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Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, chromosome fragility, and cancer susceptibility. Eight FA-associated genes have been identified so far, the products of which function in the FA/BRCA pathway. A key event in the pathway is the monoubiquitination of the FANCD2 protein, which depends on a multiprotein FA core complex. In a number of patients, spontaneous genetic reversion can correct FA mutations, leading to somatic mosaicism. We analyzed the FA/BRCA pathway in 53 FA patients by FANCD2 immunoblots and chromosome breakage tests. Strikingly, FANCD2 monoubiquitination was detected in peripheral blood lymphocytes (PBLs) in 8 (15%) patients. FA reversion was further shown in these patients by comparison of primary fibro-blasts and PBLs. Reversion was associated with higher blood counts and clinical stability or improvement. Once constitutional FANCD2 patterns were determined, patients could be classified based on the level of FA/BRCA pathway disruption, as “FA core” (upstream inactivation; n = 47, 89%), FA-D2 (n = 4, 8%), and an unidentified downstream group (n = 2, 4%). FA-D2 and unidentified group patients were therefore relatively common, and they had more severe congenital phenotypes. These results show that specific analysis of the FA/BRCA pathway, combined with clinical and chromosome breakage data, allows a comprehensive characterization of FA patients.

CD4+ T cells are most likely the ultimate effectors of chronic heart lesions in rheumatic heart disease (RHD). We have demonstrated that infiltrating CD4+ T cell clones were able to recognize several heart tissue and streptococcal antigens by molecular mimicry. Clonality analysis of the mitral valve and myocardium infiltrating T cell lines showed several oligoclonal expansions, some of which were found in both sites of the lesions. The results presented in this study showed a degenerate pattern of reactivity of intralesional T cell clones from one RHD patient. Four mitral valve and one papillar muscle-derived T cell clones, presenting the same TCR-BV13 BJ2S7 with same sequences of the CDR3 region recognized different antigens. They expressed two alpha chains at the RNA level and the AV AJ segments were the same for mitral valve T cell clones, but not for the papillar muscle-derived T cell clone. Two other intralesional T cell clones using the same TCR-BV3 JB2S1 segments with identical CDR3 sequences also recognized different antigens. These results indicate that intralesional T cell clones with common TCR usage can recognize several epitopes that probably amplify the deleterious immune reaction. These data, allow us to hypothesize that degenerate T cell recognition may lead to intramolecular degenerate reactivity against epitopes with low homology. This can be a novel mechanism of epitope spreading, of relevance in the increase of epitopes targets that can activate cross-reactive autoimmune T cells.

Les fonctions lymphocytaires T et NK sont la resultante de signaux d'activation et de signaux inhibiteurs. Parmi les recepteurs des lymphocytes Natural Killer (NK), on distingue : les KIRs (Killer cell Immunoglobulin-like Receptor) et ILTs (Immunoglobulin-Like Transcript), majoritairement inhibiteurs dont les ligands sont les molecules HLA de classe I ; les recepteurs de la famille des lectines de type C, heterodimeres formes par l'association d'une molecule CD94 avec une chaine de la famille des NKG2, dont le ligand principal est la molecule HLA-E ; les recepteurs activateurs NCR (Natural Cytotoxicity Receptors) qui permettent l'activation directe de la cellule NK. Il s'agit des recepteurs NKp30, NKp44, NKp46 et NKG2D. Le stress cellulaire (infections virales ou bacteriennes, tumeurs) peuvent moduler la fonction des lymphocytes NK par differents mecanismes : defaut d'expression des molecules HLA de classe I, levant l'inhibition des recepteurs KIR ; expression de peptides presentes par HLA-E modulant la fonction du recepteur inhibiteur CD94/NKG2A ; activation de recepteurs activateurs, en particulier NKG2D present sur les lymphocytes NK, certaines populations activees de lymphocytes T αβ et γδ. Plusieurs ligands de ce recepteur sont a present connus dont chez l'homme les molecules MIC (MHC class I related), codees dans le locus HLA, exprimees a l'etat physiologique preferentiellement par l'epithelium intestinal et dont l'expression est augmentee lors d'infections virales (CMV), bacteriennes (M. tuberculosis, E. coli) et dans des tumeurs.

A low thymic function is associated with leukemia relapse in children given T-cell-depleted HLA-haploidentical stem cell transplantation

Previous work suggested that expanded CD8+ T-cell clones in the synovial fluid (SF) of HLA-B27+ patients with reactive arthritis (ReA) preferentially use the T-cell receptor variable region (TCRBV) 1, similar CDR3 sequences, and joining region (BJ) 2S3. To determine the range of conservation and disease-specificity of CDR3-sequences, we analyzed the TCRBV1-J2S3 repertoire from 33 healthy HLA-B27+ individuals, patients with various types of spondyloarthropathies (SpA), and with rheumatoid arthritis (RA) by CDR3-spectratyping. After collection and database submission of all available TCRB-CDR3 from HLA-B27-restricted or SpA-derived T cells, we systematically screened the entire human sequence database for sequences similar to the B27/SpA-related CDR3. Spectratyping revealed expanded T cell clones using conserved TCRBV1J2S3 in the SF from 5/6 of the patients with acute ReA but not among the controls. In database searches, 50 HLA-B27 or SpA-related CDR3-sequences generated similar clusters of matched sequences, and matched reciprocally. Identical or closely related sequences were identified in 15 different individuals and a canonical ReA-associated TCRB was defined [BV1-CASSVG(V/I/L)(Y/F)STDTQYF-J2S3]. All but one patient-derived conserved sequences originated from acute stage ReA-patients, and were not present among approximately 3800 other human TCRB sequences in the database. Five of the conserved sequences originated from T cell clones that recognized uninfected cells in an HLA-B27-restricted fashion, implying a role of HLA-B27-restricted CD8+ T cells specific for a ubiquitous self- or cross-reactive microbial determinant in the early phase of ReA. Related sequences were independently identified in four different laboratories. The consensus TCRB motif could be a helpful diagnostic marker in HLA-B27-associated 'undifferentiated arthritis'.

CD1 gene (CD1A to CD1E) products are involved in non-peptide antigen presentation, such as lipids and glycolipids, to T cells. With a similar function to MHC, namely antigen presentation, these genes nevertheless displayed a much lower level of polymorphism as compared to MHC. We report here two additional CD1E variants identified in black African individuals, designated herein CD1E*05 and CD1E*06. While the former differs from the common (wild type) allele sequence by two substitutions at nucleotide positions 217 and 229 of exon 2, the latter only by a single base change at position 91 of exon 3. These substitutions lead to amino acid changes at position 73 and 77 of the alpha1 domain in the former and at position 30 of the alpha2 domain in the latter. Identification of these additional variants suggests that the CD1 locus, especially the CD1E gene, is much more polymorphic than previously assumed.

The aim of this study was to determine whether NK cell receptor (NKR) expression could modulate cytotoxicity of oligoclonal CD8+ T cells present in the synovial fluid (SF) of HLA-B27-reactive arthritis (ReA) patients, especially in a TCRBV1 population shared among different patients and cytotoxic toward HLA-B27. A CD8+ T cell line, two TCRBV1 lines and clones were isolated from the SF of an HLA-B27+ ReA patient, and tested with mAb specific for Ig-like (KIR2DL1, KIR2DL2, KIR3DL1 and ILT2) and CD94 C-type lectin NKR. Transcripts for NKG2 subunits (NKG2A-2E) associated with CD94 were also evaluated. Function was tested in a 51Cr-release cytotoxic assay. We found stable but distinct levels of CD94/NKG2 complexes at the surface of T cell lines and clones. Different NKG2 members could be associated with CD94, either inhibitory (NKG2A/B) or activating (NKG2C). The inhibitory ILT2 receptor could also be differently expressed, but other Ig-like NKR were negative. Functionally, one TCRBV1 line and clones with a high CD94/NKG2A expression did not lyse B27+ targets. Another TCRBV1 line with the same TCRBV1 rearrangement had a low expression of CD94/NKG2A, but expressed NKG2C transcripts and was cytotoxic toward HLA-B27. HLA-B27 is a ligand for ILT2 and we observed an inhibitory effect of ILT2 engagement on B*2705 targets in blockade experiments. Altogether, these data indicate a high degree of heterogeneity in the expression of NKR by intrasynovial CD8+ T cells which could modulate their cytotoxicity and play a role in the control of this HLA class I-associated autoimmune disease.

