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Introduction Acute myeloid leukemia (AML) is a high-risk malignancy with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative if it induces a potent graft versus leukemia (GvL) response. GvL responses and graft versus host disease (GvHD) are typically considered T cell mediated, because T cell depletion from hematopoietic stem cell grafts reduces the risk of GvHD at the cost of leukemia relapse. In addition, depletion of B-lymphocytes with rituximab has led to amelioration of GvHD in a number of studies suggesting that B cells are also important in the pathophysiology of GvHD and, in analogy, in GvL responses. However, the characteristics of the antibodies produced by these leukemic specific B cells have not yet been studied. Methods We selected three patients with high-risk myelomonocytic leukemia who remained disease free years after allogeneic HSCT, from whom we established clonal human B cell lines, using a unique and innovative technology that was developed in our laboratory (Kwakkenbos ea, Nat Med 2010). These B cell lines, that concomitantly express immunoglobulin on their membranes and secrete antibodies, were used to select antibodies specific for cell surface antigens on AML cell lines with similar morphologic and immunophenotypic characteristics as the patients’ leukemic blasts, using a FACS based assay. Results From each patient, several AML specific B cell clones were retrieved. Their antibodies recognized surface antigens on primary AML blasts derived from multiple patients and on AML cell lines, but not on healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. The majority of the antibodies were of the IgG3 isotype. Approximately 40% of the AML-specific antibodies induced direct death of cultured AML cell lines and of primary AML blasts. The cell death pathway induced by these cytotoxic antibodies was oncotic rather than apoptotic. Conclusion Our data demonstrate that high-risk AML patients with a potent GvL response mount robust antibody responses against surface antigens that are specifically expressed on tumor cells and binding of these antibodies induces direct cell death. The targets recognized by the recovered antibodies are also expressed on leukemic blast from other patients suggesting evidence for a common immune mechanism responsible for AML clearance. Our findings suggest that antibody responses are important in GvL and open up new ventures for specific antibody treatment of AML patients. Disclosures No relevant conflicts of interest to declare.

PurposeTo determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients.Patients and MethodsSixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1, and MAGE-A3 using human leukocyte antigen/peptide tetramers and functional assays.ResultsThe high-dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high-dose vaccine showed an increase in MART-1– or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease-free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1– and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced interferon-γ on specific antigenic stimulation.ConclusionWe conclude that vaccination with GM-CSF–transduced autologous tumor cells has limited toxicity and can enhance T-cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease-free survival of metastatic melanoma patients who are surgically rendered as having no evidence of disease before vaccination is worthy of further investigation.

There is an increasing amount of knowledge on the functional properties of regulatory T cells (Treg) in the adult immune system, but data on the generation and function of these cells during human embryonic development are scarce. In this study, we show that in the fetal thymus, double-positive cells initiate expression of CD25, GITR, CTLA4 and CD122 during their transition from the CD27(-) to the CD27(+) stage. Moreover, CD4(+)CD25(+) fetal thymocytes already have the potential to suppress proliferation of CD25(-) cells. After leaving the thymus, FoxP3(+)CD4(+)CD25(+) Treg enter the fetal lymph nodes and spleen, where they acquire a primed/memory phenotype. A model is proposed for the development of human fetal Treg that encompasses two sequential maturation steps: initiation of a regulatory phenotype and suppressive activity in the thymus; and subsequent activation within the peripheral lymphoid organs. Upon activation, FoxP3+CD4+CD25+ Treg suppress potentially deleterious responses by autoreactive lymphocytes and maintain homeostasis within the developing fetus

The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2-/- γc-/-mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of XGVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3+ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2-/- γc-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model. (Blood. 2003;102:2522-2531)

Human plasmacytoid dendritic cells (pCs), also called type 2 dendritic cell precursors or natural interferon (IFN)producing cells, represent a cell type with distinctive phenotypic and functional features. They are present in the thymus and probably share a common precursor with T and natural killer (INK) cells. In an effort to identify gene's that control pDC development we searched for genes of which the expression is restricted to human pDC using a cDNA subtraction technique with activated monocyte-derived DCs (MoDCs) as competitor. We identified the transcription factor Spi-B to be expressed in pDCs but not in Mo-DCs. Spi-B expression in pDCs was maintained on in vitro maturation of pDCs. Spi-B was expressed in early CD34(+)CD38(-) hematopoietic progenitors and in CD34(+)CD1a(-) thymic precursors. Spi-B expression is down-regulated when uncommitted CD34(+)CD1a(-) thymic precursors differentiate into committed CD34(+)CD1a(+) pre-T cells. Overexpression of Spi-B in hematopoietic progenitor cells resulted in inhibition of development of T cells both in vitro and in vivo. In addition, development of progenitor cells into B and INK cells in vitro was also inhibited by Spi-B overexpression. Our results indicate that Spi-B is involved in the control of pDC development by limiting the capacity of progenitor cells to develop into other lymphoid lineages. (C) 2003 by The American Society of Hematology

This unit describes protocols for the generation of human (allo-) antigen-specific T cell lines, and T lymphocyte subpopulations with distinct cytokine production profiles from purified peripheral or cord blood CD4+ T cells, respectively. Methods for the cloning and maintenance of these cell lines are given, as well as a protocol for freeze/thaw procedures.

