB1-specific CD4+ T-cell clones were capable of recognizing and lysing several HLA-DP-expressing myeloid and lymphoid hematological malignant cells. Since HLA-DP expression is mainly restricted to hematopoietic cells, HLA-DP may be used as a specific target for immunotherapy following T-cell-depleted URD-SCT. Therefore, in patients with HLA class II-expressing hematological malignancies HLA-DP-mismatched SCT may be preferable over fully matched SCT allowing DLI to induce a GVL effect.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4 + T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II β gene. A polyclonal CD4 + T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4 + T cells recognized and lysed the CD34 + CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-γ and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4 + T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8 + T cell immunity as helper cells.

Minor histocompatibility antigens (mHags) play an important role in both graft-versus-tumor effects and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. We applied biochemical techniques and mass spectrometry to identify the peptide recognized by a dominant tumor-reactive donor T-cell reactivity isolated from a patient with relapsed multiple myeloma who underwent transplantation and entered complete remission after donor lymphocyte infusion. A frequently occurring single nucleotide polymorphism in the human ATP-dependent interferon-responsive (ADIR) gene was found to encode the epitope we designated LB-ADIR-1F. Although gene expression could be found in cells from hematopoietic as well as nonhematopoietic tissues, the patient suffered from only mild acute GVHD despite high percentages of circulating LB-ADIR-1F–specific T cells. Differential recognition of nonhematopoietic cell types and resting hematopoietic cells as compared with activated B cells, T cells, and tumor cells was demonstrated, illustrating variable LB-ADIR-1F expression depending on the cellular activation state. In conclusion, the novel mHag LB-ADIR-1F may be a suitable target for cellular immunotherapy when applied under controlled circumstances.

Carbonate crusts in marine environments can act as sinks for carbon dioxide. Therefore, understanding carbonate crust formation could be important for understanding global warming. In the present study, the microbial communities of three carbonate crust samples from deep-sea mud volcanoes in the eastern Mediterranean were characterized by sequencing 16S ribosomal RNA (rRNA) genes amplified from DNA directly retrieved from the samples. In combination with the mineralogical composition of the crusts and lipid analyses, sequence data were used to assess the possible role of prokaryotes in crust formation. Collectively, the obtained data showed the presence of highly diverse communities, which were distinct in each of the carbonate crusts studied. Bacterial 16S rRNA gene sequences were found in all crusts and the majority was classified as alpha-, gamma-, and delta- Proteobacteria. Interestingly, sequences of Proteobacteria related to Halomonas and Halovibrio sp., which can play an active role in carbonate mineral formation, were present in all crusts. Archaeal 16S rRNA gene sequences were retrieved from two of the crusts studied. Several of those were closely related to archaeal sequences of organisms that have previously been linked to the anaerobic oxidation of methane (AOM). However, the majority of archaeal sequences were not related to sequences of organisms known to be involved in AOM. In combination with the strongly negative delta C-13 values of archaeal lipids, these results open the possibility that organisms with a role in AOM may be more diverse within the Archaea than previously suggested. Different communities found in the crusts could carry out similar processes that might play a role in carbonate crust formation.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8+ T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7–restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1–specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Low alloreactivity of umbilical cord blood (UCB) T-cells may explain diminished graft-versus-host-disease after UCB transplantation. We investigated whether UCB T-cells have an intrinsic lower capacity to become activated. T-cells from UCB or adult blood (AB) were stimulated with anti-CD3 and anti-CD28 antibodies. On days 1–3 after stimulation, T-cell activation was determined by CD25 expression, proliferation was measured, and kinetics of cell division were analyzed by staining with CFSE. UCB and AB T cells exhibited similar numbers of activated and proliferating cells, but the extent of activation was lower in UCB T-cells. Enzyme-linked immunospot analysis showed lower levels and slower kinetics of IL-2, IL-4, IL-10, and IFN-γ secreting cells for UCB T-cells. Comparison of UCB T-cells with CD45RA+ naive or CD45RO+ memory T cells purified from AB showed relatively low numbers of IL-4 and IL-10 secreting T cells in CD45RA+ AB T-cells and UCB T-cells as compared with CD45RO+ AB T cells. Numbers of IL-2 or IFN-γ secreting cells in adult CD45RA+ T-cells were lower than in CD45RO+ T-cells but higher than in UCB T-cells. Thus diminished reactivity of UCB T-cells was not caused by a lower capacity to become activated and proliferate but may be explained by a lower extent of activation in UCB T cells, the absence of memory T cells in UCB, and differences between naive T cells from UCB and AB.