MICA are distant homologs of MHC class I molecules expressed in the normal intestinal epithelium. They are ligands of the NKG2D activating receptor expressed on most γδ T cells, CD8+ αβ T cells, and natural killer cells and therefore play a critical role in innate immune responses. We investigated MICA cell-surface expression on infection of epithelial cell lines by enteric bacteria and show here that MICA expression can be markedly increased by bacteria of the diffusely adherentEscherichia colidiarrheagenic group. This effect is mediated by the specific interaction between bacterial adhesin AfaE and its cellular receptor, CD55, or decay-accelerating factor. It is extremely rapid after AfaE binding, consistent with a stress-induced signal. MICA induction on epithelial cells triggered IFN-γ release by the NKG2D expressing natural killer cell line NKL. This host–bacteria interaction pathway could play a role in the pathogenesis of inflammatory bowel disease, a condition that implicates a bacterial trigger in genetically susceptible individuals. This was supported by the increased MICA expression at the surface of epithelial cells in colonic biopsies from Crohn's disease-affected patients compared with controls.

Immune reconstitution after allogeneic stem cell transplantation is a dynamic and complex process depending on the recipient and donor characteristics, on the modalities of transplantation, and on the occurrence of graft-versus-host disease. Multivariate methods widely used for gene expression profiling can simultaneously analyze the patterns of a great number of biological variables on a heterogeneous set of patients. Here we use these methods on flow cytometry assessment of up to 25 lymphocyte populations to analyze the global pattern of long-term immune reconstitution after transplantation. Immune patterns were most distinct from healthy controls at six months, and had not yet fully recovered as long as two years after transplant. The two principal determinants of variability were linked to the balance of B and CD8(+) T cells and of natural killer and B cells, respectively. Recipient's cytomegalovirus serostatus, cytomegalovirus replication, and chronic graft-versus-host disease were the main factors shaping the immune pattern one year after transplant. We identified a complex signature of under- and over-representation of immune populations dictated by recipient's cytomegalovirus seropositivity. Finally, we identified dimensions of variance in immune patterns as significant predictors of long-term non-relapse mortality, independently of chronic graft-versus-host disease.

Using B-cell rearrangement excision circle measurements, we analyzed B-cell reconstitution in a cohort of 243 patients who underwent allogeneic stem cell transplantation. Acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively) transiently increased B-cell replication but decreased overall B-cell neogenesis with a clear difference in terms of kinetics. Moreover, the impact of aGVHD in the absence of cGVHD was transient, recovering at month 6 similar values as in patients who did not suffer from GVHD. Conversely, impact of cGVHD at month 12 in multivariate analysis was independent of the previous aGVHD effect on B-cell output. Finally, we showed in patients affected with cGVHD a higher B-cell division rate that correlates with an elevated BAFF/CD19(+) B-cell ratio, supporting a B-cell hyperactivation state in vivo.

BACKGROUND Activating anti-angiotensin type 1 receptor antibodies (AT1R-AA) have been described in patients with systemic scleroderma, an auto-immune disorder with clinical fibrotic features. Chronic graft-versus-host disease (cGvHD) after hematopoietic stem cell transplantation may have clinical fibrotic features, whose pathogenesis may be similar with systemic sclerosis. OBJECTIVE To evaluate the presence of AT1R-AA and their association with clinical and biological symptoms in cGvHD patients. MATERIAL AND METHODS Sera from 87 patients including 45 extensive cGvHD and 42 hematopoietic stem cell transplantation patients without cGvHD were retrospectively analyzed for the presence of AT1R-AA using an enzymatic immunoassay. RESULTS The frequency of AT1R-AA was significantly increased (odds ratio [OR]=3.4, P=0.04) in the cGvHD group (24.4%) compared with the non-cGvHD group (7.1%). In the cGvHD group the positivity of AT1R-AA was significantly associated with: i/ the presence of antinuclear antibodies (OR=5.9, P=0.04) ii/ a more severe global and organ-specific cGvHD scoring (P

International audience; Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin−CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24− progenitor subpopulations. CD34+lin−CD10+CD24− lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin−CD10+CD24− cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.

S.B. and N.D. contributed equally to this work.; International audience; Despite the effectiveness of immunosuppressive drugs, kidney transplant recipients still face late graft dysfunction. Thus, it is necessary to identify biomarkers to detect the first pathologic events and guide therapeutic target development. Previously, we identified differences in the T-cell receptor Vb repertoire in patients with stable graft function. In this prospective study, we assessed the long-term effect of CD8 + T-cell differentiation and function in 131 patients who had stable graft function. In 45 of 131 patients, a restriction of TCR Vb diversity was detected and associated with the expansion of terminally differentiated effector memory (TEMRA; CD45RA+CCR7-CD27-CD28-) CD8+ T cells expressing high levels of perforin, granzyme B, and T-bet. This phenotype positively correlated with the level of CD57 and the ability of CD8+ T cells to secrete TNF-α and IFN-γ. Finally, 47 of 131 patients experienced kidney dysfunction during the median 15-year follow-up period. Using a Cox regression model, we found a 2-fold higher risk (P=0.06) of long-term graft dysfunction in patients who had increased levels of differentiated TEMRA CD8+ T cells at inclusion. Collectively, these results suggest that monitoring the phenotype and function of circulating CD8+ T cells may improve the early identification of at-risk patients.

