scholarly journals 762 A combination of functional biomarkers improves identification of the tumor-specific reactive T cell repertoire

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A810-A810
Author(s):  
Arianna Draghi ◽  
Katja Harbst ◽  
Inge Svane ◽  
Marco Donia
Keyword(s):  
T Cells ◽  
T Cell ◽  
De Novo ◽  
Autologous Tumor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Simple Method ◽  
Functional Biomarkers

BackgroundDetecting the entire repertoire of tumor-specific reactive T cells is essential for investigating the broad range of T cell functions in the tumor-microenvironment. At present, assays identifying tumor-specific functional activation measure either upregulation of specific surface molecules, de novo production of the most common antitumor cytokines or mobilization of cytotoxic granules.MethodsIn this study, we combined transcriptomic analyses of tumor-specific reactive tumorinfiltrating lymphocytes (TILs), TIL-autologous tumor cell co-cultures and commonly used established detection protocols to develop an intracellular flow cytometry staining method encompassing simultaneous detection of intracellular CD137, de novo production of TNF and IFNy and extracellular mobilization of CD107a.ResultsThis approach enabled the identification of a larger fraction of tumor-specific reactive T cells in vitro compared to standard methods, revealing the existence of multiple distinct functional clusters of tumor-specific reactive TILs. Publicly available datasets of fresh tumor single-cell RNA-sequencing from four cancer types were investigated to confirm that these functional biomarkers identified distinct functional clusters forming the entire repertoire of tumor-specific reactive T cells in situ.ConclusionsIn conclusion, we describe a simple method using a combination of functional biomarkers that improves identification of the tumor-specific reactive T cell repertoire in vitro and in situ.

Spectratype Analysis of In Vitro Donor Anti-Host T Cell Repertoire Responses Predict Those of In Vivo Post-BMT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2163-2163
Author(s):  
Thea M. Friedman ◽  
Kira Goldgirsh ◽  
Jenny Zilberberg ◽  
Stephanie A. Berger ◽  
Joanne Filicko-O’Hara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Post Transplant ◽  
Cell Responses ◽  
Repertoire Analysis ◽  
Alloreactive T Cell

Abstract Immunotherapeutic strategies have gained recognition as viable alternatives to more conventional modalities for the treatment of cancer. In this regard, adoptive T cell therapy through allogeneic blood and marrow transplantation (BMT) has provided the strongest evidence that anti-tumor effects could be achieved against hematological malignancies. However, the major complications of BMT still include graft failure, opportunistic infections, disease relapse and graft-versus-host disease (GVHD). The presence of mature donor T cells in the transplant inoculum reduces the incidence of the first three complications, while unfortunately increasing the risk of GVHD, which can be directed against either HLA or minor histocompatibilty antigen (miHA) disparities. Thus, a major objective in the field has been to develop tactics that could facilitate the separation of graft-versus-tumor (GVT) effects from the deleterious effects of GVHD. One such approach would be to selectively deplete donor alloreactive T cells in the donor inoculum while allowing residual T cells to provide some protection against infection and to support a tumor-specific GVT response. For a more targeted approach, delayed donor lymphocyte infusion (DLI) of positively-selected donor GVT-reactive T cells could be used weeks to months post-transplant, if these elements were identifiable. In this regard, TCR Vβ repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T cell responses. Theoretically, molecular analysis of T cell responses in vitro, given the high sensitivity of the PCR-based spectratyping technique, should identify the most potentially critical Vβ families involved in the later development of GVHD and GVT effects in patients. To this end, we tested the hypothesis that T cell repertoire analysis of HLA-matched sibling (SIB) or matched unrelated donors (URD) from in vitro, host-stimulated, mixed lymphocyte cultures (MLC) would be predictive of the TCR-Vβ spectratype analysis of the T cell repertoire in the patient following BMT. In this study, we examined 17 patient pairs and report that for the resolvable Vβ families, we observed overall 71.2 ± 11.9% (mean ± SD.; range 40%–85%) of the in vitro anti-host T cell responses were predictive of those in the patient post-transplant. Of the 28.8% non-predictive Vβ families, 6.9 ± 6.3% (range 0%–27%) exhibited skewing in the MLC but no skewing in the patient post-transplant repertoire, 9.3 ± 6.3% (range 0%–18.8%) exhibited skewing in different peaks within the same Vβ family, and 12.5 ± 10.8% (range 0%–40%) showed skewing in the patient post-transplant and none in the MLC. Taken together, these results suggest that the in vitro MLC T cell responses show good consistency with post-transplant patient responses. Thus, in vitro spectratyping may be useful for predicting the alloreactive T cell responses involved in GVHD and could be used to guide custom-designed select Vβ family T cell-depleted transplants to improve patient outcomes. The additional advantage of this approach is that minimization of GVHD risk can be obtained without any direct knowledge of the specific miHA involved in the individual donor-patient pair.

scholarly journals Influences of antigen processing on the expression of the T cell repertoire. Evidence for MHC-specific hindering structures on the products of processing.

