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Graft-versus-host disease (GVHD) is among the most challenging complications in unrelated donor hematopoietic cell transplantation (HCT). The highly polymorphic MHC class I chain-related gene A, MICA, encodes a stress-induced glycoprotein expressed primarily on epithelia. MICA interacts with the invariant activating receptor NKG2D, expressed by cytotoxic lymphocytes, and is located in the MHC, next to HLA-B. Hence, MICA has the requisite attributes of a bona fide transplantation antigen. Using high-resolution sequence-based genotyping of MICA, we retrospectively analyzed the clinical effect of MICA mismatches in a multicenter cohort of 922 unrelated donor HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 10/10 allele-matched HCT pairs. Among the 922 pairs, 113 (12.3%) were mismatched in MICA. MICA mismatches were significantly associated with an increased incidence of grade III-IV acute GVHD (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.50-2.23; P < .001), chronic GVHD (HR, 1.50; 95% CI, 1.45-1.55; P < .001), and nonelapse mortality (HR, 1.35; 95% CI, 1.24-1.46; P < .001). The increased risk for GVHD was mirrored by a lower risk for relapse (HR, 0.50; 95% CI, 0.43-0.59; P < .001), indicating a possible graft-versus-leukemia effect. In conclusion, when possible, selecting a MICA-matched donor significantly influences key clinical outcomes of HCT in which a marked reduction of GVHD is paramount. The tight linkage disequilibrium between MICA and HLA-B renders identifying a MICA-matched donor readily feasible in clinical practice.

The MHC class I–related chain A (MICA) molecules exist as membrane-bound and soluble isoforms and are encoded by a polymorphic gene. Their genetic and phenotype characteristics have been studied in various pathologic settings but not in the context of hematopoietic stem cell transplantation (HSCT). Here, we evaluated whether MICA-related features namely MICA-129 gene polymorphism, serum levels of soluble MICA (sMICA) and anti-MICA antibodies (MICA Abs) before and after HSCT could influence the incidence of chronic graft-versus-host disease (cGVHD) and relapse of their disease in 211 HLA-identical sibling pairs and in a subset of 116 recipients, respectively. Although the MICA-129 val/val genotype and elevated sMICA serum levels after HSCT are independently associated with the incidence of cGVHD (P = .002 and .001) regardless of history of acute GVHD, the presence of MICA Abs before transplantation confers protection against cGVHD (P = .04). There is an inverse relationship between MICA Abs and sMICA, suggesting an antibody-based neutralization of deleterious effects of sMICA. Similarly, these genetic and phenotype characteristics of MICA influence the incidence of relapse. Altogether, these data suggest that the studied MICA genotype and phenotype specificities could be used as relevant biomarkers for cGVHD monitoring.

To address the role of chromatin structure in the establishment of hematopoietic stem cell (HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed histone modifications at a panel of lymphoid- and myeloid-affiliated genes in multipotent and lineage-committed hematopoietic cells isolated from human cord blood. Our results show that many B- and T-lymphoid genes, although silent in HSCs, are associated with acetylated histones H3 and H4. We also detected histone H3 lysine 4 methylation but not repressive lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure. Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific loci was high in multipotent CD34+CD38lo progenitors and decreased as they become actively transcribed in B-lineage cells. In vitro differentiation of CD34+ cells toward the erythroid, granulocyte, and T-cell lineages resulted in a loss of histone acetylation at nonlineage-associated genes. This study provides evidence that histone modifications involved in chromatin decondensation are already in place at lymphoid-specific genes in primary human HSCs, supporting the idea that these genes are “primed” for expression before lineage commitment. This permissive chromatin structure is progressively lost as the stem cell differentiates.

Despite a lack of signaling motifs in their cytoplasmic domain, major histocompatibility complex (MHC) class II molecules trigger a variety of intracellular signals that regulate antigen-presenting cell function. They thus may use associated effector molecules as demonstrated on B cells and dendritic cells. The starting point of this study comes from our previous work, which demonstrated that the ecto-enzyme CD38 is functionally linked to MHC class II molecules. We report that CD38 and human leukocyte antigen-DR (HLA-DR) are functionally and physically associated in lipid rafts microdomains of cellsurface monocytes and that the integrity of these domains is necessary for the HLA-DR and CD38 signaling events. Moreover, we identified the tetraspanin CD9 molecule as a partner of the CD38/HLA-DR complex and demonstrated that HLA-DR, CD38, and CD9 share a common pathway of tyrosine kinase activation in human monocytes. The analysis of conjugate formation between monocytes presenting superantigen and T cells shows the active participation of CD9 and HLA-DR on the monocyte surface. Together, these observations demonstrate the presence of a CD38 and HLA-DR signaling complex within tetraspanin-containing lipid rafts and the functional impact of their molecular partner CD9 in antigen presentation.

