Epitope Identification and Generation of an Antigen-Specific T-Cell Clone in a Mild Hemophilia A Inhibitor Subject with Missense Genotype R593C.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1159-1159 ◽  
Author(s):  
Eddie A. James ◽  
Simon D. van Haren ◽  
Ruth A. Ettinger ◽  
Arthur R. Thompson ◽  
William W. Kwok ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Hemophilia A ◽  
Cell Clone ◽  
Peptide Binding ◽  
Class Ii ◽  
High Affinity ◽  
T Cell Clone ◽  
A2 Domain

Abstract The development of inhibitory anti-FVIII antibodies is a major clinical problem in hemophilia A. While less common in mild/moderately severe patients, the relative risk of inhibitor formation is elevated in patients with missense mutations in the FVIII A2 domain, especially those with an R593C genotype. In this study, T-cell responses to 14 FVIII A2 domain peptides with predicted DRB1*1101 MHC binding motifs were investigated using MHC class II tetramer reagents. CD4+ T cells were isolated from a hemophilic subject (DRB1*1101, 1302) with FVIII missense genotype R593C and stimulated with pooled peptides. This subject had developed a longstanding low titer inhibitor after receiving multiple FVIII infusions. Staining with fluorescent, peptide-loaded tetramers revealed that the hemophilic subject, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide A2589–608, which contained the wild-type R593 sequence. MHC class II tetramers with bound A2589–608 were used to sort antigen-specific T cells and then generate a T-cell clone recognizing this high avidity DRB1*1101-restricted epitope. FVIII residues 594–602 (FLPNPAGVQ) comprise a predicted high-affinity binding motif. Peptide binding assays confirmed that A2589–608 bound to recombinant, monomeric DRB1*1101 protein with high affinity. A peptide with the corresponding hemophilic sequence (A2589–608, 593C) bound with affinity that was threefold lower, but in a range that should be sufficient for presentation to T cells. The other 12 A2 peptides had a wide spectrum of binding affinities for the DRB1*1101 protein. Interestingly, prediction algorithms suggest that the peptides containing residue 593 occupy the MHC Class II peptide-binding groove in a register that places this residue just outside the p1 anchor position. Thus we anticipate that residue 593 modulates T-cell recognition of peptide A2589–608. This provides a potential explanation for inhibitor development in mild hemophilia A patients with a FVIII R593C genotype who are HLA-DRB1*1101. Immunogenicity of this FVIII region with respect to other HLA types is under investigation.

scholarly journals A Class II-Restricted CD8γ13 T-Cell Clone Protects During Chlamydia muridarum Genital Tract Infection

2020 ◽  
Vol 221 (11) ◽  
pp. 1895-1906
Author(s):  
Raymond M Johnson ◽  
Norma Olivares-Strank ◽  
Gang Peng
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Genital Tract ◽  
Cell Clone ◽  
Class Ii ◽  
Class I ◽  
T Cell Clone ◽  
Tract Infections

Abstract Background The T-cell response to chlamydia genital tract infections in humans and mice is unusual because the majority of antigen-specific CD8 T cells are not class I restricted (referred to here as “unrestricted” or “atypical”). We previously reported that a subset of unrestricted murine chlamydia-specific CD8 T cells had a cytokine polarization pattern that included interferon (IFN)-γ and interleukin (IL)-13. Methods In this study, we investigated the transcriptome of CD8γ13 T cells, comparing them to Tc1 clones using microarray analysis. That study revealed that CD8γ13 polarization included IL-5 in addition to IFN-γ and IL-13. Adoptive transfer studies were performed with Tc1 clones and a CD8γ13 T-cell clone to determine whether either influenced bacterial clearance or immunopathology during Chlamydia muridarum genital tract infections. Results To our surprise, an adoptively transferred CD8γ13 T-cell clone was remarkably proficient at preventing chlamydia immunopathology, whereas the multifunctional Tc1 clone did not enhance clearance or significantly alter immunopathology. Mapping studies with major histocompatibility complex (MHC) class I- and class II-deficient splenocytes showed our previously published chlamydia-specific CD8 T-cell clones are MHC class II restricted. Conclusions The MHC class II-restricted CD8 T cells may play an important role in protection from intracellular pathogens that limit class I antigen presentation or diminish CD4 T-cell numbers or impair their function.

scholarly journals Effector and regulatory T?cells derived from the same T?cell clone differ in MHC class II-peptide multimer binding