Graft-vs-leukemia reactivity after donor lymphocyte infusion (DLI) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on recipient hematopoietic cells. To study the diversity of cells involved in this immune response, hematopoietic cell reactive T cells were directly clonally isolated from peripheral blood of patients entering complete remission after DLI. T cells were briefly stimulated with bone marrow cells from patients pretransplant, and IFNgamma-secreting T cells were directly clonally isolated, and expanded. Cytotoxic T-lymphocyte (CTL) clones from individual patients used multiple distinct HLA-restricting molecules and varied in reactivity against patient-derived normal and/or malignant hematopoietic cells. For each patient, CTL clones specific for known immunodominant mHags as well as distinct unknown mHags were found. Within individual patients, CTL clones using the same HLA-restricting element could show differential recognition patterns, indicating further diversity in mHag reactivity. CTL clones from individual patients exhibiting identical specificities could show oligoclonal origin. In conclusion, the direct cloning technique shows that the response to hematopoietic cells after DLI is directed against multiple distinct mHags, including but not limited to known immunodominant mHags, implying that immunotherapy with T cells against multiple mHag specificities may be more effective in eradicating malignant cells.

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNgamma were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.

Because a relatively low incidence and severity of graft-versus-host disease after umbilical cord blood (UCB) transplantation is observed, we investigated whether T cells from UCB or adult blood (AB) were differentially activated by antigen-presenting cells with or without human leukocyte antigen (HLA)-DR expression. T cells from UCB or AB, or CD45RA(+) naive T cells and CD45RO(+) memory T cells separated from AB, were stimulated with the HLA-DR(+) or HLA-DR(-) cell line AML193. On days 1-3 after stimulation, numbers of interleukin (IL)-2, IL-4, IL-10 or interferon gamma (IFN-gamma)-secreting cells were determined by enzyme-linked immunospot analysis. No IL-4 or IL-10 was produced. AML193-DR(+) cells induced IL-2 and IFN-gamma secretion with slower kinetics and lower levels in UCB T cells than in AB T cells. AML193-DR(+) cells induced comparable IL-2 but higher IFN-gamma secretion in CD45RA(+) T cells from AB than in UCB T cells. AML193-DR(-) cells did not induce IL-2- or IFN-gamma secretion in UCB T cells, but stimulated both CD45RA(+) and CD45RO(+) T cells from AB to secrete IL-2 and IFN-gamma. Thus, not only the absence of memory T cells but also the inability to respond to HLA-DR-negative antigen-presenting cells and the slower kinetics and level of activation found for naive T cells from UCB as compared with AB may partly explain the reduced antirecipient reactivity after UCB transplantation.

"Buffer capacities" has been defined in ecology as a holistic concept (e.g., Integration of Ecosystem Theories: A Pattern, second ed. Kluwer, Dordrecht, 1997, 388pp), but we show that it can also be worked out in mechanistic studies. Our mechanistic approach highlights that "buffering capacities" can be depleted progressively, and, therefore, we make a distinction between current and potential "buffering capacities". We have applied this concept to understand the limited "local stability" in seagrass ecosystems and their vulnerability towards structural changes into macro-algal dominated communities. We explored the following processes and studied how they confer buffering capacities to the seagrass ecosystem: (i) net autotrophy is persistent in Zostera noltli meadows where plant assimilation acts as a sink for nutrients, this contrasted with the Ulva system that shifted back and forth between net autotrophy and net heterotrophy; (ii) the Z. noltii ecosystem possesses a certain albeit rather limited capacity to modify the balance between nitrogen fixation and denitrification, i.e., it was found that in situ nitrogen fixation always exceeded denitrification; (iii) the nitrogen demand of organoheterotrophic bacteria in the sediment results in nitrogen retention of N in the sediment and hence a buffer against release of nitrogen compounds from sediments, (iv) habitat diversification in seagrass meadows provides shelter for meiofauna and hence buffering against adverse conditions, (v) sedimentary iron provides a buffer against noxious sulfide (note: bacterial sulfide production is enhanced in anoxic sediment niches by increased organic matter loading). On the other hand, in the coastal system we studied, sedimentary iron appears less important as a redox-coupled buffer system against phosphate loading. This is because most inorganic phosphate is bound to calcium rather than to iron. In addition, our studies have highlighted the importance of plant-microbe interactions in the seagrass meadows. [KEYWORDS: coastal lagoon; seagrass; macro-algae; sulfide; iron phosphate; community metabolism; rhizosphere; nitrogen fixation; 16-S RNA clone library introduction Sulfate-reduction rates; zostera-marina; nitrogen-fixation; bassin darcachon; rhizosphere; sediments; sulfide; noltii; carbon; france]