1988 ◽  
Vol 168 (1) ◽  
pp. 357-373 ◽  
Author(s):  
S J Brett ◽  
K B Cease ◽  
J A Berzofsky
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Processing ◽  
Sperm Whale ◽  
T Cell Response ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Similar Dose ◽  
Sperm Whale Myoglobin

Two lines of evidence in the current study indicate that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance. First, immunization with synthetic peptides of myoglobin sequences revealed new reactivities that had not appeared after priming with native myoglobin. For example, B10.S mice (H-2S) immune to equine myoglobin predominantly responded to peptide 102-118, whereas there was little, if any, response to this peptide in B10.BR (H-2k) mice immunized with native equine myoglobin. However, after immunization with the 102-118 peptide, both strains responded to the peptide. After in vitro restimulation, B10.BR T cells responded as well as B10.S T cells. Similarly, some individual 102-118-specific T cell clones from mice of both haplotypes showed similar dose responses and fine specificity patterns. Thus, low responsiveness to this site is due neither to a hole in the repertoire nor to a failure to bind to the appropriate MHC molecule. An alternative explanation was suggested by the observation that, whereas B10.S T cells from peptide 102-118-immune mice responded almost as well to whole myoglobin as to the peptide, the B10.BR T cells from peptide immune mice, while responding well to peptide, were poorly stimulated by whole myoglobin. Thus, the product of natural processing of equine myoglobin probably has hindering structures in the regions flanking the core epitope 102-118 that interfere with presentation by I-Ak but not I-AS. The second line of evidence that processing of native myoglobin may influence the apparent specificity of the T cell response was obtained using the I-Ad-restricted sperm whale myoglobin 102-118-specific clone 9.27. This clone discriminated readily between whole sperm whale myoglobin and equine myoglobin, but it did not distinguish between peptides corresponding to 102-118 of the sperm whale and equine sequences. This distinction between equine peptide and native equine myoglobin could be overcome by artificial "processing" of equine myoglobin with cyanogen bromide. In both sets of experiments, F1 APCs that present the same epitope well to T cells of another haplotype failed to overcome the defect, which was therefore not due to the availability of different processed cleavage fragments in APC of different haplotypes, as would be expected if there were MHC-linked processing. Thus, the differential responses to peptides versus native molecule for both I-Ad- and I-Ak-restricted clones appeared to depend on the restricting molecule used.(ABSTRACT TRUNCATED AT 400 WORDS)

Mapping T-Cell Peptide Epitopes on Activated Recombinant Factor VII (rFVIIa) Contributing to Inhibitor Antibody Response

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2295-2295
Author(s):  
Pedro Paz ◽  
Jens Schroeder ◽  
Prasad Mathew ◽  
Marion Hardtke ◽  
Fred Aswad
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Epitope Mapping ◽  
T Cell Response ◽  
Factor Vii ◽  
Amino Acid Substitutions ◽  
T Cell Repertoire ◽  
Cell Repertoire