Background: Graft-versus-host disease (GVHD) is a major cause of mortality after unrelated hematopoietic stem cell transplantations (HSCT). Despite the development of modern immunosuppressive strategies, a nearly perfectly controlled compatibility of the classical HLA genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) and availability of numerous so-called minor histocompatibility antigens (e.g. HY or HA-1), its incidence remains largely unexplained to date. MIC genes (MHC class I chain-related) - a distinct lineage of MHC class I genes – are promising candidates to explain, at least partially, the incidence of GVHD in HLA-matched transplantations. MICA and MICB are highly polymorphic (100 alleles for MICA and 40 for MICB) and encode functional cell-surface glycoproteins up-regulated by cell stress. They interact with NKG2D, an activating receptor expressed on the surface of cytotoxic αβ CD8+ and γδ T lymphocytes and natural killer cells. MIC genes are already known to have a HLA-independent effect on solid graft outcomes and may play a similar role in HSCT by triggering GVHD. Objective: The objective of the present study was to determine the impact of donor/patient matching at the MICA and MICB loci on the incidence of GVHD in patients undergoing unrelated HSCT. Methods: We retrospectively analyzed a multicenter cohort of 1072 unrelated transplantations performed between 1996 and 2013. All donor-recipient pairs were fully typed at high resolution for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 and were matched for ten of ten HLA alleles (HLA 10/10 matched). High resolution genotyping of MICA and MICB was performed by sequenced-based typing in order to define matching grades between donors and patients. The endpoints of the study were acute and chronic GVHD. Apart from HLA-DPB1 matching, statistical models were adjusted for major clinical variables which have been shown to be associated with outcome (patient’s age, patient’s and donor’s sex, patient’s and donor’s serological status for cytomegalovirus, year of transplantation, time to transplantation, transplantation center, source of stem cells, conditioning regimen, GVHD prophylaxis, treatment with anti-thymocyte globulin, disease category and severity at transplantation). Results: Of the 1072 transplantations, 134 (12.5 %) and 380 (35.4 %) were mismatched at the MICA and MICB locus, respectively. Both MICA and MICB mismatches were significantly associated with an increased incidence of severe acute GVHD (grades III-IV) in univariate and multivariate models (multivariate model: HR = 2.32, 95 % CI = 1.84-2.92; p=0.0003 for MICA and HR = 1.49, 95 % CI = 1.24-1.79; p=0.03 for MICB). At day 100 post-HSCT severe acute GVHD incidences in mismatched vs. matched transplantations were 19.62 % vs. 15.08 % and 20.00 % vs. 14.84 % for MICA and MICB, respectively (Figure 1). Chronic GVHD was associated with MICA and MICB mismatches in univariate analysis (HR = 1.55, 95 % CI = 1.27-1.89; p=0.029 for MICA and HR=1.38, 95 % CI = 1.19-1.62; p=0.03 for MICB), but showed only a trend for association in multivariate models. Figure 1 Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Figure 1. Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Conclusion: To date this is the largest reported MICA and MICB sequence analysis whether in HSCT or solid organ transplantation. Inclusion of MICA and MICB typing in the donor selection process may be a practical clinical strategy for lowering the risks of severe acute GVHD after unrelated HSCT. Disclosures No relevant conflicts of interest to declare.

To evaluate the long-term immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), we prospectively screened standard immune parameters in a series of 105 patients, at a median time of 15 months after SCT. Analysing lymphoid phenotypes, in vitro immune functions and immunoglobulin levels, we found that, more than 1 year post SCT, cellular and humoral immunity was still altered in a significant number of patients. CD4+ T cells were < 200/microl in one third of patients, and the CD4/CD8 ratio was still reversed in 78% of patients. Almost all patients showed positive T-cell responses against mitogens, but antigen-specific proliferation assays identified 20% to 80% of non-responders. B-cell counts were reconstituted in 61% of the patients, but levels of total immunoglobulins were still low in 59%. In multivariate analyses, human leucocyte antigen (HLA) disparity between donor and recipient and chronic graft-versus-host disease were the leading causes affecting immune reconstitution. Interestingly, cytomegalovirus (CMV) infections were strongly associated with normal CD8+ T-cell counts. Studying the impact of impaired immune reconstitution on the rate of infections occurring in the 6 years following screening, we identified three parameters (low B-cell count, inverted CD4/CD8 ratio, and negative response to tetanus toxin) as significant risk factors for developing such late infections.

Eighteen different HLA-B*27 alleles (B*2701-B2718) have so far been recognized by the WHO Nomenclature Committee for Factors of the HLA System. Frequency and disease association of these alleles with spondyloarthropathies differ among ethnic groups. We describe here a novel HLA-B*27 subtype identified in a Lebanese patient suffering from ankylosing spondylitis (AS). This new variant differs from the common HLA-B*2705 DNA sequence at five different nucleotide positions. These nucleotide changes lead to three amino acid differences in the alpha2 domain; Thr to Ile at position 94, Leu to Ile at position 95 and Asn to Arg at position 97. Since this novel allele is encountered in an AS patient, the associated sequence changes are not expected to affect significantly neither the presentation of a putative arthritogenic peptide nor the conformation-dependent recognition by effector cells.

Background: Post transplant immune reconstitution plays a major role in determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is currently monitored with different techniques in different Centers, with the aim of identifying clinically relevant immunological biomarkers. However, it is unclear which and how many of these immunological tests are currently performed on a routine basis, and which ones have the potential to predict patient outcome, and possibly guide patient care after allo-HSCT. Methods: The EBMT Cellular Therapy & Immunobiology Working Party (CTIWP) conducted a survey to identify current policies to monitor immune reconstitution in patients undergoing allo-HSCT and possibly reach a general consensus. This study followed the EBMT study guidelines. All EBMT Centers were invited to participate. Each participating Center received a questionnaire on the availability of specific immunomonitoring assays, specifying the use in clinical practice and/or within investigational trials. Assays were based on relatively simple and readily available parameters such as absolute lymphocyte counts (ALC) to more complex cellular and molecular tests. Moreover, the Centers were asked to define the transplant platform (HLA-identical sibling, matched unrelated donor, haploidentical and/or cord blood) on which each test is generally performed. Results: Policies for post-transplant immunomonitoring have been reported by 35 participating EBMT Centers active in 14 Countries and performing allo-HSCT from HLA identical related (35 centers), matched unrelated (33), haploidentical (34), unrelated cord blood (27). Complete blood counts and immunoglobulins are routinely tested for patients' care by all centers. Relative proportions of T cell subsets are currently tested by flow-cytometry as "standard of care" or "investigational" by 82% and 17% of centers respectively. B cell and NK cell counts are quantified routinely by 46% and 23% of Centers, and investigationally by 40% of Centers. The availability of molecular tests (STR, qPCR, Fish) to measure post-transplant engraftment are reported by all Centers, except two, as a standard of care measure. T cell receptor-expressing circles (TRECs) and/or K-deleting recombination excision circles (KRECs) are quantified within selected clinical trials by 37% of Centers. Interestingly, 60% of Centers evaluate, mostly as an investigational measure, antigen specific T cell responses by: proliferation assays (49%), interferon-gamma enzyme-linked immunospot-Elispot (49%), intracellular cytokine staining (46%) and tetramer/dextramer staining (37%). Most of these Centers test responses to Cytomegalovirus and Epstein Barr Virus, and 5 Centers use at least one of these assays on a routine basis. About half of the participating Centers (43%) commonly test antigen-specific antibodies, mainly as responses to vaccines, and not routinely. T-cell receptors (TCR) and B-cell receptors (BCR) repertoires are measured by spectratyping in 14 out of 35 Centers (4 as clinical practice and 10 in selected trials), or, in selected trials, by next generation sequencing (in 11 out of 35 the participating Centers). Conclusions: Results of this survey indicate that country- and center expertise are associated with heterogeneous and distinct protocols, and underline the clinical need to harmonize methods and to provide practical recommendations for monitoring post-transplant immune reconstitution, both for routine purposes and investigational studies. Adequate reporting and connection between individual Centers exploiting these data will foster collaborative and comparative research studies, with the ultimate goals of improving patient care and refining our understanding of the immunological correlates to clinical outcome. Acknowledgments: R. Ram, M. A. Diaz, G. McQuaker, D. Russo, E. Faber, P. Chiusolo, C. Rössig, S. M. Martin, A. Anagnostopoulos, M. Stelljes, K. Orchard, P. Jindra, A. Sampol, K. Patrick, M. A. Bekadja, J. Gayoso, A. Olivieri, J. Passweg, E. Jost, H Labussiere-Wallet, Y Koc, A. Lange, I. Garcia Cadenas, N. Kröger, A. Biondi, N. Milpied, D. Olive, E. Lanino, G. Stuhler, J.H. Dalle, J.R. Cabrera Marín, F. Ciceri, D. Uckan-Cetinkaya, R. Parody Porras, G. Kriván. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Introduction Nous avons precedemment montre la fiabilite du marqueur CD158k/KIR3DL2 pour identifier les cellules de Sezary (CS). Il nous a permis de realiser un suivi prospectif des patients atteints de maladie de Sezary (MS). Contrairement a la litterature qui rapportait les CS comme uniformement centrales memoires (TCM) dans la MS, nous avons observe l’ensemble des profils possibles, de la T naive (TN) a la T effectrice terminale (TEMRA). Nous nous sommes egalement interesses a des phenotypes nouvellement decrits : TSCM ( T stem cell memory ), et T residente memoire (TRM). Materiel et methodes Cinquante-quatre echantillons sanguins de patients Sezary ont ete analyses en cytometrie en flux pour determiner leur phenotype naif/memoire. En parallele, 16 patients ont egalement subi le meme jour une biopsie cutanee afin de comparer l’expression de marqueurs de maturation (CD45RA, CCR7, CD27, CD95), de TRM (CD69, CD103), mais aussi de recepteurs d’interleukines, de chemokines et de marqueurs d’activation. Les CS ont ete definies comme les lymphocytes T CD3 + CD158k + , et la concordance avec le clone Vβ connu du TCR a ete verifiee lorsque l’anticorps etait disponible. Resultats Cinquante-sept pour cent des patients ont un phenotype homogene de CS, correspondant soit a des TCM (CD45RA − CCR7 + CD27 + ), soit a des pseudo-naives (CD45RA + CCR7 + CD27 + ), alors que 43 % de la cohorte presente un profil complexe associant plusieurs sous-types, de la TN a la TEMRA. Grâce a l’antigene CD95, en reprenant l’analyse sur cellules mononucleees congelees de 13 patients presentant des types pseudo-naifs, nous avons pu distinguer la presence de CS de type TSCM (CD45RA + CCR7 + CD27 + CD95 + ) chez 6 d’entre eux. Dans la peau, les CS presentent une association de phenotypes exclusivement memoires. Elles expriment des marqueurs de TRM, ce qui n’est pas le cas des CS circulantes. On retrouve une expression forte mais heterogene de CCR4 et CCR10 sur les CS sanguines, egalement sur celles de la peau, de plus souvent CLA + et/ou CCR6 + . De fortes expressions de CD127 (IL-7Rα), CD25 et PD-1, sont retrouvees, en particulier a la surface des CS cutanees. Discussion Nos resultats mettent en evidence l’heterogeneite intra- et inter-individuelle des CS en termes de phenotypes de maturation, egalement entre les deux compartiments de la MS. Les CS cutanees montrent des profils plus matures que les CS circulantes. Conclusion Nous analysons actuellement le transcriptome de differentes sous-populations de CS afin de pouvoir relier fonctionnellement ces phenotypes a un comportement naif, souche memoire ou memoire de ces cellules, et potentiellement a des differences d’agressivite tumorale. La meilleure comprehension de l’origine des CS, et des interactions entre les compartiments sanguins et cutanes de la MS, pourrait permettre de definir la cible cellulaire la plus pertinente pour les immunotherapies du futur.