The production and consumption of sulfide and its influence on phosphorous cycling were studied in a hypertrophic coastal lagoon (Valle Smarlacca, Italy). Oxygen measurements revealed that the water phase was supersaturated except for the layer directly overlying the sediment. This layer was devoid of oxygen and contained sulfide at all times. Maximal rates of sulfide production, calculated from in situ profiles, were observed in the 0 to 2 cm sediment layer and the 1 cm water layer directly above. Sediment iron data suggested a moderate chemical buffering capacity towards free sulfide; however, the in situ buffering capacity was fully exploited. Stirring increased the chemical buffer, indicating that, in situ, part of the iron did not contribute to the chemical buffer. The potential rate of biological sulfide oxidation, estimated in sediment slurries amended with oxygen, was high; however, the actual rate was low due to a shortage of oxygen in the sediments and the overlying water. Evidence was obtained for enhanced release of phosphate caused by free sulfide. Under simulated natural conditions the release of phosphate exceeded the initial concentration of Fe-bound phosphate by an order of magnitude, indicating a significant contribution of non-iron- bound phosphate. The observations in Valle Smarlacca were used to shed Light on the virtually complete disappearance of Ruppia sp, from other lagoons of the Valli di Comacchio (Northern Italy). [KEYWORDS: eutrophication; phosphate mobilization; sulfide production; Ruppia; coastal lagoons Marine-sediments; bassin-darcachon; organic-matter; phosphorus; iron; ecosystems; fractionation; reduction; exchange; ferrara]

Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can be a curative treatment for patients with hematological malignancies due to the capacity of allo-reactive donor derived T cells to mediate a curative potent graft versus leukemia (GVL) effect. However, associated acute graft versus host disease (GVHD) remains a major risk. To study the role of CD8+ T cells in GVL reactivity and GVHD, we selected patients who responded to DLI (without preceding cytoreductive treatment) for recurrent disease or incomplete donor chimerism after alloSCT. The patients were grouped according to absence (7 patients) or presence (6 patients) of GVHD. To quantify the number of circulating activated CD8+ T cells before DLI and at the time of disease regression or conversion to full donor chimerism we measured the frequencies of CD8+ HLA-DR+ T cells in peripheral blood samples by flowcytometry. Before DLI, highly variable numbers of CD8+ HLA-DR+ T cells were found (37.8 ± 42.9 x106/L), that significantly increased after DLI (309±473 x106/L, p Disclosures: No relevant conflicts of interest to declare.

Patients with hematological malignancies can be successfully treated with allogeneic hematopoietic stem cell transplantation (alloSCT). Beneficial Graft-versus-Leukemia (GvL) reactivity, however, is often accompanied with undesired Graft-versus-Host Disease (GvHD). In HLA-matched alloSCT, donor T-cells can mediate GvL reactivity by recognition of minor histocompatibility antigens (MiHA) on malignant cells, but also GvHD when MiHA are recognized on non-hematopoietic tissues. To decrease the incidence and severity of GvHD, T-cells can be (partially) depleted from the graft and re-administered later as donor lymphocyte infusion (DLI). MiHA are polymorphic peptides that are presented in HLA-molecules and can be recognized as non-self by donor-derived T-cells. Only a minority of MiHA have therapeutic relevance based on hematopoietic-restricted expression and only 25% and 40% of recipients transplanted with sibling and unrelated donors, respectively, are eligible for T-cell therapies in which one of the known hematopoietic-restricted MiHA is targeted. Therefore, to increase the efficacy and limit the toxicity of T-cell therapy, more therapeutic MiHA are needed. Recently, we identified a MiHA encoded by ARHGDIB, a gene that has been described to be highly expressed on hematopoietic cells, and we here studied the therapeutic relevance of LB-ARHGDIB-1R in detail. First, we confirmed hematopoietic-restricted expression of ARHGDIB by microarray gene expression analysis and demonstrated >10-fold overexpression in the majority of malignant and healthy hematopoietic versus non-hematopoietic cells. In line with its hematopoietic-restricted gene expression profile, LB-ARHGDIB-1R in the context of HLA-B* 07:02 was specifically recognized on different hematological malignancies, but not on non-hematopoietic fibroblasts and keratinocytes cultured in the absence or presence of IFN-γ, which was added to mimick the pro-inflammatory cytokine milieu of the early post-transplantation period. Next, we investigated the cytolytic capacity of LB-ARHGDIB-1R specific T-cells, and demonstrated specific lysis of patient, but not donor, EBV-B cells and specific lysis of an ALL sample in a 10 hrs 51Cr-release assay. Specific lysis of additional ALL and AML samples could be measured after 48 hrs of co-incubation in a flowcytometry-based cytotoxicity assay. To determine the in vivo immunogenicity of LB-ARHGDIB-1R, 11 MiHA-disparate HLA-B* 07:02 positive patient-donor pairs were screened for specific CD8+ T-cells by dual color tetramer analysis. All patients received partial T-cell depleted alloSCT and sampling was done at different time points after alloSCT (and DLI). In 4 out of 11 patients, LB-ARHGDIB-1R-specific T-cells (>0.01%) could be detected directly ex vivo and in 4 additional patients after 7 days of in vitro peptide stimulation, indicating that LB-ARHGDIB-1R is highly immunogenic. High frequencies of LB-ARHGDIB-1R specific T-cells were measured ex vivo in a patient whose hematological relapse was successfully treated with DLI, without development of GvHD, further supporting the therapeutic relevance of LB-ARHGDIB-1R. In summary, we confirmed hematopoietic-restricted expression of LB-ARHGDIB-1R and demonstrated T-cell mediated lysis of primary leukemic cells in long-term co-incubation assays. Furthermore, we showed that LB-ARHGDIB-1R is highly immunogenic and that specific T-cells could be detected in a patient who responded to DLI in the absence of GvHD. Altogether, our data support the clinical relevance of LB-ARHGDIB-1R as therapeutic MiHA with the potential to shift the delicate balance between GvL and GvHD in favor of a desired anti-tumor effect. Disclosures: No relevant conflicts of interest to declare.