Abstract Bypassing agents such as activated recombinant factor VII (rFVIIa) are used to treat acute bleeding episodes in patients with hemophilia and inhibitors to coagulation factors VIII (FVIII) or IX. BAY 86-6150 is a modified rFVIIa protein with 6 amino acid substitutions in the rFVII molecule that prolong half-life and improve potency compared with the currently available rFVIIa. In a dose-escalation clinical study, 1 patient out of a cohort of 10 treated with 6.5 μg/kg BAY 86-6150 developed low-titer neutralizing antibodies that were detected after the third exposure. The patient's anti-BAY 86-6150 antibodies also cross-reacted with and neutralized wild-type FVIIa (WT-FVIIa) in vitro. T-cell epitope mapping was performed to identify BAY 86-6150 sequence(s) that may have contributed to the immunogenic response in the patient by measuring CD4+ T-cell response to individual 15-mer peptides spanning BAY 86-6150. The epitope mapping study did not identify any of the 14 peptides unique to BAY 86-6150 as epitopes recognized by the patient's T cells. However, strong responses were detected against 2 WT-FVIIa peptides, WT p6-20 (EELRPGSLERECKEE) and WT p156-170 (GKVCPKGECPWQVLL), indicating that CD4+ T helper cells recognizing these WT peptides may have contributed to the immune response that resulted in the production of anti-BAY 86-6150 antibodies during treatment. It should be noted that although the patient had no detectable anti-FVIIa antibodies before the start of the study, he had been treated with factor eight inhibitor bypassing activity (FEIBA), which contains active FVII, on 3 consecutive days 3 months before entry into the study. Hence it is possible that the patient had been primed for a response against FVIIa that was triggered by the subsequent exposure to BAY 86-6150. The fact that the patient's T cells only responded to WT-FVIIa peptides might be explained if the WT-FVIIa peptides were seen as foreign/non-self peptides by his T-cell repertoire. Unfortunately, the patient's FVII gene sequence was not obtainable, and this possibility remains unanswered. The Universal Protein Resource (UniProt) database reports 2 natural FVII gene variants that encompass the WT p6-20 sequence (Millar, et al. Hum Genet. 2000;107:327-42; Herrmann, et al. Haemophilia. 2009;15:267-280) and one for the WT p156-170 sequence (Wulff and Herrmann. Hum Mutat. 2000;15:489-496) that resulted from single amino acid substitutions. Hence the possibility exists that the patient's T-cell repertoire sees WT-FVIIa sequences as foreign and immunogenic. T cells from 40 healthy donors were also tested for reactivity against the peptide panel to assess relative immunogenicity of BAY 86-6150 vs WT-FVIIa. Seven BAY 86-6150 neoepitopes were stimulatory for 8 unrelated healthy donor T cells, but their mean stimulation indices were not statistically higher than those observed against WT FVIIa peptides. Mean % stimulation values of the positive responses against the 7 BAY 86-6150 neoepitopes versus 39 WT FVIIa peptides were 1.09 (N=13 responses) vs 1.01 (N=70 responses; P=0.5059). Statistical analysis of the in vitro T-cell response indicates that specific mutations to BAY 86-6150 do not result in BAY 86-6150 being more immunogenic than WT FVIIa. This would suggest that BAY 86-6150 will not elicit stronger or higher frequency of anti-FVIIa antibody response than WT FVIIa in patients with hemophilia. Disclosures Paz: Bayer HealthCare: Employment. Schroeder:Bayer Pharma AG: Employment. Mathew:Bayer HealthCare Pharmaceuticals: Employment. Hardtke:Bayer Pharma AG: Employment. Aswad:Bayer HealthCare: Employment.

Generation of T-cells reactive against CT-7 and BCMA peptides: Potential targets for T-cell immunotherapy of multiple myeloma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7615-7615
Author(s):  
L. D. Anderson ◽  
D. G. Maloney ◽  
S. R. Riddell
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Plasma Cell ◽  
Target Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Therapy

7615 Background: Multiple myeloma is a malignant plasma cell disorder that is incurable with chemotherapy or autologous stem cell transplantation (SCT), and novel therapies with lower toxicity are needed. There is evidence that T-cells can recognize myeloma and mediate anti-tumor effects, but the lack of defined target antigens other than idiotype has hindered the development of myeloma-specific T-cell therapy. We are investigating cancer-testis antigens and overexpressed “self”-proteins as candidate myeloma antigens, including MAGE-C1 (CT-7), which is expressed by >80% of myelomas, and B-Cell Maturation Antigen (BCMA), a plasma cell differentiation antigen commonly over-expressed in myeloma. Methods: To identify potential T-cell epitopes from CT-7 and BCMA, we scanned the protein sequences with computer algorithms and synthesized peptides predicted to bind to HLA-A2 and A3. CT-7 and BCMA are both “self” proteins to which the T-cell repertoire may be relatively tolerant, so we have utilized culture conditions that facilitate the expansion of rare myeloma-reactive T-cells. CD8+ T cells were stimulated in vitro with autologous dendritic cells pulsed with CT-7 or BCMA peptides in the presence of cytokines that avoid excessive nonspecific expansion of T-cells. Wells were screened for reactivity against peptide-pulsed target cells and myeloma cell lines. Results: A specific CD8+ T-cell response by both ELISPOT and cytotoxicity assays to at least one HLA-A2 peptide from each of the CT-7 and BCMA proteins has been identified in normal donors. CT-7 and BCMA-specific T-cells are being cloned in order to determine their ability to recognize primary myeloma cells. Experiments are also in progress to elicit responses to these peptides in myeloma patient samples and to screen HLA A3-binding epitopes. Conclusions: T-cells recognizing CT-7 and BCMA are detectable in the normal T-cell repertoire and can be isolated and expanded in vitro. We are currently pursuing the identification of additional antigenic epitopes in these proteins to define their potential utility as targets for vaccination or adoptive T-cell therapy. No significant financial relationships to disclose.