2004 ◽  
Vol 34 (12) ◽  
pp. 3359-3369 ◽  
Author(s):  
Esther?N.?M. Nolte-?t Hoen ◽  
Maria?Grazia Amoroso ◽  
Jetty Veenstra ◽  
Mayken?C. Grosfeld-Stulemeyer ◽  
Willem van Eden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
Cell Clone ◽  
Class Ii ◽  
T Cell Clone

Reduced Immunogenicity of FVIII Peptides by Rational Modifications of An Immunodominant T-Cell Epitope.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 220-220
Author(s):  
Ruth A. Ettinger ◽  
Joseph A. Liberman ◽  
Douglas C. Bolgiano ◽  
Arthur R. Thompson ◽  
Kathleen P. Pratt
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Mhc Class Ii ◽  
Hemophilia A ◽  
Peptide Binding ◽  
Cell Epitope ◽  
Class Ii ◽  
Amino Acid Substitutions ◽  
T Cell Epitope

Abstract Abstract 220 Introduction. The development of antibodies that interfere with factor VIII (FVIII) pro-coagulant activity, often referred to as “inhibitors”, can complicate the treatment of hemophilia A. These alloimmune responses, as well as the rare development of autoimmune FVIII inhibitors, are associated with significant morbidity and mortality. The production of anti-FVIII antibodies follows stimulation of helper T cells by epitopes in FVIII. An immunodominant HLA-DRB1*0101-restricted T-cell epitope was recognized by CD4+ T cells from a mild hemophilia A inhibitor subject and from his brother, who had a sub-clinical inhibitor (James et al., J Thromb Haemost 5: 2399-2407, 2007). Their CD4+ T cells recognized overlapping synthetic peptides with sequences corresponding to FVIII residues 2186-2205, 2187-2205 and 2194-2213. Nineteen T-cell clones recognizing this epitope were isolated, with phenotypes representing four distinct T-cell lineages. Aims: (1) to evaluate the promiscuity/immunodominance of an HLA-DRB1*0101-restricted T-cell epitope in FVIII; (2) to introduce amino acid substitutions that will prevent presentation of this epitope to the immune system by DR0101 and by other DR alleles. Methods. The minimal epitope and MHC Class II (DR0101) “anchor” residues were determined using a competition assay measuring displacement of a labeled peptide having high affinity for recombinant DR0101 by a series of FVIII peptides. Peptide concentrations at which 50% inhibition of the labeled peptide binding occurred (IC50) were obtained by regression analysis. Binding of the peptides to five additional DR alleles was evaluated directly using recombinant proteins; predicted binding of peptides to additional DR alleles was evaluated using the program ProPred. Proliferation and cytokine production by the clones in response to wild-type and modified peptides were measured, and the concentrations at which half-maximal T-cell responses (EC50) to the FVIII peptides occurred were determined. Results. Binding of truncated peptides to DR0101 identified FVIII2194-2205 as the minimal epitope. Binding of FVIII2194-2205 peptides with single Arg substitutions identified F2196, M2199, A2201 and S2204 as anchor residues at positions 1, 4, 6 and 9, respectively, corresponding to peptide-binding pockets seen in the crystal structure of a DR0101-peptide complex. The relative binding of Ala-substituted peptides confirmed that F2196 and M2199 are anchor residues. T-cell stimulation requires recognition of peptides by both the Class II receptor and the T-cell receptor (TCR). Sequences of TCR variable regions (TCRBVs) expressed by the clones were identified as TCRBV20-1*01 (3 VDJ combinations), TCRBV6-6*01, TCRBV5-1*01, and TCRBV6-1*01, indicating at least six different T-cell progenitors recognized this epitope. The clones were next stimulated with peptides having modified epitopes. Strikingly, none proliferated or secreted cytokines when stimulated by FVIII2194-2205, F2196A, which also showed an IC50 > 10 μM when tested for binding to DR0101, DR0301, DR0401, DR1101, DR1104, and DR1501. Substitutions at other anchor positions affected binding to some but not all of the DR proteins. Predicted binding of the F2196A variant to 51 DR alleles was analyzed using ProPred; none bound at a threshold stringency of 10% (low stringency, thus the predicted epitopes included those with lower calculated affinities). In preparation for directly testing the immunogenicity of additional substitutions, all possible amino acid substitutions at position 2196 were evaluated using ProPred. 13 of 19 possible substitutions were predicted to prevent FVIII2194-2205 binding to all 51 DR alleles included in the algorithm (with a 3% threshold = intermediate stringency). Conclusions. MHC class II anchor residues and TCR contact sites for an immunodominant HLA-DRB1*0101-restricted T-cell epitope have been mapped precisely. Both measured and predicted effects of amino acid substitutions indicated that this F2196 is essential for effective presentation of this epitope by multiple DR alleles. Effects of various sequence modifications on FVIII function, conformation and immunogenicity are currently being evaluated using recombinant FVIII and FVIII C2 domain proteins to indicate their possible therapeutic potential. Disclosures: Pratt: CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxter: Honoraria; Grifols: Honoraria.

scholarly journals HLA-DQB1 codon 57 is critical for peptide binding and recognition.