Abstract 4116 Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can mediate curative Graft-versus-Leukemia (GVL) reactivity although frequently at the cost of Graft-versus-Host Disease (GVHD). We previously illustrated that donor CD8 T lymphocytes recognizing HLA class-I restricted minor histocompatibility antigens (MiHAs) that are broadly expressed on tissues of the recipient cause GVL associated with GVHD, whereas T lymphocytes recognizing MiHAs selectively expressed on hematopoietic cells, including the malignant cells, can selectively mediate GVL without GVHD. Since in contrast to HLA class-I, expression of HLA class-II molecules is predominantly restricted to hematopoietic cells, we hypothesized that infused purified donor CD4 T lymphocytes may selectively recognize and eliminate hematopoietic cells from the recipient resulting in GVL without GVHD. We treated a patient with CML in blastic phase in remission after intensive chemotherapy with T cell depleted alloSCT from his HLA-identical sibling donor after myelo-ablative conditioning. After donor engraftment, recipient hematopoiesis reoccurred within 3 months to 90% of CD8 T lymphocytes, 13% of CD4 T lymphocytes and 5% of myelopoiesis. As part of a clinical trial, the patient was treated with 106/kg positively selected purified donor derived CD4 T lymphocytes resulting within 19 weeks in conversion into full donor chimerism in all hematopoietic cell lineages in the total absence of GVHD. To characterize the nature of this hematopoiesis restricted immune response, in vivo activated HLA-DR positive CD4 and CD8 T lymphocytes were clonally isolated by flowcytometric cell sorting at the time of the clinical response, expanded and tested for alloreactivity on patient and donor derived hematopoietic target cells using IFNγ ELISA. From the 204 expanding CD4 T lymphocyte clones 31 clones were alloreactive, whereas none of the 66 expanding CD8 T lymphocyte clones showed alloreactivity. To further identify the fine specificity of this hematopoiesis directed HLA class-II restricted immune response, target molecules of several T lymphocyte clones were molecularly characterized using whole genome association scanning. We first performed blocking studies with HLA class-II restricted monoclonal antibodies and identified HLA-DR to be the restriction molecule. Next, a large panel of third party EBV-LCLs was retrovirally transduced with each of the possible restriction molecules being HLA-DRB1*11:01, HLA-DRB1*15:01, HLA-DRB3*02:02 and HLA-DRB5*01:01. By comparing the recognition pattern of the transduced EBV-LCLs with the 1.1 million single nucleotide polymorphisms in each EBV-LCL, we identified 3 novel MiHAs. Synthesis and analysis of the patient and donor derived allelic peptide variants further confirmed the specificity of the MiHAs as LB-KHNYN-1K in the context of HLA-DRB5*01:01, LB-CTSB-1G in HLA-DRB1*11:01 and LB-ZDHHC13-1K in HLA-DRB1*15:01. Gene expression profiles of KHNYN (located on chromosome 14), CTSB (chromosome 8) and ZDHHC13 (chromosome 11) illustrated that the genes encoding these MiHAs were not only transcribed in hematopoietic cells, but also in other tissues including GVHD target tissues. These results further illustrated that the hematopoietic specificity of the CD4 T lymphocyte response was mainly defined by the restricted expression of the HLA-DR molecules on hematopoietic cells. We conclude that purified CD4 DLI can lead to GVL without GVHD by a selective HLA class-II restricted immune response against patient hematopoiesis. By molecular characterization of 3 novel HLA-DR restricted MiHAs we illustrated that the relative specificity of HLA class-II molecules on hematopoietic cells under non inflammatory conditions was probably responsible for this effect. Since HLA class-II is predominantly expressed on hematopoietic cells only, infusion of donor CD4 T lymphocytes under non inflammatory conditions after HLA identical alloSCT can result in efficient induction of GVL without the toxicity of GVHD. Disclosures: No relevant conflicts of interest to declare.