Senescence limits the proliferative capacity of primary cells in culture. We describe here a genetic screen to identify genes that allow bypass of this checkpoint. Using retroviral cDNA expression libraries, we identify BCL6 as a potent inhibitor of senescence.BCL6 is frequently activated in non-Hodgkin's lymphoma, but its mechanism of action has remained unclear. BCL6 efficiently immortalizes primary mouse embryonic fibroblasts and cooperates with RAS in oncogenic transformation. BCL6 overrides the senescence response downstream of p53 through a process that requires induction ofcyclin D1 expression, as cyclin D1 knockout fibroblasts are specifically resistant to BCL6 immortalization. We show that BCL6 expression also dramatically extends the replicative lifespan of primary human B cells in culture and induces cyclin D1 expression, indicating that BCL6 has a similar activity in lymphoid cells. Our results suggest that BCL6 contributes to oncogenesis by rendering cells unresponsive to antiproliferative signals from the p19ARF–p53 pathway.

Background & Aims: Systemic treatment of Crohn's disease patients using recombinant interleukin (rIL)-10 has not resulted in significant therapeutic benefit presumably because of limited bioavailability and unexpected proinflammatory effects of high-dose rIL-10. Ex vivo gene transfer of the interleukin (IL)-10 gene to gut-homing CD4 + cells may lead to improved long-term management. Methods: Peripheral blood mononuclear cells (PBMCs) were transduced with a retroviral vector containing the IL-10 and green fluorescent protein (GFP) gene or a control vector containing GFP only. Transduced CD4 + cells were sorted and maintained in culture for phenotypic and functional analysis. Results: Stimulated IL-10–GFP CD4 + cells produced significantly higher levels of IL-10 than control cells for at least 4 months. The IL-10 transgene was biologically active and decreased proliferation of IL-10–GFP CD4 + cells as well as expression of major histocompatibility class (MHC) class II, proliferation of autologous responder cells, and IL-12 production by dendritic cells (DCs). The majority of transduced CD4 + cells had a gut-homing potential because they expressed the mucosal integrin α4β7, and displayed efficient binding to MAdCAM-1–expressing cells in vitro. Conclusions: Transduction of peripheral blood CD4 + lymphocytes with IL-10 results in a regulatory phenotype. The use of regulatory gut-homing human CD4 + cells may provide a novel approach to local delivery of immunomodulatory signals to the intestine in Crohn's disease. GASTROENTEROLOGY 2002;123:1877-1888

The murine thymus contains two types of dendritic cells (DCs)-like cells. These two DC types can be distinguished on the basis of expression of MHC class II antigens: one expresses high levels and the other low levels of MHC class II antigens. Both DC types express CD8α and another DC marker, DEC-205, which is not expressed on CD8α – splenic DCs. There are also morphological differences between these subsets: MHC class II lo cells are round while the MHC class II hi cells are irregular-shaped veiled cells. Human CD34 + CD1a – thymic precursors have the capacity to develop into two types of DC, suggesting that different DC subsets can exist in human thymus. Upon culture in cytokine mixtures, DCs are generated that express high levels of CD13 and CD33. Many of these cells also express CD1a. When the CD34 + CD1a – cells are co-cultured with the S17 stromal cells, some of the cells develop into CD123 hi DC precursors that develop into mature DC following culture with IL-3 and CD40L.

The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generatedin vitroand introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequentin vitroselection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resultingin vitroselected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than thein vivogenerated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes.

Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8+ T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA+) and memory (CD45RO+) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA+ T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

The thymus is populated by hematopoietic cells that have the capacity to develop into at least three different hematopoietic lineages, T, NK and dendritic cells. While developing into T cells these cells pass a series of developmental stages that can be discriminated on the basis of expression of a number of antigens. The availability of a myriad of monoclonal anti- bodies against human differentiation antigens has permitted a detailed analysis of the various cellular stages in the human thymus. This analysis not only comprised investigation of molecular but also of functional features of purified thymocyte subsets, since more recently assays were set up that allowed investigation of the hematopoietic precursor activities of human thymic progenitor cells. Here we review the current status of knowledge with regard to early developmental stages in the human thymus. In addition, we discuss recent data on later developmental stages, in particular concerning positive selection and maturation of T cells.

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.

The superior ability of dendritic cells (DC) in triggering antigen-specific T cell responses makes these cells attractive tools for the generation of antitumor or antiviral immunity. We report here an efficient retroviral transduction system for the introduction of antigens into DC. A retroviral vector encoding several CTL epitopes in a string-of-beads fashion in combination with the marker gene green fluorescence protein (GFP) was generated. Polyepitope transduced EBV-LCL could be isolated on the basis of GFP expression and were found to be sensitive to lysis by antigen-specific cytotoxic T cells, demonstrating that antigens encoded by the retroviral construct were stably expressed, processed, and presented in the context of HLA class I molecules. CD34+ cells isolated from G-CSF mobilized peripheral blood were transduced with high efficiency (40–60%) with this retroviral construct. These cells could be considerably expanded in vitro and differentiated into mature DC without loss of the transduced antigen. DC transduced with the polyepitope constructs were able to mount a CTL response against an influenza epitope in the context of HLA-A2, demonstrating the antigen-specific CTL priming capacity of retrovirally transduced DC. Staining of the T cells with tetramers of HLA-A2 and the influenza virus peptide demonstrated a marked antigen-specific CTL enrichment after 2 in vitro stimulations using DC transduced with the polyepitope. However, additional in vitro stimulations of the T cells with transduced DC did not result in a further enrichment of tetramer staining cells.