Umbilical cord blood (CB) constitutes a promising alternative to bone marrow for allogeneic transplantation and is increasingly used because of the reduced severity of graft-versus-host disease after CB transplantation. We have compared the T-cell receptor β chain (TCRB) diversity of CB lymphocytes with that of adult lymphocytes by analyzing the complementarity determining region 3 (CDR3) size heterogeneity. In marked contrast to adult samples, we observed bell-shaped profiles in all of the 22 functional β-chain variable (BV) subfamilies that reflect the lack of prior antigenic stimulation in CB samples. However, the mean CDR3 size and BV usage were comparable between CB and adult samples. BJ2 (65%) segments were used preferentially to BJ1 (35%), especially BJ2S7, BJ2S5, BJ2S3, and BJ2S1, in both CB and in adult lymphocytes. We therefore conclude that although naive as reflected by the heterogeneity of the CDR3 size, the TCRBV repertoire appears fully constituted at birth. The ability to expand TCRB subfamilies was confirmed by stimulation with staphylococcal superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. This study provides the basis for future analysis of the T-cell repertoire reconstitution following umbilical CB transplantation.

In an acute promyelocytic leukemia (APL)-transplantable mouse model, we previously reported the presence of antibodies recognizing PML-RARalpha and RARalpha in the sera of ATRA-treated mice. To evaluate this immune response, we determined the prevalence of anti-RARalpha antibodies in a cohort of 48 APL mice, treated by ATRA (n = 24) or by placebo pellets (n = 24), and in a preliminary subset of 9 patients with APL using a specific enzyme-linked immunosorbent assay (ELISA). In APL mice, significantly higher antibody levels were observed at the latest time points (day 48 to 58 levels superior to day 15 to 18 or day 28 to 38 levels). Antibody levels were higher in ATRA-treated mice than in placebo-treated mice and were also predictive of better survival. In the patients with APL, anti-RARalpha antibodies were detected at diagnosis and after maintenance therapy, reminiscent of the ATRA-treated APL mice. Antinuclear or antineutrophil cytoplasmic autoantibodies were also detected. These data reveal for the first time that in patients with APL an immune response may be detected at diagnosis and enhanced after maintenance therapy.

HLA class II molecules, expressed on the surface of antigen-presenting cells, are responsible for the presentation of antigen-derived peptides to CD4+ helper T lymphocytes. Signaling via these molecules initiates the generation of second messengers leading to programed cell death (PCD) of activated B lymphocytes. The present study examined the mechanism of HLA class II–mediated apoptosis and describes the essential role of the molecule Fas and its ligand (FasL). FasL was expressed in B lymphocytes after stimulation via HLA class II or with phorbol esters. Expression of FasL protein was significantly increased in 50% of B lymphocytes after stimulation via HLA class II, and the level of FasL mRNA was also increased either by activation with phorbol esters and ionomycin or by signaling via HLA class II. Although HLA class II signaling did not change the expression of the Fas molecule, it did lead to increased sensitivity to Fas-mediated apoptosis. The crucial role of Fas/FasL interactions was confirmed by the absence of cell death via HLA class II in B cells lacking Fas expression, and by the significant inhibition of HLA class II–mediated apoptosis in the presence of either an antagonistic anti-Fas or anti-FasL antibody. These data demonstrate FasL expression on activated human B lymphocytes and support the idea that antigen presentation could contribute to the regulation of lymphocyte populations via Fas and FasL interactions.

The rules governing natural killer (NK)–cell education in the allogeneic environment created by unrelated hematopoietic stem-cell transplantation (HSCT) are still largely elusive, especially in an unrelated donor setting. NK-cell inhibitory receptors for self-human leukocyte antigen (HLA) play a central role in the acquisition or maintenance of NK-cell functional competence. Therefore, the responsiveness of different NK-cell subsets was assessed as a function of their expression or absence of expression of self-HLA–specific inhibitory receptors, in a large cohort (n = 60) of unrelated HSCT recipients. A fully effective NK-cell education process was achieved within the first year after allogeneic HSCT and lasted for at least 3 years thereafter. In addition, HLA-mismatched HSCT led to a stable education pattern that was determined by the donor's HLA ligands. These data suggest that the NK cell's education partner could be of hematopoietic rather than extrahematopoietic origin. This donor-ligand–driven NK-cell education model would suggest a sustained graft-versus-leukemia effect after HLA-mismatched HSCT.

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4+ or CD8+ cells abolished this effect. CD4+ depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4+ and CD8+ subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4+ cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester–based assays showed major histocompatibility complex–restricted APL-specific T cell–mediated immune responses. Sorted APL-specific CD8+CD107a+ T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA–treated mice were shown to secrete interferon-γ when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARα vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-γ–producing and cytotoxic T cells in the leukemic mice.