Clinical studies indicated an enhanced antileukemic effect of allogeneic bone marrow transplantation (BMT), as compared with autologous BMT. After allogeneic HLA-identical BMT, donor-derived cytotoxic T lymphocytes (CTLs) directed at minor histocompatibility (mH) antigens on the recipients, tissues can be shown. To evaluate the antileukemic reactivity of mH antigen-specific CTLs, we analyzed the expression of mH antigens on circulating lymphocytic and myeloid leukemic cells. We show that the defined mH specificities HA-1 through HA-5 and H-Y are present on leukemic cells, indicating that mH antigen- specific CTLs are capable of HLA class I-restricted antigen-specific lysis of leukemic cells. Compared with interleukin-2-stimulated normal lymphocytes, leukemic cells of lymphocytic origin are less susceptible to T-cell-mediated cytotoxicity by the HA-2 mH antigen-specific CTL and the anti-HLA-A2 CTL clone. A possible explanation for this phenomenon is impaired expression of the LFA-1 adhesion molecule. Our study suggests that mH antigen-specific HLA class I-restricted CD8+ CTLs may be involved in the graft-versus-leukemia reactivity after allogeneic BMT.

Allogeneic bone marrow transplantation (BMT) has been associated with a graft-vs.-leukemia (GVL) reactivity. Since T cell depletion of the bone marrow graft has decreased the risk of graft-vs.-host disease (GVHD), but has been associated with higher rates of leukemia relapse, GVL reactivity is probably caused by donor-derived T lymphocytes. Previously, we demonstrated that minor histocompatibility (mH) antigen-specific cytotoxic T lymphocyte (CTL) clones, generated from patients after BMT, are capable of major histocompatibility complex-(MHC) restricted lysis of (clonogenic) myeloid leukemic cells. Here, we investigated whether donor-derived leukemia-specific CTL clones can be generated in vitro, before BMT, using irradiated leukemic cells from a patient with acute myeloid leukemia as stimulator cells, and peripheral blood or bone marrow from the HLA genotypically identical sibling donor as responder cells. Several CTL lines were generated that showed specific lysis (> 50%) of the recipient leukemic cells in a 51Cr-release assay. Two of these CTL lines were cloned by limiting dilution in the presence of the irradiated recipient cells. Multiple leukemia-reactive, HLA class I and II-restricted clones with various specificities could be established. These alloreactive, antileukemic CTL clones may cause GVL reactivity after BMT, and may be used as adjuvant immunotherapy in the treatment of leukemia.

Abstract 4084 Poster Board III-1019 Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T-cells recognizing minor histocompatibility antigens (mHags) encoded by single nucleotide polymorphisms (SNPs) on malignant cells of the recipient. Donor T-cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues. The aim of this study was to investigate the specificity and diversity of mHags recognized by T-cells in Graft-versus-Leukemia (GvL) reactivity. Activated (HLA-DR+) CD8+ and CD4+ T-cell clones were isolated from a patient successfully treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. GvL reactivity in this patient was accompanied with mild GvHD of the skin. Isolated T-cell clones were shown to recognize 13 different mHags. CD8+ T-cell clones were specific for HA-1 and HA-2 in HLA-A*0201, one unknown mHag in B*0801 and 4 unknown mHags in B*4001. CD4+ T-cell clones were specific for one unknown mHag in HLA-DQ and 5 unknown mHags in DR. By screening plasmid (class I) and bacteria (class II) cDNA libraries, we identified a mHag in HLA-DQ encoded by the PI4K2B gene (Griffioen et al., PNAS 2008), 4 mHags in HLA-DR encoded by the PTK2B, MR-1, LY75 and MTHFD1 genes (Stumpf et al., Blood 2009) and a mHag in B*4001 encoded by the TRIP10 gene. For the 3 T cell clones recognizing unknown mHags in B*4001, we performed Whole Genome Assocation scanning (WGAs). A panel of 60 EBV-LCL was retrovirally-transduced with B*4001 and tested for T-cell recognition. In parallel, genomic DNA was isolated and more than one million single nucleotide polymorphisms (SNPs) were determined by the Illumina beadchip array. Statistical analysis revealed significant association between T-cell recognition of EBV-LCL and the presence of coding SNPs in the SON DNA-binding protein and SWAP-70 genes. To get more insight into the role and potential use of the mHags in GvL reactivity and GvHD, all T-cell clones were analyzed in detail for reactivity against hematopoietic and non-hematopoietic cells. Hematopoietic cells included peripheral blood cells (monocytes, B-cells and T-cells), professional antigen presenting cells (APC) and leukemic cells (CML, ALL and AML). All CD8+ T-cell clones recognized (subsets of) peripheral blood cells as well as CML cells, except for the T-cell clone for TRIP10. Recognition of (subsets of) peripheral blood cells was also observed for all CD4+ T-cell clones, but CML cells were differentially recognized. CML cells were strongly recognized by the T-cell clones for MTHFD1 and the unknown mHag in HLA-DR, whereas no or low reactivity was observed for all other CD4+ T-cell clones. All CD8+ and CD4+ T-cell clones strongly recognized professional APC, including monocyte-derived dendritic cells and in vitro differentiated CML cells with APC phenotype. All T-cell clones were also capable of recognizing AML and ALL, except for the T-cell clone for TRIP10, which showed restricted recognition of AML-M4 and -M5 of monocytic origin. As non-hematopoietic cells, patient-derived fibroblasts were cultured with and without IFN-γ and tested for T-cell recognition. In the absence of IFN-γ, all T-cell clones failed to recognize fibroblasts, except for the T-cell clone for the unknown mHag in B*0801. After treatment with IFN-γ, additional reactivity was observed for the T-cell clones for SON DNA-binding protein and the unknown mHag in B*4001. Our data showed the specificity and diversity of mHags recognized by T-cells induced in a patient successfully treated with DLI for relapsed CML. The T-cell response was directed against 13 different mHags, of which 10 mHags in HLA class I and class II have now been identified by different techniques. Detailed analysis of T-cell recognition of hematopoietic and non-hematopoietic cells provides evidence that the mHags played different roles in the onset and execution of GvL and GvHD. Moreover, only one of the 10 identified mHags was expressed on fibroblasts after treatment with IFN-γ, indicating the characterization of mHags with potential relevance for T-cell based immunotherapy. Disclosures: No relevant conflicts of interest to declare.