The majority of lymphomas induced in Rag-deficient mice by Moloney murine leukemia virus (MoMuLV) infection express the CD4 and/or CD8 markers, indicating that proviral insertions cause activation of genes affecting the development from CD4(-)8(-) pro-T cells into CD4(+)8(+) pre-T cells. Similar to MoMuLV wild-type tumors, 50% of CD4(+)8(+) Rag-deficient tumors carry a provirus near the Pim1 protooncogene. To study the function of PIM proteins in T cell development in a more controlled setting, a Pim1 transgene was crossed into mice deficient in either cytokine or T cell receptor (TCR) signal transduction pathways. Pim1 reconstitutes thymic cellularity in interleukin (IL)-7- and common gamma chain-deficient mice. In Pim1-transgenic Rag-deficient mice but notably not in CD3gamma-deficient mice, we observed slow expansion of the CD4(+)8(+) thymic compartment to almost normal size. Based on these results, we propose that PIM1 functions as an efficient effector of the IL-7 pathway, thereby enabling Rag-deficient pro-T cells to bypass the pre-TCR-controlled checkpoint in T cell development.

Cytokine synthesis inhibitory factor (CSIF; interleukin-10), a product of mouse TH2 T cell clones that inhibits synthesis of cytokines by mouse TH1 T cell clones, exhibits extensive sequence similarity to an uncharacterized open reading frame in the Epstein-Barr virus BCRF1. Recombinant BCRF1 protein mimics the activity of interleukin-10, suggesting that BCRF1 may have a role in the interaction of the virus with the host's immune system.

It is firmly established that cytokines are involved in development of hematopoietic cells. Until recently it was not known whether cytokines are involved in development of lymphoid cells. Initially, interleukin (IL)-2 and IL-4 were considered to be candidates essential for lymphoid development. However, this turned out not to be the case since mice deficient for IL-2 [1], IL-4 [2] or both [3] had no obvious defects with regard to development of T and B cells. Nonetheless, it became clear that cytokines do play an essential role in T cell development when the molecular defect underlying the X chromosome linked severe combined immunodeficiency (X-SCID) was determined [4]. These patients have no T or NK cells but B cells are present in normal numbers. It turned out that these patients have mutations in the gamma chain of the IL-2 receptor complex [4]. As it was already known that IL-2 is not essential for T cell development, it was suspected that this component of the IL-2R was also part of receptors for other cytokines. This suspicion was confirmed soon thereafter when it was found that the IL-2Rγ chain is also a component of the receptors of IL-4, IL-7, IL-9 and IL-15 [5–7]. Therefore, the IL-2Rγ chain is now commonly referred to as the gamma common (γc) chain. Studies with IL-7 [8] and IL-7Rα chain deficient mice [9] havve made clear that IL-7 is the critical γc ligand involved in T cell development. Like the γc deficient mice [10], these mice have a thymus that is only 10% in size of the wild type thymus.

Background Innate lymphoid cells (ILCs) represent a novel family of effector and regulatory cells in innate immunity and tissue remodelling [1]. The family comprises several phenotypically and functionally distinct subsets that produce various cytokines such as IL-22, IL-17, IFNγ, TNF, IL-13 and IL-5, of which many have been shown to be important in arthritis pathogenesis. Objectives The IL-17 and IL-22 producing ILCs are of major interest as they are implicated in chronic gut inflammation [2]. Based on the broad clinical overlap between inflammatory bowel disease and spondyloarthritis (SpA) and the clinical importance of IL-17 in SpA we hypothesize that IL-17 and IL-22 producing ILCs contribute to inflammation and remodelling in SpA synovitis. As these cells have never been described in the joint we aimed at characterising ILCs in chronic inflammatory arthritis. Methods ILCs (lineage negative, CD45 + CD127 + ) were analysed and sorted by flow cytometry from synovial tissue and fluid from rheumatoid arthritis (RA) and SpA patients as well as in blood from SpA patients and healthy donors. mRNA expression of sorted and expanded cells was analysed by qPCR. Results ILCs were identified in blood as well as in synovial tissue and fluid from both RA and SpA patients. The frequency of ILCs was higher in the inflamed joint (0,5-3,3% of the lymphocyte population) than in the peripheral blood compartment (0,1%). In blood, there was no marked difference in the frequency of the different ILC subset between healthy controls (n=10) and SpA patients (n=5). In the inflamed joint, the ILC3 (CRTH2 - NKp44 + ckit + ) and ILC1 (CRTH2 - NKp44 - ckit - ) populations, previously shown to express IL-22 and IFNγ respectively in other tissues, were present in all samples whereas the Th2 cytokine expressing ILC2s (CRTH2+) were found in very low frequencies. Frequencies of ILC subpopulations varied considerably between patients and no differences could be detected between RA and SpA patients. qPCR analysis of expanded cells revealed that ILC1s expressed TBX21 whereas ILC3s expressed RORC . Accordingly, stimulated ILC3s expressed transcripts for both IL-23R and IL-22 but not IL-17. Conclusions ILC1s and ILC3s are present in the chronically inflamed joint and express the key transcription factors associated with specific cytokine profiles. These data indicate that ILCs could contribute to local cytokine-driven immune alterations in SpA and RA. References Spits H, Cupedo T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function. Annu Rev Immunol. 2012;30:647-75. Geremia, A., Arancibia-Carcamo, C. V., Fleming, M. P., Rust, N., Singh, B., Mortensen, N. J., Travis, S. P. & Powrie, F. IL-23-responsive innate lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011;208:1127-1133. Disclosure of Interest H. Hreggvidsdottir: None Declared, J. Mjosberg: None Declared, J. Bernink: None Declared, D. Baeten: None Declared, H. Spits Employee of: AIMM Therapeutics