Long-term T-cell reconstitution after hematopoietic stem cell transplantation (HSCT) is dependent on patient thymic function and affected by graft-versus-host disease (GVHD). To assess the impact of acute GVHD (aGVHD) on thymic function, we followed a cohort of 93 patients who received HSCT from a human histocompatibility leukocyte antigen-identical sibling, mainly for hematologic malignancies. Thymic output was measured by signal-joint T-cell receptor excision circles (sjTREC) real-time polymerase chain reaction. Absolute sjTREC number was lower at 6 months in patients with aGVHD (P = .014), associated with lower absolute counts of naive CD4 T cells at 6 and 12 months (P = .04 and .02), and persistent abnormalities in T-cell repertoire diversity. Age and aGVHD affected thymic function independently in multivariate analysis. In patients less than 25 years of age, thymic function recovered almost totally at 1 year. As a marker of thymocyte proliferation, we quantified the βTREC generated during the T-cell receptor β-chain recombination, in a group of 20 age-matched patients. Mean βTREC level was reduced at 6 months in patients with aGVHD, indicating an impact on early thymic differentiation rather than on intrathymic proliferation. These data show that aGVHD or its treatment has a transient impact on thymic function in younger patients in the first months after HSCT.

Thymic function is critical for immune reconstitution after hematopoietic stem cell transplantation (HSCT). We evaluated recipient thymic function before HSCT by quantifying T-cell receptor excision circles (TRECs) in pretransplantation peripheral blood lymphocytes from 102 patients who received HSCs from an HLA-identical sibling for malignant (n = 87) or nonmalignant diseases (n = 15). Median TREC value before transplantation was 257 TRECs per 150 000 CD3+ cells (range, 0-42 746). We assessed 172 TRECs per 150 000 CD3+ cells as the most discriminating TREC value for survival in a first cohort of patients (n = 62). This cut-off was validated in a second independent prospective group of 40 patients. In the 102 patients, a TREC value greater than or equal to 172 was associated with a better survival (P < .000 01), a decreased incidence of grade II-IV acute graft-versus-host disease (GVHD; P = .017), chronic GVHD (P = .023), and bacterial (P = .003) and cytomegalovirus (CMV) infection (P = .024). In a multivariate analysis, low pretransplantation TREC values were associated with a higher incidence of CMV infection (hazard ratio [HR] = 2.0, P = .06) and severe bacterial infections (HR = 2.8, P = .036). Finally, high TREC values (HR = 6.6, P = .002) and ABO compatibility (HR = 2.7, P = .02) were associated with a better survival. Therefore, recipient host thymic function assessment could be helpful in predicting HSCT outcome and identifying patients who require a close immunologic monitoring.

Cord blood (CB) is used increasingly as a source of hematopoietic stem cells because of a lower risk of acute and chronic graft-versus-host disease (GVHD). However, there is some concern regarding the ability to adequately reconstitute host immune response due to the immaturity and naivety of CB T cells. This study was designed to evaluate T-cell reconstitution using combined approaches of phenotyping, analysis of αβ T-cell receptor (TCR) diversity, and assessment of ex vivo thymic function by measuring TCR rearrangement excision circles (TRECs). Ten patients who underwent CB transplantation for high-risk hematologic disorders were compared to a reference group of 19 age- and GVHD-matched patients who underwent transplantation with non-T cell-depleted bone marrow from an HLA-identical sibling donor. TREC values correlated with the relative number of naive T cells and with TCR repertoire polyclonality. During the first year after transplantation, TCR repertoires were highly abnormal and TREC values low in both groups. Notably, 2 years after transplantation onward TREC values as well as TCR diversity were higher in CB recipients than in recipients of bone marrow transplants. These data indicate an efficient thymic regeneration pathway from CB lymphoid progenitors despite the low number of cells infused compared to bone marrow, arguing for a complete clinical immune recovery after CB transplantation.

Human immunodeficiency virus (HIV)-1-infected long-term nonprogressors (LT-NP) represent less than 5% of HIV-1-infected patients. In this work, we tried to understand whether combined genotypes of CCR5-triangle up32, CCR2-64I, SDF1-3'A and HLA alleles can predict the LT-NP status. Among the chemokine receptor genotypes, only the frequency of the CCR5-triangle up32 allele was significantly higher in LT-NP compared with the group of standard progressors. The predominant HLA alleles in LT-NP were HLA-A3, HLA-B14, HLA-B17, HLA-B27, HLA-DR6, and HLA-DR7. A combination of both HLA and chemokine receptor genotypes integrated in a multivariate logistic regression model showed that if a subject is heterozygous for CCR5-triangle up32 and homozygous for SDF1 wild type, his odds of being LT-NP are increased by 16-fold, by 47-fold when a HLA-B27 allele is present with HLA-DR6 absent, and by 47-fold also if at least three of the following alleles are present: HLA-A3, HLA-B14, HLA-B17, HLA-DR7. This model allowed a correct classification of 70% of LT-NPs and 81% of progressors, suggesting that the host's genetic background plays an important role in the evolution of HIV-1. The chemokine receptor and chemokine genes along with the HLA genotype can serve as predictors of HIV-1 outcome for classification of HIV-1-infected subjects as LT-NPs or progressors.