Mismatched unrelated donor (MMUD) or umbilical cord blood (UCB) can be chosen as alternative donors for allogeneic stem cell transplantation but might be associated with long-lasting immune deficiency. Sixty-six patients who underwent a first transplantation from either UCB (n = 30) or 9/10 MMUD (n = 36) and who survived beyond 3 months were evaluated. Immune reconstitution was prospectively assessed at sequential time points after transplantation. NK, B, CD4(+), and CD8(+) T cells and their naïve and memory subsets, as well as regulatory T cells (Treg), were studied. Detailed analyses on infections occurring after 3 months were also assessed. The 18-month cumulative incidences of infection-related death were 8% and 3%, and of infections were 72% and 57% after MMUD and UCB transplantation, respectively. Rates of infection per 12 patient-month were roughly 2 overall (1 for bacterial, .9 for viral, and .3 for fungal infections). Memory, naïve CD4(+) and CD8(+)T cells, naïve B cells, and Treg cells reconstitution between the 2 sources were roughly similar. Absolute CD4(+)T cells hardly reached 500 per μL by 1 year after transplantation and most B cells were of naïve phenotype. Correlations between immune reconstitution and infection were then performed by multivariate analyses. Low CD4(+) and high CD8(+)T cells absolute counts at 3 months were linked to increased risks of overall and viral (but not bacterial) infections. When assessing for the naïve/memory phenotypes at 3 months among the CD4(+) T cell compartment, higher percentages of memory subsets were protective against late infections. Central memory CD4(+)T cells protected against overall and bacterial infections; late effector memory CD4(+)T cells protected against overall, bacterial, and viral infections. To the contrary, high percentage of effector- and late effector-memory subsets at 3 months among the CD8(+) T cell compartment predicted higher risks for viral infections. Patients who underwent transplantation from alternative donors represent a population with very high risk of infection. Detailed phenotypic analysis of immune reconstitution may help to evaluate infection risk and to adjust infection prophylaxis.

The nonpolymorphic soluble HLA-G1 (sHLA-G1) isoform has been reported to be secreted by trophoblast cells at the materno-fetal interface, suggesting that it may act as immunomodulator during pregnancy. In this paper, we report that affinity-purified β2-microglobulin-associated sHLA-G1 triggered apoptosis in activated, but not resting CD8+ peripheral blood cells. We demonstrate by Western blotting that sHLA-G1 enhanced CD95 ligand expression in activated CD8+ cells. Cytotoxicity was inhibited by preincubation of the cells with a CD95 antagonist mAb (ZB4) or a soluble recombinant CD95-Fc, indicating that apoptosis is mediated through the CD95/CD95 ligand pathway. Finally, we show that such sHLA-G1-induced apoptosis depends on the interaction with CD8 molecules, with cell death being blocked by various CD8 mAbs.

A novel HLA-B∗39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B∗3916. It differs from HLA-B∗3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of α 2 domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with α 2 m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B∗3916, or its expression or both.

Spondyloarthropathies constitute a group of autoimmune diseases of special interest because of their tight association with the MHC class I molecule HLA-B27 and the bacterial triggering of some clinical forms called reactive arthritis (ReA). One current hypothesis is the presentation by HLA-B27 of a so-called arthritogenic peptide to T cells. To better focus on the relevant T cell populations within the joint, we performed an extensive β-chain T cell repertoire analysis of synovial fluid compared with PBL in seven patients, four of whom were characterized as having ReA triggered by Yersinia enterocolitica, Chlamydia trachomatis, or Shigella sonnei. Analysis of the size diversity of the β-chain complementarity-determining region 3 (CDR3) allowed us to evaluate the degree of T cell clonality in the samples. Oligoclonal T cell expansions were frequently observed in the joint. In one patient, CDR3 amino acid sequences of major expansions using two different BV genes were identical. One dominant T cell expansion and several CDR3 amino acid sequences were identical in two different patients. Furthermore, one sequence was identical with a sequence reported independently in a Salmonella-induced ReA patient. Together, these data indicate a surprisingly high degree of conservation in the T cell responses in recent-onset ReA triggered by different micro-organisms. A CD8+ synovial line expressing shared clonotypes was established and reacted toward several B*2705 lymphoblastoid cell lines, therefore supporting a molecular mimicry phenomenon at the T cell level in the disease mechanism.

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.