Background Rheumatoid arthritis (RA) is a prototypic autoimmune disease characterised by a prominent humoral autoimmunity. Of particular relevance is the local production of autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies in the inflamed synovial tissue. The mechanisms underlying break of B cell tolerance and local autoantibody production remains poorly understood. Objectives To identify cellular and molecular pathways implicated in RA-specific humoral autoimmunity. Methods Synovial tissue samples were obtained by arthroscopy from untreated individuals with RA (n = 33) and inflammation matched SpA controls (n = 58). Gene expression profiling was performed on tissue samples of patients with established arthritis using 44K. Whole Genome Human microarrays (Agilent). Top differentially expressed genes were validated on three independent cohorts by Taqman based RT-qPCR and immunohistochemistry. Collagen-induced arthritis (CIA) and Experimental autoimmune encephalomyelitis (EAE) experiments were conducted using Bob1 knockout mice and their littermate controls. Results Microarray screening for genes differentially expressed in the inflamed synovium, the key target of the disease process in RA, revealed a prominent and disease-specific B cell/plasma cell signature with the B cell-specific transcriptional co-activator Bob1 and its transcriptional target BCMA among the most upregulated genes. Validation by RT-qPCR on two independent cohorts representing early and established arthritis confirmed microarray data and demonstrated elevated expression of Bob1 and BCMA not only in established RA, but also at the early phase of the disease. Quantitative evaluation of immunohistochemical stainings of synovial tissue with monoclonal antibody for Bob1 revealed significant increase in Bob1 positive cells in RA synovium (p − / − mice were fully resistant to CIA induction compared to their wild-type littermates. This remarkable protection from CIA is explained by decreased antigen-presentation/costimulatory capacity of B cells and by failure to produce pathogenic anti-collagen autoantibodies in the absence of Bob1. Conclusions The specific increase in Bob1 expressing cells in RA synovitis and the resistance of Bob1-deficient mice to development of CIA indicate that Bob1/BCMA axis may contribute to humoral autoimmunity in RA. The relationship between an aberrant Bob1 expression and the break of peripheral tolerance in RA is currently under investigation.

The discovery of the lymphokine interleukin 2 (IL-2), which induces growth of T cells, set the stage for establishing methods for in vitro cloning of human T cells. More recently, it has become clear that in addition to IL-2, other T-cell growth factors, such as IL-4 and IL-7, can be used for generation and expansion of T-cell clones. In the past it was shown that it is possible to grow T cells differing in function and phenotype. In vitro-expanded T-cell clones have been instrumental in studies on the antigen specificities and biological properties of these cells. Cloned lines of natural killer (NK) cells can also be established using IL-2, permitting detailed studies on the biology of these cells. Here we describe the methods we use to generate and to expand human T-cell and NK cell clones. We discuss the differences between IL-2 and IL-4 as growth factors to expand T-cell clones. In addition, we summarize some recent findings of studies using T- and NK cell clones.

CD50 (ICAM-3) has been identified as the third CD11a/CD18 (LFA-1) counter receptor. We investigated the expression and possible role of this molecule in the induction of early and late activation events in human thymocytes. We observed that CD50 expression is acquired by early T cell progenitors (CD34+) and maintained during thymic development, reaching the highest levels in the most mature population of thymocytes (CD3high). Neither basal nor cytokine-induced expression of CD50 was observed on untransformed human thymic epithelial cell lines. Cross-linking of CD50 expressed on the surface of human thymocytes, by using mAbs recognizing epitopes not related to the CD11a binding site, transduced transmembrane signals leading to an increase of intracellular calcium concentration. This calcium mobilization was inhibited when CD50 was co-cross-linked with CD45, suggesting that tyrosine phosphorylation is also involved in CD50 signaling. The same anti-CD50 mAbs that were able to affect intracellular calcium levels were shown to induce CD69 but not CD25 expression on human thymocytes. This effect was preferentially observed on CD3low/CD3high thymocyte subpopulations. Cross-linking of CD50 also significantly increased activation-induced cell death of human thymocytes. These results support the idea that CD50 molecule can play a role in developing functionally mature T lymphocytes.