In unrelated donor hematopoietic stem cell transplantation (URD-SCT) patients are preferably transplanted with stem cells from a fully HLA matched donor, usually defined as identical for HLA-class I, -DR and -DQ. Since HLA-DPB1 is often not taken into consideration in donor selection, 80–90% of URD-SCTs are mismatched for HLA-DPB1. The role of HLA-DPB1 as transplantation antigen has been unclear, since clinical reports on the impact of matching for HLA-DPB1 on transplant outcome showed conflicting results. HLA-DPB1 mismatching has been associated with an increased risk of graft versus host disease (GVHD). However, we recently demonstrated that HLA-DPB1 specific T cells can mediate a potent graft versus leukemia effect without inducing GVHD. It has been suggested that the controversial effects of matching for HLA-DPB1 in URD-SCT could partly be explained by the assumption that not all HLA-DPB1 differences are immunogenic. This theory was based on the cross-reactive recognition of two HLA-DPB1* 09 specific T cell clones that recognized other HLA-DPB1 alleles sharing amino acids (aa) in position 8–11 of HLA-DPB1 (Zino et al, blood 2004). It was hypothesized that there would be no induction of T cell responses between individuals expressing HLA-DPB1 molecules sharing this aa sequence. This was translated into a classification of permissive and non-permissive HLA-DPB1 mismatches in order to allow a broader donor selection. To investigate whether cross-reactive recognition of other HLA-DPB1 molecules by our previously generated HLA-DPB1*02 or *03 specific CD4+ T cell clones depended on the presence of specific aa sequences we tested recognition of a panel of 14 EBV-LCL expressing 9 different HLA-DPB1 molecules. All HLA-DPB1*02 as well as all *03 specific T cell clones showed cross-reactivity with other HLA-DPB1 alleles and each T cell clone exhibited its own pattern of cross-reactivity. Two HLA-DPB1*0201 specific T cell clones with different TCR-Vβ showed also recognition of EBV-LCL expressing HLA-DPB1*1001 and *1701 or HLA-DPB1*1001, *0901 and *1601 respectively. Five HLA-DPB1*03 reactive T cells clones with different TCR-Vβ showed differential cross-recognition of EBV-LCL expressing HLA-DPB1*0101, *0601, *1101, *1301 and *1401. To identify immunogenic differences the aa sequences of the HLA-DPB1 molecules recognized by the various T cell clones were compared. The HLA-DPB1 molecules recognized by the HLA-DPB1*02 specific T cell clones shared an aa substitution at position 69 compared to the responder cell. However, HLA-DPB1*0601,*0901 and *1901 with the same substitution were not recognized by both T cell clones. This phenomenon was also observed for the HLA-DPB1*03 specific T cell clones, indicating that the cross-reactive recognition of HLA-DPB1 could not be predicted by aa sequences. Next, we analyzed the immunogenicity of various HLA-DPB1 alleles in different stimulator/responder combinations to verify the classification of permissive and non-permissive mismatches. We developed a model to generate allo-HLA-DP responses by transducing HLA-class II negative HELA cells with various HLA-DP molecules and used these cells to stimulate purified CD4+ T cells from HLA-DPB1 homozygous donors. HELA cells transduced with HLA-DPB1*0101, *0201, *0301, *0401, *0402, *0501, *0601, *0901, *1101, *1301, *1401 or *1701 were used as stimulator cells. Responder CD4+ T cells were typed HLA-DPB1* 0201, *0301, *0401 or *0402. 14 days after stimulation, CD4+ T cells were tested for recognition of the stimulator cells and of HELA cells transduced with the responder HLA-DPB1 molecule as a negative control. For these 4 responders, stimulation with 12 different HLA-DP transduced HELA cell lines resulted in specific IFN-γ production in response to the stimulator cells in 47 out of 48 stimulations. 28 CD4+ T cell lines also showed cross-reactive recognition of HELA cells transduced with at least one other HLA-DPB1 molecule. In conclusion, we showed that cross-reactive recognition of various HLA-DPB1 molecules by HLA-DPB1 specific T cells is a common observation. However, we demonstrated that cross-reactivity between HLA-DPB1 molecules by allo-HLA-DPB1 specific T cells does not exclude the generation of immune response between individuals expressing these HLA-DPB1 molecules. By generating multiple allo-HLA-DP specific T cell lines, we showed that all HLA-DPB1 mismatch combinations are immunogenic.