Introduction Les infections fongiques invasives (IFI) sont associees a une mortalite elevee en reanimation. Elles surviennent surtout en cas d’association de troubles de l’immunite et d’une rupture de la barriere cutaneo-muqueuse. Les patients brules graves apparaissent donc particulierement a risque. Une meilleure caracterisation de leurs deficits immunitaires pourrait permettre d’identifier ceux les plus a risque et de proposer des therapeutiques immmunomodulatrices. L’objectif etait de decrire le profil immunologique de patients brules graves ayant presente une IFI. Materiel et methodes Les patients brules > 20 % de surface cutanee brulee (SCB) admis entre mars et octobre 2014 ont ete inclus. Prelevements sequentiels a J0, J2, J7, J14, J28 ou jusqu’a la sortie ou le deces du patient. Les marqueurs de l’immunite innee ont ete explores en cytometrie de flux sur sang total : Natural killer (NK), Mucosal Associated Invariant T cells (MAIT), quantification des molecules HLA-DR sur les monocytes, polynucleaires neutrophiles (PNN) et sous populations monocytaires (ces dernieres definies selon des marquages membranaires classiques (CD14 + CD16−), intermediaires (CD14 + CD16 + ) et non classiques (CD14lowCD16 + )). Les patients avec IFI (IFI + ) ont ete compares avec ceux sans (IFI−). Resultats exprimes en medianes (25–75 percentiles). Analyse par test de ANOVA pour mesures repetees analyse post-hoc (Bonferroni) ou Mann-Whitney. p Resultats 26 patients ont ete inclus, 7 (27 %) etaient IFI+. La SCB des IFI+ etait de 40 (33–85) % vs 33 (27–42) % pour les IFI− (p = 0,14). L’IGS 2 et l’ABSI etaient respectivement de 41 (36–49) et 9 (9–13) chez les IFI+ et 23 (17–33) et 7 (6–8) chez les IFI− (p = 0,002 et p = 0,009 respectivement). La mortalite a 90 jours etait de 57 % chez les IFI+ et de 11 % chez les IFI−. Le compte absolu de PNN n’etait pas statistiquement different entre les deux. Le compte absolu de monocytes etait plus bas a J2 chez les IFI + (p = 0,03). Le nombre de site d’HLA-DR par monocyte etait plus bas des J3 chez les IFI+ et le restait par la suite (p = 0,03). Les cellules NK etaient plus basses chez les IFI+ a partir de J7 et les MAIT plus bas chez les IFI+ a J0 (p = 0,01) mais cette difference s’estompait ensuite. Une population particulierement representee chez les IFI+, notamment a mucormycose ( Fig. 1 ), possede des caracteristiques morphologiques de monocytes et exprime faiblement CD14. Elle presente cependant un profil de PNN (CD45low, CD16hi, CD66 + ). Ce phenotype composite est evocateur de cellules myeloides suppressives MDSC. Discussion Les brules graves presentent une alteration profonde de leur immunite innee. Cette alteration est d’autant plus marquee chez les patients IFI+. L’augmentation de cellules myeloides suppressives chez ces patients pourrait contribuer a l’immunosuppression favorisant l’IFI.

Shigella flexneri is a triggering agent for reactive arthritis in HLA-B27-susceptible individuals. Considering the intracellular multiplication of bacteria, it seems likely that bacterial peptides may be presented by the major histocompatibility complex (MHC) class I pathway. To examine this hypothesis, we infected HLA-B*2705- and/or human β2-microglobulin-transfected murine L-cell lines with M90T, an invasive strain of S. flexneri . Bacterial infection induced no detectable modifications in the biosynthesis and expression level of HLA-B27, as assessed by immunoprecipitation, Northern blot analysis, and flow cytometry. Using confocal microscopy, we observed that bacterial infection induced a clustering of HLA-B27 molecules during macropinocytosis and before bacterial dissemination from cell to cell. Peptides naturally bound to HLA-B27 molecules were acid eluted from infected cells and separated by high-performance liquid chromatography. Major differences were observed in high-performance liquid chromatography profiles and in the nature of peptides presented following bacterial infection. Although most of the antigens presented were not accessed by Edman degradation, we obtained two sequences partially homologous to bacterial proteins. These peptides lacked the major HLA-B27 peptide anchor (Arg) at position 2, and one had an unusual length of 14 amino acids. These data suggest that alterations in the peptide presentation by HLA-B27 occur during infection, which could be relevant to the pathogenesis of HLA-B27-related arthritis.

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.

Crohn's disease (CD) is an inflammatory pathology of the mucosal intestine that results from uncontrolled immune response towards commensal microbes. Clonal expansions of T cells have been found in patients with CD suggesting an antigen-specific stimulation of pathogenic T cells. Here we show, using T-cell receptor repertoire analysis by real-time PCR, that oligoclonal expansions are found in both CD8+ and CD4+ T cells in the blood and intestinal mucosa of CD patients. The majority of CD4+ T-cell-expanded clones are CD4+NKG2D+ T cells. These clonal expansions were found in both inflamed and neighboring healthy tissue and were persisting during the course of the disease. The presence of these CD4+NKG2D+ T-cell clones at the macroscopically normal edge of the surgical resection might be predictive of inflammation relapse post surgery.

Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.

Use of alternative donors/sources of hematopoietic stem cells (HSC), such as cord blood (CB) or HLA-haploidentical (Haplo)-related donors, is associated with a significant delay in immune reconstitution after transplantation. Long-term T-cell immune reconstitution largely relies on the generation of new T cells in the recipient thymus, which can be evaluated through signal joint (sj) and beta T-cell-Receptor Excision Circles (TREC) quantification. We studied two groups of 33 and 24 children receiving, respectively, HSC Transplantation (HSCT) from an HLA-haploidentical family donor or an unrelated CB donor, for both malignant (46) and non-malignant disorders (11). Relative and absolute sj and beta-TREC values indicated comparable thymic function reconstitution at 3 and 6 months after the allograft in both groups. Compared to children with non-malignant disorders, those with hematological malignancies had significantly lower pre-transplantation TREC counts. Patients who relapsed after HSCT had a significantly less efficient thymic function both before and 6 months after HSCT with especially low beta-TREC values, this finding suggesting an impact of early intra-thymic T-cell differentiation on the occurrence of leukemia relapse.

Telomere attrition induces cell senescence and apoptosis. We hypothesized that age-adjusted pretransplantation telomere length might predict treatment-related mortality (TRM) after hematopoietic stem cell transplantation (HSCT). Between 2000 and 2005, 178 consecutive patients underwent HSCT from HLA-identical sibling donors after myeloablative conditioning regimens, mainly for hematologic malignancies (n = 153). Blood lymphocytes' telomere length was measured by real-time quantitative PCR before HSCT. Age-adjusted pretransplantation telomere lengths were analyzed for correlation with clinical outcomes. After age adjustment, patients' telomere-length distribution was similar among all 4 quartiles except for disease stage. There was no correlation between telomere length and engraftment, GVHD, or relapse. The overall survival was 62% at 5 years (95% confidence interval [CI], 54-70). After a median follow-up of 51 months (range, 1-121 months), 43 patients died because of TRM. The TRM rate inversely correlated with telomere length. TRM in patients in the first (lowest telomere length) quartile was significantly higher than in patients with longer telomeres (P = .017). In multivariate analysis, recipients' age (hazard ratio, 1.1; 95% CI, .0-1.1; P = .0001) and age-adjusted telomere length (hazard ratio, 0.4; 95% CI; 0.2-0.8; P = .01) were independently associated with TRM. In conclusion, age-adjusted recipients' telomere length is an independent biologic marker of TRM after HSCT.