To the editor: The CTLA-4 gene encodes a molecule providing a negative signal for T-cell activation. CTLA-4 +49A/G and CT60 polymorphisms have been associated with auto-immune diseases (AID).[1][1],[2][2] A recent study suggested that donor genotype AA of CT60 was associated with better survival

HLA class II molecules, expressed on the surface of antigen-presenting cells, are responsible for the presentation of antigen-derived peptides to CD4+ helper T lymphocytes. Signaling via these molecules initiates the generation of second messengers leading to programed cell death (PCD) of activated B lymphocytes. The present study examined the mechanism of HLA class II-mediated apoptosis and describes the essential role of the molecule Fas and its ligand (FasL). FasL was expressed in B lymphocytes after stimulation via HLA class II or with phorbol esters. Expression of FasL protein was significantly increased in 50% of B lymphocytes after stimulation via HLA class II, and the level of FasL mRNA was also increased either by activation with phorbol esters and ionomycin or by signaling via HLA class II. Although HLA class II signaling did not change the expression of the Fas molecule, it did lead to increased sensitivity to Fas-mediated apoptosis. The crucial role of Fas/FasL interactions was confirmed by the absence of cell death via HLA class II in B cells lacking Fas expression, and by the significant inhibition of HLA class II-mediated apoptosis in the presence of either an antagonistic anti-Fas or anti-FasL antibody. These data demonstrate FasL expression on activated human B lymphocytes and support the idea that antigen presentation could contribute to the regulation of lymphocyte populations via Fas and FasL interactions.

Fetal mononuclear cells are increasingly used in transplantation of hematopoietic cells due to a reportedly lower incidence of graft-versus- host disease. Previous studies of immune responses of fetal lymphocytes have indicated a general hyporesponsiveness in response to polyclonal stimulation. Fetal B lymphocytes display many features typical of the resting state such as a low level of HLA class II expression, but a large proportion of cells also carry the activation-associated CD23 antigen. We show here that despite a low cell surface level of all three HLA class II isotypes on fetal B cells, their allogeneic capacity, measured as the ability to elicit a mixed lymphocyte reaction, is similar to that of adult B cells. Allogeneic stimulation is believed to be peptide-dependent. Surprisingly, the majority of the HLA class II molecules on cord blood B cells appeared to be devoid of stably bound peptide as detected by the binding of a recombinant and soluble invariant chain, as well as by the absence of sodium dodecyl sulfate (SDS) stable alpha beta heterodimers in whole cell lysates. Immunoblot experiments showed that HLA class II molecules of fetal B cells were predominantly present in high molecular weight aggregates in stark contrast to B cells of adult origin. However, a sensitive cell surface labeling technique followed by immunoprecipitation enabled us to detect an SDS-stable 120-kD molecule on fetal B cells. We propose that the 120-kD molecules could correspond to HLA class II doubledimers or superdimers. We hypothesize that the 120-kD HLA class II molecule functions as the antigen-presenting molecule in the mixed lymphocyte reaction of fetal B cells, as it is the major species detected on the surface. Secondly, we suggest that a high level of empty HLA class II molecules may be indicative of a particular stage in B-cell ontogeny.

Abstract 4046 Treatment with lenalidomide, an immunomodulatory drug, increases the time to progression in relapsed/refractory multiple myeloma. However, due to its pleiotropic effect, it is not known whether the efficacy of this drug is due only to direct tumor toxicity or also to immunomodulatory effects. We assessed in vivo the changes in T-cell reconstitution induced by lenalidomide consolidation and maintenance treatment following autologous peripheral blood stem cell transplantation (ASCT) in a cohort of multiple myeloma patients. Twenty-nine newly diagnosed myeloma patients were treated with the induction combination bortezomib plus dex followed by high dose melphalan (140–200 mg/m2) and ASCT. A first group of 11 patients were treated with lenalidomide consolidation initiated 3 to 6 months post transplantation: 25 mg/day, days 1–21 of a 28 day cycle for 2 months, followed by maintenance (10 mg/day) until disease progression. This group was compared with the 18 patients who did not receive any treatment after ASCT. Blood samples were collected at diagnosis, before the transplant and 1, 3, 6, 9, 12 and 18 months after ASCT. Thymic function was assessed by real-time PCR quantification of T cell receptor excision circles (sjTREC) and percentages and absolute counts of T lymphocyte subpopulations were determined by multicolor flow cytometry. Statistics were performed using the Log-Rank or Mann-Whitney test. The two cohorts had similar baseline characteristics and all 29 patients were in remission after ASCT. With a median follow-up of 4 years, progression-free survival (PFS) was superior with lenalidomide treatment (69% vs 36%, p=0.05) while overall survival (OS) was similar (82% vs 75%, p=0.5). Lenalidomide treatment induced a progressive decrease in sjTREC (median at 18 months, 0.25/μL vs 1.61/μL, p We confirm an increase in PFS with lenalidomide consolidation/maintenance following ASCT. However, our data also suggest that in myeloma patients, the effect of lenalidomide on the myeloma tumor may not be T cell mediated and this treatment may have a negative impact on the T cell immune surveillance. Disclosures: No relevant conflicts of interest to declare.