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLI). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, mHag specific CD4+ T cells may selectively mediate Graft-versus-Leukemia (GvL) reactivity without Graft-versus-Host Disease (GvHD). Clinical studies have shown that adoptive transfer of CD4+ donor lymphocytes after HLA-matched alloSCT may lead to clinical remissions with a reduced incidence of GvHD, emphasizing the relevance of CD4+ T cells and HLA class II associated mHags for development of effective anti-tumor T cell therapies after alloSCT with a low risk for GvHD. The aim of this study was to identify the HLA class II associated mHag that is recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLA-matched alloSCT who developed strong GvL reactivity with mild GvHD of the skin after treatment with DLI. We previously developed recombinant bacteria cDNA expression libraries based on delivery of exogenous antigens for identification of HLA class II antigens and used this method for identification of the first autosomal HLA class II (HLA-DQB1*0603) associated mHag LB-PI4K2B-1S. LB-PI4K2B-1S has a population frequency of 40–50% and is encoded by the broadly-expressed phosphatidylinositol 4-kinase type II β gene. In the patient with CML, a polyclonal CD4+ T cell response against LB-PI4K2B-1S and simultaneous mHag specific CD8+ T cells were demonstrated. LB-PI4K2B-1S specific CD4+ T cells were shown to recognize the CD34+ CML cells of the patient as well as other leukemic cells. Recognition and lysis of normal hematopoietic cells by LB-PI4K2B-1S specific CD4+ T cells critically depended on the number of HLA-DQ molecules expressed at the cell surface and was restricted to high HLA-DQ-expressing B cells, mature dendritic cells (DC) and EBV-transformed B cells. HLA-DQ expression on T cells, PHA-stimulated blasts, monocytes and immature DC was absent or low and not sufficient for T cell recognition. We also demonstrated that HLA-DQ expression on normal cells of non-hematopoietic origin after extensive culturing with IFN-γ was moderately upregulated as compared to HLA-DR and -DP and not sufficient for recognition by LB-PI4K2B-1S specific CD4+ T cells. In conclusion, the data suggest that LB-PI4K2B-1S specific CD4+ T cells mediated tumor rejection by directly eliminating the malignant cells of the patient as effector cells and stimulating the induction and maintenance of CD8+ T cell immunity as helper cells, and HLA-DQ associated mHags may be appropriate targets for T cell therapies with the aim to selectively stimulate GvL after HLA-matched alloSCT with a low risk for GvHD.