Retroviral vectors have been used in most human gene therapy trials that have been undertaken. Many of these therapies have focused on the introduction of genes into hematopoietic stem cells with the goal of obtaining expression in the mature T lymphocytic progeny. It has proven difficult to achieve expression in the lymphoid lineage, although several groups have demonstrated low expression of transduced genes in the myeloid lineage. In this study we used an in vitro thymic organ culture in which stem/progenitor cells can develop into T cells and all intermediate stages can be studied and manipulated to investigate the fate of a retrovirally introduced Escherichia coli LacZ gene in this system. Here we show that certain conditions can transduce Jurkat T cells, three different antigen-specific T cell clones and CD34+CD3-CD4-CD8- thymocytes (progenitor T cells) with high (> 80%) efficiency. Moreover, retroviral transduction with the LacZ gene does not inhibit T and NK cell differentiation of progenitor cells in fetal thymic organ cultures (FTOC). The LacZ gene also is functionally expressed at all stages of development, although the expression decreases somewhat during differentiation. This experimental system, combining FTOC and retroviral transduction, provides a genetic tool for the study of human T cell development.

Abstract 4104 Introduction Patients undergoing hematopoietic stem cell transplantation (HSCT) are at increased risk of developing (severe) opportunistic infections, which can be attributed to the combination of a decimated immune system and disrupted epithelial barriers. Innate lymphoid cells (ILCs) represent a novel family of regulator and effector cells that share phenotypic and functional characteristics with both NK cells and T lymphocytes, but are non-cytotoxic and lack antigen specific receptors. ILCs serve important roles in the maintenance of epithelial integrity and - as part of the first line of defence - in protection against microbes. ILCs are mainly tissue-resident and can be identified by the absence of lineage markers (Lin-) and bright expression of the alpha chain of the IL-7 receptor (CD127). Extensive research has been carried out focussing on the relative contribution of different immune cells to immune deficiency and graft-versus-host disease (GvHD) following HSCT. However, little is known about ILCs in this context. We studied reconstitution dynamics of ILCs in order to explore their function in tissue repair and mucosal immunity following HSCT. Methods Blood samples of adult patients (n=15) receiving an allogeneic HSCT after reduced-intensity conditioning (RIC) for acute myeloid leukemia (AML) were obtained at sequential time points during induction, consolidation and conditioning chemotherapy, at the day of HSCT and up to 6 months following HSCT. Peripheral blood ILCs were analyzed using flow cytometry and defined as Lin-CD127hi. The ILC population was further divided into various subpopulations using the markers NKp44, CRTH2 and CXCR3. Results Induction and consolidation chemotherapy led to a significant decline in the number of circulating ILCs. Recovery of ILCs in between chemotherapy cycles was partial as ILC numbers improved without returning to healthy control values (n=10; reference values defined as upper and lower boundaries of 95% CI), in contrast to other innate cells such as neutrophils and NK cells. Following allogeneic HSCT, recovery dynamics of ILCs parallelled those of adaptive immune cells, rather than of innate cells: recovery was relatively slow, as normal numbers of circulating ILCs had not been reached 6 months after HSCT. In addition, we observed enrichment of a particular ILC subset, defined as Lin-CD127hiCD117+NKp44+ cells, in subgroups of patients after chemotherapy and HSCT. These RORγt-dependent ILCs have been found to comprise an important innate source of IL-22 (hence referred to as ILC22s), a cytokine crucial for epithelial homeostasis and induction of epithelial antimicrobial peptides. Discussion and conclusion Our observation of a relatively slow reconstitution of ILCs is notable since innate immune cells are generally known to recover within weeks to months after HSCT. In fact, ILC reconstitution following HSCT bears resemblance to post-transplantation adaptive lymphocyte recovery dynamics. Fitting with this notion, there is a certain degree of analogy between ILCs and T helper cells as ILCs with cytokine profiles resembling those of the various T helper subsets are found, i.e. ILCs producing mainly IL-13/5, IL-22, IL-17 or IFN-γ. Furthermore, proliferation and differentiation of the ILC subpopulations stands under the control of transcription factors known to be master regulators of the different T helper subsets (i.e. GATA3, Tbet, RORγt and AHR). Hence, our data show that the analogy between the ILC and T helper systems in terms of factors that control differentiation and function is matched by their similarities in reconstitution dynamics following chemotherapy and HSCT. Interestingly, following chemotherapy and allogeneic HSCT, ILC22s, known to be primarily dedicated to the maintenance of epithelial homeostasis, were found to be enriched in subgroups of patients. Damage to mucous membranes lining the gastrointestinal tract, due to chemotherapy-induced mucositis, conditioning radiotherapy or graft-versus-host disease, is a common problem that constitutes a significant limiting factor in HSCT. Therefore, improvements in the prevention or management of such epithelial damage are eagerly awaited. Our data suggest that ILCs may be involved in protection against and/or recovery of epithelial damage in patients with a hematological malignancy receiving chemotherapy and HSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract SCI-19 Innate lymphoid cells (ILC) are immune cells that lack a specific antigen receptor, yet possess the capacity to produce an array of effector cytokines that in variety matches that of T-helper-cell subsets. Innate lymphoid cells function in lymphoid organogenesis, tissue remodeling antimicrobial immunity and inflammation, particularly at barrier surfaces. The ability of ILCs to promptly respond to insults inflicted by stress-causing microbes strongly suggests that ILCs are critical in first-line immunological defenses. ILCs are also involved in repair of tissue damage inflicted by pathogenic microbes. The scientific session presentation will include data on developmental requirements, lineage relationship and effector functions of human ILCs. Two families of innate lymphoid cells will be discussed: Rorγt-expressing cells involved in lymphoid tissue formation, mucosal immunity and inflammation, and Type 2 innate lymphoid cells that are important for helminth immunity. In addition, evidence will be presented for the existence of a novel ILC population that is dedicated to producing interferon-γ and which we call type 1 ILC. The potential roles of ILC in the pathology of immunity-mediated inflammatory and infectious diseases, including allergic diseases, will be discussed. Disclosures: No relevant conflicts of interest to declare.