Abstract 3135 Background: When an HLA-matched donor is not available for allogeneic Hematopoietic Stem Cell Transplantation (HSCT), the use of an alternative HLA-mismatched stem cell source may be considered. In Europe, HLA compatibility for HSCT is calculated for 10 alleles (HLA-A, -B, -C, -DRB1, -DQB1). The aim of this study was to retrospectively compare outcomes after transplantation from 10/10 HLA-MUD, 9/10 HLA-MMUD and UCB performed at the Hospital Saint Louis (Paris, Fr). Methods: Patients receiving a first allogeneic transplantation from a 10/10, 9/10 UD or UCB from 2000 to 2011 were included. High resolution HLA typing was performed by PCR SSO and SSP for HLA loci: A, B, C, DRB1 and DQB1. The following variables were studied as risk factors for transplant outcomes: disease, disease risk (standard/high risk), age at transplant, gender, donor/recipient sex-matching, ABO matching, donor/recipient CMV status, conditioning regimen, and use of anti-thymoglobulin (ATG). Results: 355 consecutive patients with hematologic malignancies were analyzed. One hundred and ninety-six were transplanted with MUD, 84 with MMUD (mismatches for HLA-A: 16%, -B: 16%, -C: 39%, -DRB1: 8%, -DQB1: 21%) and 75 with UCB (52% with single and 48% with two UCB unit; 87% of all UCB transplants were 4–6/6 HLA-matched). Median patient age was 31 (range: 5–55). Patient characteristics differed between the 3 groups: (i) median age at transplant: 36 in MUD, 31 in MMUD, 22 in UCB (p Cumulative incidences of grade II-IV and grade III-IV acute GvHD disease (aGvHD) were 61% (66% for MUD, 60% for MMUD and 48% for UCB) and 17% (17%, 24% and 15%), respectively. Three-year cumulative incidence of chronic GvHD (cGvHD) was 46% (51% for MUD, 49% for MMUD and 29% for UCB). Three-year NRM was 34% (28% for MUD, 31% for MMUD and 51% for UCB). Graft failure occurred in 15% of UCB patient, 8% in MMUD group and 3% in MUD group (significant difference between UCB and MUD: OR=5.44, 95%CI 1.93–15.3, p=0.001). Multivariate analysis is summarized in table 1. It showed that MMUD tented to have a higher incidence of aGvHD III-IV than MUD and UCB. UCB had a lower incidence of cGvHD than MMUD. No significant effect of HSCT source on NRM was demonstrated. Conclusion: Compared to MMUD, UCB-HSCT induces less cGvHD and non significantly increased NRM. Compared to MUD, MMUD and UCB-HSCT did not result in a clear increase of NRM. UCB and 9/10 HLA-MMUD are both suitable stem cell sources for patients who cannot benefit from 10/10 HLA-MUD transplant. Disclosures: No relevant conflicts of interest to declare.