Impact of clonal competition for peptide-MHC complexes on the CD8+ T-cell repertoire selection in a persistent viral infection

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4283-4292 ◽  
Author(s):  
Katherine K. Wynn ◽  
Zara Fulton ◽  
Leanne Cooper ◽  
Sharon L. Silins ◽  
Stephanie Gras ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Infections ◽  
Cd8 T Cell ◽  
Structural Features ◽  
T Cell Response ◽  
Helical Conformation ◽  
T Cell Repertoire ◽  
Cell Repertoire

AbstractCD8+ T-cell responses to persistent viral infections are characterized by the accumulation of an oligoclonal T-cell repertoire and a reduction in the naive T-cell pool. However, the precise mechanism for this phenomenon remains elusive. Here we show that human cytomegalovirus (HCMV)–specific CD8+ T cells recognizing distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T-cell repertoire or a private, diverse T-cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public T-cell receptor (TCR) was coincident with an atypical major histocompatibility complex (MHC)–peptide structure, in that the epitope adopted a helical conformation that bulged from the peptide-binding groove, while a diverse TCR profile was observed in response to the epitope that formed a flatter, more “featureless” landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared with the T cells expressing diverse TCR. These findings provide new insights into our understanding on how the biology of antigen presentation in addition to the structural features of the pMHC-I might shape the T-cell repertoire and its phenotype.

scholarly journals Negative Selection during the Peripheral Immune Response to Antigen

2000 ◽  
Vol 193 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Stephen M. Anderton ◽  
Caius G. Radu ◽  
Pauline A. Lowrey ◽  
E. Sally Ward ◽  
David C. Wraith
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clonal Expansion ◽  
Cell Interaction ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Vitro Assay ◽  
High Affinity

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen–major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 Au-restricted, acetylated NH2-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for Au. In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

In vitro expansion of antigen-specific CD8+ T cells distorts the T-cell repertoire

2014 ◽  
Vol 405 ◽  
pp. 199-203 ◽  
Author(s):  
Dan Koning ◽  
Ana I. Costa ◽  
Raiza Hasrat ◽  
Bart P.X. Grady ◽  
Sanne Spijkers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
In Vitro Expansion

scholarly journals Nondeletional T-Cell Receptor Transgenic Mice: Model for the CD4+ T-Cell Repertoire in Chronic Hepatitis B Virus Infection

2000 ◽  
Vol 74 (16) ◽  
pp. 7587-7599 ◽  
Author(s):  
M. Chen ◽  
M. Sällberg ◽  
S. N. Thung ◽  
J. Hughes ◽  
J. Jones ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hepatitis B ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
B Virus

ABSTRACT Chronicity after infection with the hepatitis B virus (HBV) can occur for a variety of reasons. However, once established, chronicity may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of coexisting with the secreted hepatitis B e antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To ensure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR α- and β-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas produced from immunizing HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency (∼67%) of CD4+ T cells that expressed a Tg TCR specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4+ T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4+ T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg, as measured by cytokine production in vitro and T-helper-cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th1 subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4+ T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo. The 11/4-12 TCR-Tg lineage may serve as a monoclonal model for the HBe/HBcAg-specific CD4+ T-cell repertoire present in chronically infected HBV patients.

scholarly journals Identification of an autologous insulin B chain peptide as a target antigen for H-2Kb-restricted cytotoxic T lymphocytes.

1992 ◽  
Vol 175 (2) ◽  
pp. 545-552 ◽  
Author(s):  
J M Sheil ◽  
S E Shepherd ◽  
G F Klimo ◽  
Y Paterson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic T Cells ◽  
Mammalian Species ◽  
Peptide Sequence ◽  
Target Antigen ◽  
T Cell Repertoire ◽  
Cell Repertoire

We have examined the CD8+ peripheral T cell repertoire of C57BL/6 (H-2b) mice for cytotoxic T lymphocyte (CTL) reactivities to insulin, using in vitro immunization with a chymotryptic digest of reduced bovine insulin. The results presented in this study demonstrate that potentially autoreactive H-2Kb-restricted cytotoxic T cells specific for an autologous insulin B chain peptide are present in the preimmune splenic T cell repertoire. The immunogenic peptide comprises residues 7-15 from the insulin B chain and has features in common with naturally processed Kb-restricted peptides identified by others. The minimal peptide sequence recognized by these cytotoxic T cells is 10-15, which is highly conserved in mammalian species and constitutes a self-peptide in mice. The presence of class I major histocompatibility complex-restricted CTLs with potentially autoreactive specificities in preimmune animals raises the possibility of a role for such cells in autoimmune disease states. Possible mechanisms for the in vivo expansion of insulin peptide-specific CTLs are discussed.