Lymphoid-committed CD34(+)lin(-)CD10(+)CD24(-) progenitors undergo a rebound at month 3 after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the absence of acute graft-versus-host disease (aGVHD). Here, we analyzed transcriptional programs of cell-sorted circulating lymphoid-committed progenitors and CD34(+)Lin(-)CD10(-) nonlymphoid progenitors in 11 allo-HSCT patients who had (n = 5) or had not (n = 6) developed grade 2 or 3 aGVHD and in 7 age-matched healthy donors. Major upregulated pathways include protein synthesis, energy production, cell cycle regulation, and cytoskeleton organization. Notably, genes from protein biogenesis, translation machinery, and cell cycle (CDK6) were overexpressed in progenitors from patients in the absence of aGVHD compared with healthy donors and patients affected by aGVHD. Expression of many genes from the mitochondrial oxidative phosphorylation metabolic pathway leading to ATP production were more specifically increased in lymphoid-committed progenitors in the absence of aGVHD. This was also the case for genes involved in cell mobilization such as those regulating Rho GTPase activity. In all, we found that circulating lymphoid-committed progenitors undergo profound changes in metabolism, favoring cell proliferation, energy production, and cell mobilization after allo-HSCT in humans. These mechanisms are abolished in the case of aGVHD or its treatment, indicating a persistent cell-intrinsic defect after exit from the bone marrow.

Purpose: Therapy targeting CTLA-4 immune checkpoint provides increased survival in patients with advanced melanoma. However, immunotherapy is frequently associated with delayed and heterogeneous clinical responses and it is important to identify prognostic immunological correlates of clinical endpoints. Experimental design: 77 patients with stage III/IV melanoma were treated with ipilimumab alone every 3 weeks, during 9 weeks. Blood samples were collected at the baseline and before each dose for in depth immune monitoring. Results: The median follow-up was 28 mo with a median survival of 7 mo. Survival and clinical benefit were significantly improved when absolute lymphocyte count at the baseline was above 1 × 109/L. Notably, ipilimumab had a global effect on memory T cells, with an early increase of central and effector subsets in patients with disease control. By contrast, percentages of stem cell memory T cells (TSCM) gradually decreased despite stable absolute counts and sustained proliferation, suggesting a process of differentiation. Higher proportions of eomes+ and Ki-67+ T cells were observed, with enhanced skin homing potential and induction of cytotoxic markers. Conclusion: These results suggest that CTLA-4 blockade is able to reshape the memory subset with the potential involvement of Eomes and memory subsets including TSCM.

Resumen Muchos reumatismos inflamatorios se desarrollan sobre un terreno de predisposicion genetica. Asi lo indica la asociacion significativa de la espondiloartritis con HLA B27* 05 y de la artritis reumatoide con HLA DRB1*0401 y DRB 1*1001. Los estudios inmunogeneticos forman parte del diagnostico y podrian tener un valor pronostico predictivo, en particular en la artritis reumatoide.

BACKGROUND Preemptive rituximab (R) treatment decreases the incidence of Epstein-Barr virus (EBV) posttransplantation lymphoproliferative disease, but the extent of immune deficiency related to R in patients who received allogeneic hematopoietic stem-cell transplantation is unclear. The aim of our study was to evaluate the incidence of late infections and immune reconstitution after preemptive R treatment of EBV infection. METHODS Seventy-eight patients receiving preemptive R between January 2005 and January 2010 were studied. Fifty-two of them could be matched with controls (not receiving R) according to administration of antithymoglobulin, stem-cell source and donor type, age and grade of acute graft-versus-host disease. RESULTS Among the 78 patients with EBV reactivation treated with R, the 36-month cumulative incidence of bacterial, viral, and fungal infections was 64%, 59%, and 23%, respectively. When compared with controls, bacterial infection incidence was significantly higher in R patients (55% vs. 35%), and a slower reconstitution of B cells was observed. R patients had modest but not significantly higher nonrelapse mortality (35% vs. 15%) than controls. CONCLUSION R has dramatically decreased risks of posttransplantation lymphoproliferative disease but is followed by a prolonged and profound B-cell deficiency associated with an excess risk of bacterial infection and higher mortality. R should be given with caution, and immunoglobulin replacement should be provided to limit these excess risks.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma in adults. It is generally treated by a combination of chemotherapy and CD20-specific mAbs, such as rituximab, which act, at least partially, by activating antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves NK cells, particularly the CD56(dim) NK-cell subset expressing CD16, the low affinity Fcγ receptor. Here, we show that CD16 expression levels are decreased in a cohort of 36 newly diagnosed DLBCL patients compared with those in 20 healthy controls (HCs). CD137, a co-stimulatory molecule expressed on activated NK cells, was also expressed at lower levels in patients compared with controls. Cells sampled from our cohort also showed severely reduced degranulation activity when challenged with rituximab-coated tumor cells, which could not be corrected by stimulation with high doses of IL-2. These results suggest that rituximab-induced NK-cell ADCC could be defective in some DLBCL patients at diagnosis. These patients should be closely monitored and attempts made to improve their NK-cell function.

Assessment of the host immune status is becoming a key issue in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to persistent immune defects. In allo-HSCT, T-cell differentiation of donor progenitors within the recipient thymus is required to generate naive recent T-cell emigrants (RTE). These cells account for a durable T-cell reconstitution, generating a diverse T-cell receptor (TCR) repertoire and robust response to infections. It is now possible to quantify the production of RTE by measuring thymic T-cell receptor excision circles or 'TREC' which are small circular DNA produced during the recombination of the genomic segments encoding the TCR alpha chain. Here we discuss the role of thymic function in allo-HSCT. The pre-transplant recipient thymic function correlates with clinical outcome in terms of survival and occurrence of severe infections. Post-transplant, TREC analysis showed that the thymus is a sensitive target to the allogeneic acute graft-versus-host disease (GvHD) reaction but is also prone to recovery in young adult patients. In all, thymus is a key player for the quality of immune reconstitution and clinical outcome after allo-HSCT. Thymic tissue is plastic and it is a future challenge to halt or reverse thymic GVHD therapeutically by acting at the level of T-cell progenitors generation, thymic homing and/or epithelial thymic tissue preservation.