Abstract 1258 Hematopoietic Stem Cell Transplantation (HSCT) is an effective treatment for many malignant and non malignant diseases in children. Since less than 30% of the patients have an HLA identical related donor, alternative donor/sources of Hematopoietic Stem Cells need to be considered, such as unrelated bone marrow (BM) and Cord Blood (CB) donors or HLA-haploidentical related donors. However, these types of HSCT are associated with a significant delay in immune reconstitution that favors both a high incidence of opportunistic infection and, in the absence of an alloreactive, natural killer cell-mediated effect, disease relapse. Indeed, T-cell depletion of the allograft in the haploidentical setting or T-cell naivety in CB, as well as HLA-disparity and use of serotherapy before HSCT, contribute to this impaired immune reconstitution. In view of the role played by the thymus in immune reconstitution post-allogeneic HSCT, it would be informative to compare thymic-dependent immune reconstitution after these types of HSCT. We have studied immune reconstitution and thymic function through signal joint (sj) and beta T cell Receptor Excision Circles (TREC) quantification in a group of 33 haplo-HSCT pediatric patients and in a group of 24 unrelated CB-HSCT. Patients were transplanted mainly for hematological malignancies (n=46, including 31 Acute Lymphocytic Leukemia and 9 Acute Myeloid Leukemia). All children received a myeloablative conditioning regimen, including the combination of total body irradiation and chemotherapy (n=33) or chemotherapy alone (n=24). In haplo-HSCT, no pharmacological immune-suppression was given after the allograft, while patients given CB-HSCT received cyclosporine-A and steroids as Graft vs. Host Disease (GvHD) prophylaxis. In haplo-HSCT patients the median number of CD34+ cells infused/kg was 20.7 × 106 (range 8.7–41), while in patients given CB-HSCT the median number of nucleated cells infused/kg was 7.1) × 107 (range 1.4–12.5). The only significant difference between the 2 groups was that haplo-HSCT patients were older (p=.0008) than CB-HSCT patients (median age being 7.7 and 3.4 yrs, range 3–17 and 0.75–16 yrs, respectively). Patients treated for an hematological malignancy had a significant lower pre-transplant TREC value (p = .0002 and .004 for sj and beta TREC respectively) than those affected by non-malignant diseases in both groups. Number of sj and betaTREC per 150 000 PBMC or absolute counts per μl of blood, showed a very similar thymic function in both groups despite the older age of haplo-HSCT patients. TREC levels were comparable to those found before transplantation starting from 6 months after HSCT. Cumulative incidence of acute or chronic GvHD was low in both groups, with no significant impact on thymic function. In haplo-HSCT, but not in CB transplantation, patients treated for hematological malignancies (n=27) who relapsed (n=8) had a significantly lower level of thymic function (sjTREC) than those who did not relapse (n=19), before (p=0.03), after 3 (p=0.014) and 6 months (p=0.015) from the allograft. In this group of patients, relapse was not associated with age, conditioning regimen or number of CD34+ cells infused. In conclusion, we demonstrate the crucial importance of thymic function in the Graft-vs. Leukemia effect in the haplo-HSCT setting. Monitoring thymic function could be of predictive value in these patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2412 Poster Board II-387 Acquired idiopathic thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy (TMA) resulting from excessive von Willebrand factor (VWF)-mediated platelet clumping and capillary occlusion in relation with an autoantibody-mediated defect in the VWF-cleaving protease ADAMTS13. So far, the mechanisms leading to the loss of tolerance of the immune system against ADAMTS13 remain unknown. The usually switched isotype of the secreted anti-ADAMTS13 antibodies implies a cooperation between T lymphocytes and B lymphocytes and thus a recognition by T cell receptors of ADAMTS13 epitopes bound to human leukocyte antigen (HLA) molecules. We therefore hypothesized that particular HLA alleles may be involved in the process leading to a loss of tolerance of the immune system against ADAMTS13. Particularly, alleles within class I A and B loci and class II DRB1 and DQB1 loci are those most frequently associated with susceptibility to autoimmune diseases. We have compared medium resolution typing of HLA class I (HLA-A, B) and II (HLA-DRB1, DQB1) loci in 61 Caucasian patients with idiopathic TTP and a documented severe ( The frequency of HLA-A, -B and -DQB1 alleles was comparable between patients with acquired idiopathic TTP, healthy individuals and patients with other forms of TMA. By contrast, the HLA DRB1*11 allele was observed in 62% of patients with acquired idiopathic TTP, whereas the usually reported incidence of this allele ranges from 13.5% to 24.5%, with the lower incidence in Asian/Pacific Islanders and Caucasians and the greater incidence in Afro-Caribbeans (http://www.allelefrequencies.net). Comparison to our group of healthy individuals confirmed this striking difference, which was statistically significant (20%, P = 10-7, odd ratio [OR] = 6.43, CI95% =2.3-12.7). The increased incidence of DRB1*11 in acquired idiopathic TTP was also significant when compared to patients with a diagnosis of atypical HUS (17%, P= 6×10-5). By contrast, DRB1*11 frequency in this latter group was comparable to this observed in healthy individuals (P = n.s). All patients were heterozygous for this allele. We also observed a decreased incidence of the DRB1*04 allele in acquired idiopathic TTP when compared to healthy individuals (10% versus 30%, respectively, Pcorr = 0.025). DRB1*04, resulting in the DR53 antigen when associated with DRB4, could thus be involved as a protective allele in acquired idiopathic TTP, as suggested in a previous work. We found no association between DRB1*11 and clinical features of autoimmunity, central nervous system involvement, antinuclear antibodies, anti-ADAMTS13 inhibitors, flare-up and relapse episodes and survival. Of note, the association between DRB1*11 with acquired idiopathic TTP was not observed in Afro-Caribbeans (3/12, 25%, p=0.04). High resolution typing of DRB1*11 allele revealed that both DRB1*1101 and DRB1*1104 alleles were significantly over-represented, suggesting that the generic DRB1*11 (and not the DRB1*11 alleles) is the predisposing factor. The DRB1*11 was reported to be a risk factor for systemic sclerosis, early-onset juvenile chronic arthritis and sarcoidosis, and protective against multiple sclerosis and pemphigus vulgaris. Accordingly, we found no patient with one of those 2 latter diseases in our French National TTP registry, whereas a past history of systemic sclerosis, sarcoidosis and juvenile chronic arthritis was observed in 1 case each. Our findings propose DRB1*11 as a strong risk factor for acquired idiopathic TTP in Caucasians. Indeed, the autoimmune response in acquired idiopathic TTP may involve the recognition by T cells of a class II region of DRB1*11, bound to a peptide fragment of ADAMTS13. We also suggest that acquired idiopathic TTP represents a subset of TMA with specific genetic risk factors, distinguishing it from the other forms of idiopathic TMA. The incomplete rate of concordance may be explained by other yet unexplored mechanisms, including additional predisposing genes and environmental triggers. Forthcoming genomewide association studies should lead to the identification of additional alleles associated with the risk of acquired idiopathic TTP. Disclosures: No relevant conflicts of interest to declare.