Aberrant Repertoire and Deficient Proliferation of TCR γδ T Cells in Myelodysplastic Syndromes (MDS).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2639-2639
Author(s):  
Jean-Jacques Kiladjian ◽  
Géraldine Visentin ◽  
Emilie Viey ◽  
Sameh Ayari ◽  
Jean-Henri Bourhis ◽  
...  
Keyword(s):  
Innate Immunity ◽  
T Cells ◽  
T Cell ◽  
Γδ T Cells ◽  
Proliferation Index ◽  
T Cell Repertoire ◽  
Γδ T Cell ◽  
Cell Repertoire ◽  
Color Flow

Abstract Background: We recently showed that MDS natural killer cells display altered cytolytic function and proliferation in response to IL-2 (Leukemia, 2006;20:463–70). TCR γδ T cells are other important components of innate immunity, recently shown to be involved in the immune response against various tumors. Furthermore, a TCR γδ agonist synthetic phosphoantigen, the BrHPP molecule, is able to expand and activate in vitro Vδ2 cells (the peripheral blood circulating subset of γδ T cells). We studied γδ T cell population in MDS patients. Methods: PBMCs from 31 MDS patients (WHO: 3 pure RA, 2 pure RAS, 1 RCMD-RS, 6 RCMD, 2 del5q, 8 RAEB1, 4 RAEB2, 5 MDS-U; IPSS: 9 low, 12 Int-1, 6 Int-2, 4 High) and 15 controls were stimulated with BrHPP and IL-2 during 8 to 15 days. Immunophenotyping of αβ and γδ T cells was determined by multi-color flow cytometry. Cytolytic capacity of activated Vδ2 T cells was assessed using 51Cr release and CD107a degranulation assays. IFNγ was measured by ELISA after TCR γδ triggering of expanded γδ T cells. Results: % and absolute numbers of γδ T cells from MDS were comparable to those of controls, but MDS Vδ2 T cell repertoire was significantly skewed: 10/19 MDS Vδ2 T cells were characterized by a “naive” predominant population, 5/19 had a majority of “effectors” cells, and only 4 of 19 had a predominant “central memory population”, which was always the main Vδ2 compartment in healthy donors. Stimulation with BrHPP induced a significant increase of MDS Vδ2 T cells subset, indicating their specific activation. However, MDS Vδ2 T cells poorly proliferated in 2 week cultures in response to IL-2, in contrast with the high proliferation index of controls derived cells. On day 8 of culture, only 6 of the 29 MDS samples studied clearly responded to BrHPP+IL-2 (i.e. >60% of Vδ2 cells in culture) compared to 13/15 in controls, while 12/29 were intermediate responders (20 to 60% of Vδ2), and 11/29 non-responders. We found no correlation between response to BrHPP and WHO, cytogenetics or IPSS category. However, coexistence of immunologic abnormalities (rheumatoid arthritis in 5 patients, temporal arteritis in 1, antiphospholipid syndrome in 1, Raynaud’s phenomenon in 2, and thyroiditis in 2) was more frequent in non-responders. The 3 IL-2 receptor (IL-2-R) chains were normally expressed in MDS Vδ2 T cells and IL-2-Rα expression was normally induced in response to IL-2. We are currently investigating downstream signaling molecules of the IL-2-R pathway. Functional activity of MDS γδ T cells (cytotoxicity, degranulation, and IFNg secretion) was similar to that of healthy donor cells. In particular, those cells were cytotoxic against the MDS-derived P39 cell line. Conclusion: in MDS, γδ T cell repertoire is profoundly skewed, and those cells do not proliferate in response to potent agonists, although their numbers and function seem normal. Those alterations did not correlate with MDS characteristics, but were more frequent in patients with associated immunological abnormalities. Our results further support the existence of important alterations of innate immunity effectors in MDS that could play a role in disease pathophysiology or progression.

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