Umbilical cord blood transplantation (UCBT) is an alternative in the absence of related or unrelated HLA-matched donor. Defects in the mechanisms of immuno-surveillance, due to the absence of antigen-experienced lymphoid subsets transferred in the cord blood unit (CBU), render recipients more prone to viral infections. Our center conducted a clinical trial testing the benefit of ex vivo amplification of the CD34+ fraction (using SCF, FLT3L, TPO and GCSF) enriched from one CBU on hematopoietic reconstitution, while the CD34- fraction was infused at the same time as the expanded fraction (NCT01034449). We intended to simultaneously evaluate the potential impact of such a procedure on the immune reconstitution following freeze-thaw cycle for the CD34- fraction and on the expansion potential of lymphoid progenitors from CD34+ fraction. We prospectively analyzed fresh patient samples from the clinical trial (called the EVEX), at different time points (Day 42, D100, D180, D360), and, in parallel, from patients receiving one or two un-manipulated CBU during the same period (Control group - CTRL). Immune monitoring included flow cytometry analysis of T-cells (CD3+, CD4+, CD8+) and sub-compartments, B-cells (CD19+), NK cells (CD3-CD16+CD56+) and Dendritic cells (DC). Sixteen patients were included in EVEX, of which 12 were analyzed (4 excluded: 1 not grafted, 1 graft infection, 2 primary rejections before Day 42) and 12 patients in CTRL. In the EVEX group, all patients received reduced intensity conditioning, compared to 6 out of 12 in the CTRL group. GVHD prophylaxis was similar. EVEX showed a shorter duration of neutropenia compared to the CTRL (8.5 versus 17 days, p=0.02). NK and B-cell subsets recovered earlier than T-cells, reaching median normal values at 3 and 6 months, respectively. NK cell count tended to be lower in EVEX, starting at 3 months until 1 year. B-cell counts were lower for EVEX at D360. Both groups recovered T-cells within 12 months, but, interestingly, EVEX recovered earlier, reaching Healthy Donor (HD) median values as soon as D180. The CD4+ T-cells (CD4+T) recovered within 12 months for both groups, with EVEX values tending to plateau at low normal values (Fig 1). CD8+ T-cells (CD8+T) reached HD values within 12 months with an earlier recovery for EVEX (Fig 1). We characterized CD4+T and CD8+T compartments with CD45RA and CD27 delineating: naïve (TN: CD45RA+CD27+), central memory (TCM: CD45RA-CD27+), effector memory (TEM: CD45RA-CD27-) and late effector (TEMRA: CD45RA+CD27-) T-cells. Among the CD4+T, TN and TCM gradually increased until D360 for CTRL, while EVEX reached a plateau at D180. EVEX showed a higher TEMRA CD4+T count at D360. Thus, the plateau observed in the whole CD4+T at D180 in EVEX suggested an altered capacity to induce new CD4+T (thymic output or peripheral expansion), or an exhaustion phenomenon. To this end, we evaluated thymic function via CD31 expression among TN CD4+T and TREC analysis by qPCR. Both methods showed a decreased thymic function in EVEX after D180, probably related to the age difference observed (median: 52 vs. 26 years old for EVEX and CTRL, respectively). TEM and TEMRA CD8+T subsets were higher starting at D180 for the EVEX group. There was no difference between groups for myeloid or plasmacytoid DC. Group outcomes differed in terms of chronic GVHD (cGVHD) (1 vs. 6 patients for EVEX and CTRL respectively, p=0.024). Among the subsets playing a role in cGVHD pathogeny, no difference was found in Treg/Tcon ratio (Treg = CD25hiCD127lo CD4+T, Tcon = non-Treg CD4+T), but memory B-cell subset tended to be greater in CTRL from D42 to D180 (median time of cGVHD onset = 186.5 days). CMV reactivation rate was similar in both groups, occurring around 42 days post transplant. Vdelta-2 negative γd T-cells were greater in EVEX early after infection (D42), suggesting a comparable anti-CMV innate function. However, when looking at adaptive anti-CMV immunity with ELISpot, we found reduced IFNg-secreting cells in EVEX, although group size was small. In conclusion, patients receiving ex vivo expanded CBU showed faster hematopoietic reconstitution than un-manipulated CBU and comparable recovery of the main immune subsets. Baseline clinical differences observed between groups may have impacted on some of the immune findings. A prospective and randomized trial would help to better analyze if the expansion procedure has an impact on immune reconstitution. Disclosures Milpied: Celgene: Honoraria, Research Funding.

Background. Wiskott Aldrich Syndrome (WAS) is a rare primary immunodeficiency associated with thrombocytopenia, eczema, severe infectious and autoimmune complications, and lymphomas. Mismatched allogeneic hematopoietic stem cell transplantation (HSCT) is an alternative for patient lacking an HLA-matched donor but is associated with an increased frequency of complications. Moreover low lymphoid and myeloid chimerism is related to a higher rate of autoimmunity and thrombocytopenia. Recent gene therapy (GT) trials showed that gene-corrected autologous CD34+ cells infusion could be an appropriate therapeutic approach for these patients. It has been recently shown that B cell homeostasis is altered in WAS. As the B cell reconstitution participates to the restoration of immunological competence, a comprehensive study of this compartment after GT and the comparison with mismatched allogeneic HSCT is crucial. Objective. To perform a longitudinal study of B cell reconstitution in WAS patients after lentiviral vector-mediated GT, compared to mismatched allogeneic HSCT. Methods. Five patients (age 0.8-15.5 years) underwent GT at our center since 2011(follow-up 1.5-4.2 years) after near-myeloablative and immunosuppressive conditioning regimen with (n=3) or without (n=2) anti-CD20 administration. Patient 2 (P2) died 7 months after GT from a pre-existing infectious complication. Eleven patients undergoing mismatched allogeneic HSCT (age 0.6-10.9 years) at the same center were studied (follow-up 5.1-14.7 years). Longitudinal B cell assessment included B cell count before and after treatment, and the following subsets: switched memory (SM, CD19+ CD27+ IgD-), marginal zone (MZ, CD19+ CD27+ IgD+), naives (CD19+ CD27- IgD+), transitional (CD19+ CD27- IgD+ CD24high CD38high), circulating plasma cells (CD19+ CD27+ IgD- CD27high CD38high) and CD21low B cells (CD19+ CD21low CD38-), a subset abnormally expanded in WAS. Quantification of the B cell replication history was assessed through k-deleting recombination excision circles (KRECs). Analyses were compared to age-matched controls. WAS protein (WASP) expression and vector copy number (VCN) were measured in sorted B cells. Results. All alive GT patients show stable engraftment of functionally corrected lymphoid cells, without adverse events. Transduced B cells number and WASP expression increased progressively after GT. Absolute B cell count attained normal values in all the patients, and correlates with WASP expression and VCN in B cells. IgM levels are below normal ranges in four patients. P3 and P4 attained a B-cell phenotype within normal ranges; P3 discontinued intravenous immunoglobulin (IvIg) replacement. No expansion of CD21low B cells was observed. P1 and P5 (follow-up 18 months) present a variable defect in SM, naives and/or MZ B cells. P1 recently developed autoimmune manifestations; no significant changes were observed concomitantly. A defect in B cell lymphopoiesis was observed before GT as measured by KRECs analysis, normalizing after GT (P1, P3 and P4). Several complications were recorded in patients undergoing mismatched allogeneic HSCT, including dysimmunity, arthritis, developmental deficit and infections. Total B cell count normalized in eight patients, IgM levels were low in three. Among patients with available information, four still remain under IvIg replacement. Four patients developed a mixed lymphoid and myeloid chimerism, variably associated with low B cell count, low IgM and IvIg replacement. A complete B cell assessment for these patients is ongoing. Conclusions. B cell transgene expression is obtained after lentiviral vector-mediated GT in WAS patients and is associated with improved B cell lymphopoiesis. A correct B cell phenotype is observed in two patients who did not receive rituximab prior GT. The question whether this is related to the treatment will need a longer follow-up to be answered. Patients undergoing mismatched allogeneic HSCT present a higher frequency of complications. Although a higher proportion of these patients discontinued IvIg replacement, B cell reconstitution is not optimal. Analysis of patients in particular with mixed chimerism will provide important information in the setting of GT. The analysis of B cell reconstitution after GT and mismatched allogeneic HSCT deserves particular attention in the assessment of immunological reconstitution. Disclosures No relevant conflicts of interest to declare.

BACKGROUND Interleukin-7 (IL-7) is a hematopoietic cytokine essential for T-cell development in the thymus and for the maintenance of peripheral T cells. A previous study of single nucleotide polymorphisms in the exons of IL-7 receptor α-chain (IL-7Rα) in a Danish cohort of patients undergoing allogeneic stem-cell transplantation (SCT) identified donor genotype GG at rs1494555 as a risk factor for treatment-related mortality (TRM) after SCT. METHODS In this validation study, 116 British and French SCT patients and their donors were investigated by sequence-specific primer polymerase chain reaction. RESULTS Both donor rs1494555GG genotype and the tightly coupled rs1494558TT genotype were significantly associated with grade 3 to 4 acute graft versus host disease. Although both genotypes tended to be associated with increased TRM, this did not translate into altered overall survival. CONCLUSION The present data indicate that the IL-7 pathway is associated with alloreactivity in SCT, which is in line with the previously observed association between IL-7Rα single nucleotide polymorphisms and TRM.