Abstract 1138 Poster Board I-160 Background The MHC class I-related chain A (MICA) molecules is of particular in non T-cell receptor (TCR)-mediated immune function. MICA engages NKG2D, a C-type lectin expressed on effector cells including NK, and T cells. Such engagement triggers NK cells and co-simulates T lymphocytes to mount adequate immune response. Further, a soluble isoform of MICA (sMICA) has been implicated in the pathogenesis of a variety of cancers, auto-immune and other organ transplant-related disorders through NKG2D receptor down regulation. Functionally relevant polymorphism of the MICA gene may also contribute to inter-patients variability in allo-immune responses as observed in various clinical settings including organ-transplantation. A methionine (met) to valine (val) change at position 129 of the alpha 2-heavy chain domain categorized the MICA alleles into strong (MICA-129 met) and weak (MICA-129 val) binders of NKG2D receptor. Given the demonstrated importance of MICA in immune pathways, in this study, we explored the MICA-related parameters namely MICA-129 gene polymorphism, pre- and post-transplant serum levels of soluble MICA (sMICA) and anti-MICA antibodies (MICA Abs) in a HLA-matched sibling HSCT setting. Results We evaluated whether MICA-129 gene polymorphism, pre- and post-HSCT sMICA and MICA Abs could influence the incidence of chronic graft-versus-host disease (c.GvHD) and relapse of their disease in 211 HLA-identical sibling pairs. In multivariate analysis on chronic GvHD risk, 3 factors reached significance: recipient MICA-129 val/val genotype (HR = 1.52; 95% confidence interval [95%CI] = 1.02-2.24; P =.04), older recipient age (3 15 years) (HR = 3.36; 95%CI = 1.65-6.84; P =.001) and source of stem cells (PBSC vs BM) (HR = 1.67; 95%CI = 1.10-2.53; P =.017). Since acute GvHD (aGvHD) is a major risk factor of subsequent c.GvHD, we then introduced aGvHD as a time-dependant co-variate in the multivariate analysis model. This analysis confirmed that the risk conferred by the MICA-129 val/val genotype is independent from aGvHD. Elevated post-HSCT sMICA serum levels were also independently associated with c.GvHD (p =.0001) regardless of history of acute GvHD. On the contrary, the presence of pre-transplant MICA Abs confers protection against c.GvHD (p =.04). There was an inverse relationship between MICA Abs and sMICA suggesting an antibody-based neutralization of deleterious effects of sMICA. Similarly, these genetic and phenotype characteristics of MICA influence the incidence of relapse. Conclusion Altogether, these data suggest that the studied MICA genotype and phenotype specificities could be used as relevant biomarkers for c.GvHD monitoring. Disclosures No relevant conflicts of interest to declare.