Background HIV-1 is still a major public health problem and one of the priorities of the World Health Organization. The development of HAART against HIV was a considerable advance for infected individuals, but this life-long treatment does only block virus replication, and no viral eradication is obtained. Furthermore, HAART may exhibit long-term toxicity and may eventually lead to the emergence of drug-resistant viral variants. We explore a new durable therapeutic intervention based on a gene therapy that induces RNA interference (RNAi) against HIV1. In this pre-clinical research setting, “humanized” experimental mouse models are of interest considering the relative ease of handling and relatively low cost as compared to non-human primates. Methods

We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway.

B7/BB1 is a membrane differentiation antigen expressed on activated B cells, macrophages, and dendritic cells that binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T cell activation initiated via the CD3/T cell receptor complex. We now report that B7 is also expressed on activated human peripheral blood T cells, CD4 T cell clones, CD8 T cell clones, and natural killer cell clones. B7 appears relatively late after T cell activation, can be detected on both CD4 and CD8 T cell subsets, and is present on antigen-specific, major histocompatibility complex-restricted CD4 and CD8 T cell clones. Expression of B7 on activated T cells was confirmed by immunoprecipitation from 125I-labeled activated T cells and by detection of B7 transcripts. A B7+ CD4+ T cell clone was able to stimulate a primary allogeneic mixed lymphocyte response using small, resting peripheral blood T cells as responders. The alloantigen-induced proliferative response and cytokine production was partially inhibited by anti-B7 monoclonal antibody. Since activated T cells can coexpress both CD28 and its counter-receptor, B7, this suggests that activated T cells may be capable of autocrine costimulation via the CD28 activation pathway.

Abstract 3725 Deficient thymopoiesis, due to epithelial injury by chemo- and/or radiotherapy, age-associated thymic involution and by graft-versus-host disease, is an important determinant of the impaired immune competence following allogeneic transplantation. Therefore, strategies to improve thymopoiesis are considered pivotal to improve T cell recovery and immune competence after transplantation. As SCF is a cytokine produced by thymic stroma and its receptor, c-kit, is expressed by the earliest thymocytes, we evaluated whether SCF administration would improve thymic recovery following stem cell transplantation in immuno-deficient mice receiving: (1) a T-cell depleted bone marrow (BM) graft of congenic mice, or (2) a CD34+CD38low-selected xenogenic hematopoietic stem cell (HSC) graft of human fetal liver origin (HIS) mice model). In the mouse-mouse model, 10–12 week old rag-1−/− mice were 3 Gy irradiated (137Cs -source) and received 2×105 T-cell depleted C57Bl/6 (CD45.1) congeneic bone marrow cells intravenously. Recipient mice received either PBS or recombinat rat SCF (Amgen, USA, 100μg/kg per injection) by subcutaneous injection 3 times a week from day 1 until the end of the experiment. In this model, SCF enhanced thymopoiesis and peripheral T-cell recovery. BM lymphoid progenitor recovery was not affected. Median thymic cellularity increased from 0.9 in PBS- to 266 × 104/thymus in SCF-treated mice (p=0.05), which increase was similarly distributed over the thymocyte subsets of double negative (DN), double positive (DP), and CD4+, CD8+ single positive thymocytes. Next, we assessed whether SCF-induced improved thymic recovery also translated into improved T-cell recovery in the periphery. Absolute numbers of donor-derived newly developed CD4+ and CD8+ T-cells were quantified in peripheral blood, spleen and lymph nodes at weeks 4 and 6 post-transplantation. T-cell numbers were low at 4 weeks after transplantation and did not differ between PBS or SCF treated animals. However, at 6 weeks T-cell numbers were significantly increased in spleen and lymph nodes of SCF treated animals (p Next, we studied the effect of recombinant human (rh) SCF in our HIS mouse model. In short, newborn (days 3–7) Rag-2−/−gc−/− mice were 3.5 Gy irradiated and transplanted with 5–10 × 104 CD34+CD38low human fetal liver (FL) cells intra-hepatically. CD34+CD38low cells were isolated via a two step procedure. CD34+− FL cells were isolated using a CD34 human progenitor cell-isolation kit and further sorted as CD38low using a FACS Aria (BD biosciences). In HIS mice, PBS or rhSCF (Amgen, USA) 100μg/kg per injection) was administered intraperitoneally (i.p.) 3 times weekly as of day 14 following transplantation. Similar to the murine BMT model, a higher thymic cellularity was observed in SCF treated mice (Fig. 1) in the HIS model. DN and early DP thymocyte subsets were enhanced, albeit not significantly. In contrast, lateDP, CD4SP and CD8SP thymocyte subset recovery was significantly enhanced in thymi of SCF-treated HIS mice. As the HIS model remains a hybrid human–mouse system with limited cytokine cross reactivity and in which MHC-HLA mismatch compromises peripheral T-cell survival (Legrand et al J IMMUNOL 2009), the model did not allow us to study the effect of SCF on T-cells in the peripheral lymphoid organs. Figure 1: SCF improves thymic recovery following human SCT in an HIS mouse model. Newborn rag-2−/− γc−/− mice were 3.5 Gy irradiated and received 5–10 × 104 CD34+CD38low human fetal liver cells intra-hepatically and were treated with PBS or SCF. At indicated times post transplantation, thymi were harvested analyzed for the human thymocyte subsets: TN (CD3-CD4-CD8-), early DP (CD3- CD4+CD8+), late DP (CD3+CD4+CD8+), CD4SP and CD8SP (CD3+CD4-CD8+). Figure 1:. SCF improves thymic recovery following human SCT in an HIS mouse model. Newborn rag-2−/− γc−/− mice were 3.5 Gy irradiated and received 5–10 × 104 CD34+CD38low human fetal liver cells intra-hepatically and were treated with PBS or SCF. At indicated times post transplantation, thymi were harvested analyzed for the human thymocyte subsets: TN (CD3-CD4-CD8-), early DP (CD3- CD4+CD8+), late DP (CD3+CD4+CD8+), CD4SP and CD8SP (CD3+CD4-CD8+). Collectively, these results show that SCF may significantly improve post-transplant thymopoiesis after both experimental murine BMT and human fetal liver HSC transplantation in HIS mice. While peripheral T-cell recovery was significantly enhanced in the murine BMT model, it remains to be established whether improved human thymopoiesis will also translate into better peripheral T-cell recovery and enhanced immune competence towards opportunistic infections as well as a better recovery of regulatory T cells. These studies now set the stage for studying SCF, either alone or combined with other thymopoietic cytokines, in a larger pre-clinical animal model. Disclosures: No relevant conflicts of interest to declare.