The pioneering Times Although the concept of Transplantation could be traced back as early as Saint Cosmas and Damian most famous miracle, grafting of a leg to replace a patient’s ulcered leg, it is not before the 50’s that it entered reality. Important steps were made before, from sewing vessels to understanding the basis of the major histocompatibility complex (MHC) in mice (Snell, 1986). This medical field was pioneered in renal transplantation with nearly concomitant attempts in Paris, London, Edinburgh, Boston. The first successful attempt was carried out in Boston by Joseph Murray in 1954. The donor and recipient were identical twins, showing indeed the importance of Genetic compatibility. Organ transplantation (OT) then became one of the most exciting “success story” of modern Medicine, admixing advances in Surgery and intensive care Medicine, Immunology, Genetics, and Pharmacology. The crucial point was first to avoid acute graft rejection which quickly appeared to be immune-mediated. The development of potent immunosuppressive (IS) drugs (corticosteroids, calcineurin inhibitors) opened the way to clinical transplantation. Most critical landmarks were in 1957 the first hepatic transplantation followed by transplantation of other organs, in 1967 the first successful heart transplantation, lung (1968), trachea (1979), pancreas, and more recently hands (2000) and facial tissue (2005). In hematopoietic stem-cell transplantation (HSCT), the initial report of the use of bone marrow transplantation in cancer treatment was made by Thomas in 1957. Several Nobel prizes were awarded to some of the transplantation “founding fathers”: Medawar and Burnet (1960), Benacerraf, Dausset and Snell (1980), Murray and Thomas (1990).

The envelope (Env) protein from HIV-1 is the focus of several vaccine trials in humans. It could be considered for the optimization of Env vaccinal preparations to add within the molecule defined epitopes, for instance epitopes conserved among viral isolates from HIV-1, such as from Gag or Nef proteins. As a first step to this approach, we have constructed by in vitro mutagenesis HIV-1(LAI) Env gp120 molecules in which a 12-amino acid sequence in the first (V1) or the third (V3) hypervariable region was substituted by the hemagglutinin (HA) 307-318 peptide from the influenza virus, a dominant T helper cell epitope in humans. The proteins were produced by recombinant vaccinia viruses. They had kept their structural properties in terms of serological recognition and binding to CD4. Of note, we observed that the gp120 protein substituted in the V1 domain elicited a stronger serological immune response in mice compared to native or V3 substituted gp120. This indicates that gp120 can accommodate large substitutions without major structural perturbations and that, on the contrary, some of them could prove beneficial in terms of immunogenicity.

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.

Background & Aims The natural killer group 2 member D (NKG2D) is a stimulatory receptor expressed on a subset of mucosal and peripheral CD4 + T cells in patients with Crohn's disease (CD) and other inflammatory diseases. Ligand activation of NKG2D in patients induces CD4 + T cells to release T-helper (Th) 1 cytokines and become cytotoxic. We investigated the Th17 cytokines produced by T cells that express NKG2D in blood and intestinal mucosa samples from patients with CD. Methods We isolated CD4 + T cells from peripheral blood and lamina propria samples of patients with CD or ulcerative colitis (UC) and healthy individuals (controls). We analyzed the phenotype and functions of the CD4 + NKG2D + T cells and the cytokines they produce in response to NKG2D stimulation. Results In patients with CD, CD4 + T cells that express NKG2D produced high levels of interleukin (IL)-17 and IL-22 and expressed high levels of CCR6, the IL-23 receptor, CD161, and RORC (a transcription factor that regulates expression of Th17 cytokines). CD4 + T cells that produced IL-17 expressed high levels of NKG2D and CD161. Costimulation of NKG2D and the T-cell receptor (TCR) significantly increased production of IL-17 and tumor necrosis factor α by CD4 + T cells, compared with activation of only the TCR. CD4 + NKG2D + T cells also responded to Th17 polarization. Conclusions NKG2D is a functional marker of CD4 + T cells that produce IL-17 in patients with CD, via costimulation of the TCR and NKG2D. Reagents developed to block NKG2D might reduce gastrointestinal inflammation in patients with CD.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, drug-induced reaction that involves both the skin and the viscera. Evidence for reactivation of herpes family viruses has been seen in some DRESS patients. To understand the immunological components of DRESS and their relationship to viral reactivation, we prospectively assessed 40 patients exhibiting DRESS in response to carbamazepine, allopurinol, or sulfamethoxazole. Peripheral blood T lymphocytes from the patients were evaluated for phenotype, cytokine secretion, and repertoire of CD4+ and CD8+ and for viral reactivation. We found Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), or HHV-7 reactivation in 76% of the patients. In all patients, circulating CD8+ T lymphocytes were activated, exhibited increased cutaneous homing markers, and secreted large amounts of tumor necrosis factor-alpha and interferon-gamma. The production of these cytokines was particularly high in patients with the most severe visceral involvement. In addition, expanded populations of CD8+ T lymphocytes sharing the same T cell receptor repertoire were detected in the blood, skin, liver, and lungs of patients. Nearly half of these expanded blood CD8+ T lymphocytes specifically recognized one of several EBV epitopes. Finally, we found that the culprit drugs triggered the production of EBV in patients' EBV-transformed B lymphocytes. Thus, cutaneous and visceral symptoms of DRESS are mediated by activated CD8+ T lymphocytes, which are largely directed against herpes viruses such as EBV.

Immune reconstitution was analyzed in 140 consecutive patients who were 2-year disease-free and who underwent myeloablative allogeneic transplantation. A CD4 and CD8 defect was observed involving naive, terminally differentiated, memory and competent cells and above limits values for activated subsets. Natural killer cells normalize at six months while we observed expansion of CD19(+)/CD5(+) B cells after three months and a persisting defect of memory B cells. Chronic graft-versus-host disease did not influence significantly those parameters for CD8 subsets while the naïve and competent CD4 subsets were strongly affected. But the most profound impact of chronic graft-versus-host disease was on B-cell subsets, especially on the memory B population. The cumulative incidence of late severe infections was low (14% at four years). Using Cox's models, only low B-cell counts at 12 (P=0.02) and 24 (P=0.001) months were associated with the hazard of developing late infection, in particular if patients did not develop chronic graft-versus-host disease.

The new human-human hybridoma TrD3 secretes a cytotoxic IgM mAb, which reacted with 28 of a panel of 56 HLA-typed lymphoblastoid cells. All 28 TrD3+ cells expressed the HLA-B supertype Bw6, whereas 10 Bw6+ cells were not recognized by the mAb. None of the 17 Bw4 homozygous cells were positive with TrD3. Thus, TrD3 divided the Bw6+ HLA-B specificities of the cell lines into two subgroups, namely, Bw6+TrD3+ and Bw6+TrD3-, and therefore defines a new HLA-B supertype. TrD3 reacted strongly with some B8+ cell lines and weakly or not at all with others, suggesting a new split of HLA-B8. Compared with cell lines, TrD3 reacted more weakly with freshly isolated T cells from blood. The Bw6-specific rat mAb SFR8-B6 partially blocked the binding of 125I-labeled TrD3 to a Bw6+ cell line. By using cell lines transfected with hybrid genes between HLA-B7 (Bw6+) and HLA-B27 (Bw6-) as targets in flow cytometry, critical residues for the TrD3 epitope could be mapped to the amino acid region 24-62 of the HLA class-I alpha 1 domain. Comparison of deduced amino acid sequences of TrD3-positive and -negative cells indicated that a tryptophane residue at position 95 destroyed the TrD3 epitope, and that one or more of the residues in positions 24, 45, and 46 may be critical, suggesting that it is a discontinuous epitope. It is notable that none of these residues are located on alpha-helixes.(ABSTRACT TRUNCATED AT 250 WORDS)