Abstract 1152 Poster Board I-174 Introduction Human-keratinocyte growth factor 1(KGF) is currently being evaluated in clinical trials for its safety and efficacy in preventing mucosal damage from irradiation and after bone marrow transplantation (BMT). It has been suggested that, besides having preventive effects on oral mucositis, it may also have immunomodulating effects by uniquely protecting thymic epithelial cells. A potential clinical application of KGF is the amelioration of prolonged post BMT-immune deficiency in BMT recipients. In animal models of autologous and allogeneic BMT, KGF administration during BMT resulted in enhanced thymopoiesis and increased peripheral T-cell numbers. Therefore, a potential clinical application of KGF is the improvement of prolonged post BMT-immune deficiency in BMT recipients. Thymopoiesis can be assessed by analysis of T-cell receptor excision circles (TREC). This marker for recent thymic emigrants might be a novel prognostic factor for outcome after transplantation in multiple myeloma patients. We investigated the potential influence of KGF on immune reconstitution after autologous transplantation for myeloma using this marker. Materials and Methods Twenty-four myeloma patients from a single institution were treated homogeneously with the induction combination bortezomib (Velcade®) plus dexamethasone (DXM), followed by high dose melphalan (140-200 mg/m2) and an autologous transplantation with peripheral blood stem cells. Patients were randomized for KGF treatment in 2 groups, 11 having received three doses of palifermin (Kepivance®) (60 μg/kg once daily i.v.) pre- and post-conditioning regimen (total six doses). Blood samples were drawn at diagnosis, before BMT, and at 1, 3, 6, 9, 12 and 18 months post BMT. We analysed signal joint (sj) TREC in peripheral blood lymphocytes using quantitative RT-PCR. The percentage and absolute numbers of lymphocyte populations were monitored by flow cytometry. Values were expressed as means ± SEM. Patient groups were compared by the Mann-Whitney test. Results Both CD4+ and CD8+ naïve T cells (CD45RA+ CCR7+) were strongly decreased by pretransplantation bortezomib plus DXM treatment. SjTREC decreased sharply after transplantation and returned to baseline after 1 year without differences between the two groups of patients. Natural regulatory T lymphocytes (phenotypically assessed as CD4+ CD25 high, CD127 low) were not modified by KGF treatment either. Notably, the CD3+ cell population was significantly higher during the first 3 months post BMT in KGF treated patients (1518 ± 380 vs 821 ± 105 CD3+/blood mL, p = 0.032 at 1 month post BMT). This was related to a higher CD8+ cell counts, specifically in the CD8 effector memory cells (assessed as CD45 RA- CCR7-) (790 ± 320 vs 292 ± 55, p< 0.05 at 1 month post BMT). No correlation was found with documented infectious complications, relapse or survival. Conclusions These data suggest that, unexpectedly, the main effect of KGF on immunity in that autologous BMT setting, is not to be accounted by changes in thymic function but rather by the peripheral expansion of CD8+ effector memory cells which could be favoured by the treatment prior BMT and/or by the post-BMT lymphopenia. Disclosures No relevant conflicts of interest to declare.

OBJECTIVES: DNA vaccines can be effective in the acquisition of humoral and cellmediated immune responses. Our studies on an acute promyelocytic leukemia (APL) mouse model show that DNA vaccination combined with all-trans retinoic acid (ATRA) results in a survival advantage with a significant increase in the Th1 cytokine IFNg. ATRA alone can act as an adjuvant to induce immune responses as measured by an increase in anti-RARa antibody production, which correlated with improved survival in mice. Similar increases in antibody production have been observed in our patients after maintenance therapy. The aim of this study is to use immunomonitoring and functional assays to evaluate the presence of activated T-cells and to demonstrate APL-specific killing and to determine if the protective effect of DNA vaccination is CD4+ and CD8+ mediated. METHODS: Using an APL transplant model in FVB/N mice, CD107a, expressed on the surface of lytic granules of activated T-cells, was measured by flow cytometry. A flow based CFSE assay was used to measure APL specific cell killing by cytotoxic T-cells (CTLs). As FVB/N mice have H2q haplotyes, blocking anti-H2q antibodies were used to determine if the cytotoxic activity was MHC restricted. Immunophenotyping by measuring CD4+ and CD8+ absolute counts were conducted. Mice injected with APL cells were depleted of CD4+ or CD8+ cells with anti-CD4 or anti-CD8+ antibody treatment initiated the day after DNA vaccination (early) and continued every 5 days and assayed for efficacy of the DNA+ATRA combined therapy or CD4+ or CD8+ cells were depleted in long term survivors (>100 days, late). RESULTS: Th1 cytokines TNFa and IFNg were increased indicative of DNA effects and specific activated CD3/CD8 T cells were detected and observed to release cytotoxic granules in the presence of APL cells in long term survivors. A dose dependent decrease in CFSE positive cells was observed assaying effectors from spleens of ATRA alone, ATRA+DNA treated mice and CD107a+ sorted cells from the latter using APL cells as targets. This effect was MHC restricted as anti-H2q antibodies reduced the specific cytotoxic activity. CD4+ absolute numbers measured on day 38 significantly correlated with survival (p=0.005). Although not significant a similar trend was observed for CD8+ counts. The CD4+ or CD8+ depleted mice treated with DNA + ATRA died earlier compared with the undepleted animals. When DNA + ATRA treated long term survivors were depleted of CD4+ or CD8+ cells, the CD4+ depleted mice relapsed and died in 3 months whereas the CD8+ depleted mice survived for a further 3 months when the experiment was terminated. These data are consistent with an increase in anti-RARa antibody production previously measured in other protocols. Interestingly the late depletions show that CD4+ cells are required for the maintenance of the remissions and show that memory T-cells are required. CONCLUSION: Therefore we have been able to detect protective cellular and humoral responses in mice with the combined treatment of DNA+ATRA, which correlates with outcome.