NK cells and T cells share many features, suggesting that they have a common origin. Here, Lewis Lanier and colleagues describe recent studies of fetal ontogeny and thymic development that provide support for this hypothesis, and propose an integrated scheme for NK-cell and T-cell development.

In normal T cell progenitors, phosphoinositide-dependent kinase l (PDK1)–mediated phosphorylation and activation of protein kinase B (PKB) is essential for the phosphorylation and inactivation of Foxo family transcription factors, and also controls T cell growth and proliferation. The current study has characterized the role of PDK1 in the pathology caused by deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN). PDK1 is shown to be essential for lymphomagenesis caused by deletion of PTEN in T cell progenitors. However, PTEN deletion bypasses the normal PDK1-controlled signaling pathways that determine thymocyte growth and proliferation. PDK1 does have important functions in PTEN-null thymocytes, notably to control the PKB–Foxo signaling axis and to direct the repertoire of adhesion and chemokine receptors expressed by PTEN-null T cells. The results thus provide two novel insights concerning pathological signaling caused by PTEN loss in lymphocytes. First, PTEN deletion bypasses the normal PDK1-controlled metabolic checkpoints that determine cell growth and proliferation. Second, PDK1 determines the cohort of chemokine and adhesion receptors expressed by PTEN-null cells, thereby controlling their migratory capacity.

Long recognized as essential for proper lymph node organogenesis during fetal development, recent evidence has suggested that LTi cells also play an important role in the adult. Recent work from our laboratory has identified the previously unknown human LTi as lineagenegCD127+RORc+ cells. In vitro studies suggest a direct relationship of LTi cells with CD127+RORc+ NK cells. Both LTi and CD127+ NK cells isolated from human tonsil produce IL-22, IL-17, TNFalpha, and lymphotoxin, but not IFNgamma. LTi cells are present in a low frequency in human tonsils, thus making detailed analysis challenging. Using a rigorous selection strategy, we have successfully purified LTi and CD127+ NK cells and expanded these cells 30-100 fold using a feeder cell mixture and IL-2. These expanded LTi and CD127+ NK cell lines maintain RORc expression and production of IL-22 and IL-17. Interestingly, following ex vivo expansion both LTi and CD127+ NK cells produce IFNgamma upon stimulation. An outstanding question remains the cytotoxic potential of LTi and CD127+ NK cells, which we can now investigate. Further, we are investigating the role of stimulatory cytokines in regulating IL-22 production from LTi and CD127+ NK cells. As aberrant regulation of IL-22 can contribute to inflammatory diseases, further understanding of this novel population of IL-22 producing cells is of great relevance.

We investigated whether staphylococcal exotoxins (SEs), in addition to their capacity to induce T-cell activation restricted by the T-cell receptor (TCR) beta-chain variable region, can deliver an activation signal to human T-cell clones through major histocompatibility complex (MHC) class II molecules. Eleven human T-cell clones (9 alpha beta TCR and 2 gamma delta TCR clones) of different antigenic specificities were tested for their capacity to proliferate in response to toxic shock syndrome toxin 1 (TSST-1) and two SEs, SEA and SEB. In the absence of accessory cells, only 4 alpha beta TCR clones were stimulated to proliferate, each by a single SE, and to mobilize intracellular free Ca2+ in response to that SE, events indicative of TCR engagement and, presumably, recognition restricted by the beta-chain variable region. In the presence of accessory cells, each of the 11 T-cell clones was stimulated to proliferate by any one of the three SEs tested. This apparently TCR-unrestricted SE-mediated polyclonal proliferation of T-cell clones occurred in the absence of an increase in intracellular free Ca2+ and was not dependent on the presence of MHC class II expression on accessory cells. In contrast, SE-mediated polyclonal proliferation did not occur in 3 alpha beta TCR clones derived from an MHC class II-deficient patient. Furthermore, all of the three SEs induced the proliferation of 4 natural-killer-cell clones, suggesting that expression of TCR/CD3 complex is not essential for SE-mediated polyclonal proliferation of activated lymphocytes. These results indicate that MHC class II molecules transduce activation signals to human T- and natural-killer-cell clones.