Mismatching for HLA-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a significant decreased risk of disease relapse, indicating that HLA-DP might be a target for a graft versus leukemia (GVL) effect in HLA-class II expressing hematological malignancies. To determine whether a specific GVL effect could be caused by allo-HLA-DP specific T cells, we analyzed the immune response in a patient with a refractory immunocytoma responding to donor lymphocyte infusion (DLI) after HLA-DP mismatched URD-SCT. Patient and donor were fully matched for HLA-A, -B, -C, -DR and -DQ, but differed for both HLA-DP alleles (donor HLA-DPB1*0402/0501; patient HLA-DPB*020102/0301). The patient received a T cell depleted URD-SCT after a non-myeloablative conditioning regimen, resulting in mixed chimerism (75% donor) without GVHD. Because of a hematological relapse, a single DLI was given 6 months after SCT, resulting in a profound anti-leukemic effect with only grade I GVHD, treated with topical corticosteroids. 6 weeks after DLI, malignant cells in peripheral blood (PB) had dropped from 72% to 47%. 7 weeks later, only 3% malignant cells were present, and after 4 months, complete remission and conversion to full donor chimerism in the absence of GVHD was observed. To determine whether allo-HLA-DP specific T cells were involved in the immune response, leukemia-reactive donor T cell clones were isolated from PB or bone marrow at different time points during the response to DLI. Patient derived T cells were overnight stimulated with irradiated leukemic cells harvested before transplantation, and clonal IFNγ producing T cells were sorted and expanded. 21 CD4+ T cell clones, 19 CD8+ T cell clones and 6 NK cell clones were tested for recognition of patient or donor derived cells as measured by IFNγ production and cytotoxic activity. The CD8+ or NK clones did not recognize patient leukemic cells. However, all 21 CD4+ clones produced INFγ in response to patient leukemic cells but not to donor cells. To determine whether these CD4+ T cell clones were capable of killing the leukemic cells, a CFSE based cytotoxicity assay was performed. 8 clones showed 30–90% lysis of the leukemic cell population. To further analyze the specificity of these CD4+ clones, blocking and panel studies were performed. Blocking with the HLA-DP specific mAb B7.21 abrogated IFNγ production by all clones, confirming HLA-DP restricted recognition. A panel study using 12 unrelated EBV-LCL expressing different HLA-DP alleles identified 18 clones specific for HLA-DPB1*0301, and 3 clones specific for HLA-DPB1*0201. To analyze the polyclonality of the immune response, the distribution of TCR Vβ chains was characterized by RT-PCR and sequence reactions. 7 different Vβs were found within the HLA-DPB1*0301 specific clones and 3 different Vβs within the HLA-DPB1*0201 specific clones. T cells using the same Vβ could be isolated at different time points during the clinical response, demonstrating the significance of this anti-HLA-DP response. In conclusion, we observed in a patient with an HLA-class II positive B cell malignancy a profound GVL effect without GVHD, caused by a polyclonal immune response comprising both T helper and cytotoxic CD4+ HLA-DP specific T cell clones directed against both HLA-DP alleles. These data indicate that in HLA-class II expressing hematological malignancies HLA-DP mismatched SCT may be preferable over a fully matched SCT making use of HLA-DP as a specific target for immunotherapy.

Donor lymphocyte infusion (DLI) induces complete remissions in 70–80% of patients with a relapse of CML after allogeneic stem cell transplantation (SCT). However, in some patients molecular persistence of bcr-abl positive stem cells can be found. To determine possible mechanisms of this persistence we studied in detail a patient who received DLI for a hematological relapse after HLA identical sibling SCT. After obtaining a hematological and cytogenetic remission in the absence of any graft versus host disease persistence of molecular disease was observed despite multiple additional escalating doses of DLI. To determine the nature of this T cell response, donor T cells isolated from bone marrow of the patient at the time of clinical response were stimulated with CML cells from the patient. T cells responding to the leukemia by secreting IFN-γ were isolated, directly cloned by cell sorting and expanded. Four different types of CD8+ HLA class I restricted CTL clones were obtained. All clones were cytolytic against EBV-LCL of the patient, but did not lyse EBV-LCL of the donor, indicating recognition of a minor histocompatibility antigen (mHag). PHA blasts of patient and donor were not lysed, indicating that the clones recognized an antigen present on B-cells but not on T-cells. To further study the lineage restriction of the clones, bone marrow and peripheral blood CML cells were stained with CFSE and incubated with the clones. The survival of different cell populations was measured by counterstaining with different monoclonal antibodies (Jedema, Blood2004; 103: 2677). Two clones only lysed CD14 positive monocytic cells, two other clones lysed CD14 positive monocytic cells as well as CD13–33 positive myeloid cells. Interestingly, immature myeloid cells were not lysed, whereas recognition by the T cells did occur after maturation of these cells in vitro by growth factors. By morphological examination it was demonstrated that the cell clones are capable of lysing myelocytes but not myeloblasts. These results indicate that the clones recognize a mHag expressed during myeloid maturation. Subsequently, we investigated whether the CTL clones could lyse CML progenitor cells using a hematopoietic progenitor cell inhibition assay (van der Hoorn, Methods2003; 31: 113). In the presence of the CTL clones persistent proliferation of 20–40% was found, in contrast to the complete proliferation inhibition of CML progenitors observed when mHag clones HY-A1 and HA-1 were used as control. Secondly, survival studies showed that the CTL clones were not able to lyse CFSE labelled CD34 positive CML cells, in contrast to the control clones HY-A1 and HA-1. In conclusion, different types of CD8+ mHag-specific CTL clones recognizing lineage specific antigens have been isolated from a relapsed CML patient with molecular persistence of bcr-abl after repeated administrations of DLI. These clones recognize mHag’s present on B-cells, monocytic cells and mature myeloid CML cells, but not on CML progenitor cells. The isolation of these CTL’s in the absence of T cells against CML progenitor cells explains the clinical hematological remission but molecular disease persistence of this patient after DLI. These findings suggest that for adequate eradication of CML a cytotoxic T cell response against leukemic progenitor cells is needed.