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.

Human triple negative (CD3- CD4- CD8-) thymocytes and double negative (CD4- CD8-) thymocytes purified from post-natal thymus were cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells and PHA and expanded with IL-2. The cloning efficiency of triple negative thymocytes was less than 1% and the majority of the clones were triple negative. One out of 11 clones was TCR αβ+ CD4+. No TCR γδ + clones were isolated. On the other hand, the cloning efficiency of double negative thymocytes was about 10% and most of the clones isolated were TCR γδ +. We could not find any evidence of in vitro differentiation of triple negative thymocytes into TCR γδ + cells. Some of the triple negative clones expressed CD16- brightly and were apparently NK cells. All CD 16-clones isolated from triple negative thymocytes, however, expressed NKH1, which is also an NK cell marker. Cytoplasmic CD3-δ and CD3-e Ag which have been reported to be expressed in the most immature thymocytes were not detected in any of these clones. Furthermore, the CD 16- triple negative clones exhibited significant cytolytic activity against K562. Pheno-type of the clones seems to be stable under various conditions in vitro including coculture with human thymic epithelial cells. These data indicate that the CD16- triple negative clones isolated from triple negative thymocytes are similar to a minor subset of NK cells which is CD16- NKH-1+. It is not clear whether they originated from a distinct subset of mature or immature NK cells resident in the thymus tissue or from common precursors of both T and NK lineage.

We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N-glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil-derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Deficient thymopoiesis and a retarded or absent recovery of newly developed CD4+ T-cells has become one of the most important determinants of impaired immune competence in the later time period after allogeneic transplantation. We previously showed that Interleukin-7 (IL-7) may enhance peripheral T-cell expansion without affecting thymopoiesis after BMT in immunodeficient mice (Broers et al. Blood 2003). In order to improve thymopoiesis, we evaluated whether the cytokine Flt3L alone or combined with IL-7 would affect thymopoiesis and/or the generation of lymphoid progenitors following BMT in immunodeficient RAG-2−/−γc, −/− mice, lacking T-, B- and NK-cells. Following 3 Gy irradiation and transplantation of graded restricted numbers of T-cell depleted (TCD) BM, mice received Flt3L (3 x 20μg/week), Il-7 (35 x 5 μg/week) or the combination of IL-7 and Flt3L until 80 days after BMT. Hematopoietic recovery was evaluated weekly by flowcytometry. While B-cell and NK-cell recovery were moderately enhanced, Flt3L strongly accelerated and enhanced the recovery of T-cells, especially in the setting of BMT with a low dose of 4 x 104 TCD-BM. In contrast, T-cell recovery was still insufficient in control mice treated with PBS (p < 0.01) or mice treated with IL-7 alone by day 80 after BMT with 4 x 104 TCD-BM. The combination of Flt3L and IL-7 did not result in better recovery as compared to Flt3L alone. As early concurrent enhanced T-cell, B-cell, NK-cell and myeloid recovery may suggest an effect on lymphoid progenitors, the number of common lymphoid progenitors (CLP) as characterized by lineage-, IL-7 Rα+, sca-1low and AA4.1+ was assessed. At day 20 post-BMT, 13.2 x 103 (±10) CLP were harvested from the BM of Flt3L treated mice versus 3.9 x 103 (±0.6) from control PBS treated mice (p = 0.2). Furthermore, thymic cellularity was increased (18.6 (±8) x 106 thymocytes versus 6.6 (±0.4) x 106, p = 0.1) and especially the number of double positive CD4/CD8 thymocytes were increased in Flt3L treated mice (14.3 x 106 (±7) versus 2.3 x 106 (±1), p = 0.03) at day 20 post-BMT. Next, we studied whether enhanced hematopoietic recovery following Flt3L would result in better immune competence by evaluating survival and clearance of viral load after opportunistic murine cytomegalovirus (mCMV) infection. RAG-2−/−γc, −/− mice were transplanted with 4 x104 TCD-BM and subsequently infected intraperitoneally with 104 PFU mCMV at day 28 post-BMT. All Flt3L treated mice survived and rapidly cleared their viral load as assessed by quantitative real-time Taqman PCR in plasma. T-cell numbers were inversely correlated with viral load (r = −0, 467, p = 0.04), while numbers of NK-cells, B-cells or granulocytes were not associated with viral load. A 100% mortality was observed in control mice developing a viral load > 2 x 105 geq/ml. These results suggest that Flt3L may restore T-cell recovery and immune competence especially in the setting of transplantation with restricted numbers of progenitor cells by promoting thymopoiesis. Enhanced thymopoiesis directly mediated by Flt3L or expansion of lymphoid progenitors by Flt3L may account for the higher numbers of newly developed T-cells.