1996 ◽  
Vol 183 (3) ◽  
pp. 1253-1258 ◽  
Author(s):  
W W Kwok ◽  
M E Domeier ◽  
M L Johnson ◽  
G T Nepom ◽  
D M Koelle
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Cell Clone ◽  
Peptide Binding ◽  
Class Ii ◽  
Binding Studies ◽  
Peptide Complex ◽  
Mhc Molecules ◽  
Hla Dq ◽  
T Cell Clone

The association of specific HLA-DQ alleles with autoimmunity is correlated with discrete polymorphisms in the HLA-DQ sequence that are localized within sites suitable for peptide recognition. The polymorphism at residue 57 of the DQB1 polypeptide is of particular interest since it may play a major structural role in the formation of a salt bridge structure at one end of the peptide-binding cleft of the DQ molecules. This polymorphism at residue 57 is a recurrent feature of HLA-DQ evolution, occurring in multiple distinct allelic families, which implies a functional selection for maintaining variation at this position in the class II molecule. We directly tested the amino acid polymorphism at this site as a determinant for peptide binding and for antigen-specific T cell stimulation. We found that a single Ala-->Asp amino acid 57 substitution in an HLA-DQ3.2 molecule regulated binding of an HSV-2 VP-16-derived peptide. A complementary single-residue substitution in the peptide abolished its binding to DQ3.2 and converted it to a peptide that can bind to DQ3.1 and DQ3.3 Asp-57-positive MHC molecules. These binding studies were paralleled by specific T cell recognition of the class II-peptide complex, in which the substituted peptide abolished T cell reactivity, which was directed to the DQ3.2-peptide complex, whereas the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57. This structural and functional complementarity for residue 57 and a specific peptide residue identifies this interaction as a key controlling determinant of restricted recognition in HLA-DQ-specific immune response.

Induction and Break of Immune Tolerance to Human FVIII in a HLA-DRB1*1501 Humanized Hemophilic Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2280-2280
Author(s):  
Katharina Nora Steinitz ◽  
Brigitte Binder ◽  
Christian Lubich ◽  
Rafi Uddin Ahmad ◽  
Markus Weiller ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Mouse Model ◽  
Mhc Class Ii ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
Class Ii ◽  
T Cell Epitopes

Abstract Abstract 2280 Development of neutralizing antibodies against FVIII is the major complication in the treatment of patients with hemophilia A. Although several genetic and environmental risk factors have been identified, it remains unclear why some patients develop antibodies while others do not. Understanding the underlying mechanisms that drive the decision of the immune system whether or not to make antibodies against FVIII would help to design novel therapeutics. We used a new humanized hemophilic mouse model that expresses the human MHC-class II molecule HLA-DRB1*1501 on the background of a complete knock out of all murine MHC-class II genes. Initial studies had indicated that only a fraction of these mice developed antibodies when intravenously (i.v.) treated with human FVIII. These findings which resemble the situation in patients with severe hemophilia A, evoked the question if the lack of antibody development in non-responder mice reflects the induction of specific immune tolerance after i.v. application of FVIII or represent non-responsiveness for other reasons. We addressed this question by choosing another application route (subcutaneous, s.c.) and by combining i.v. application with a concomitant activation of the innate immune system applying LPS, a well characterized ligand for toll-like receptor 4, together with FVIII. Both strategies resulted in the development of antibodies in all mice included in the study what suggested that non-responsiveness against i.v. FVIII does not reflect an inability to develop antibodies against FVIII. Next, we asked if i.v. FVIII does induce immune tolerance in non-responder mice. We pretreated mice with i.v. FVIII, selected non-responder mice and challenged them with s.c. FVIII. None of the mice developed antibodies what indicated that i.v. pretreatment had induced immune tolerance in non-responder mice. Currently, we test the hypothesis that immune tolerance after i.v. application is induced and maintained by FVIII-specific regulatory T cells. The differences in responder rates after i.v. and s.c. application of FVIII raised the question if there are differences in FVIII T-cell epitopes involved in the initial activation of FVIII-specific CD4+ T cells. We obtained spleen cells from mice treated with either i.v. or s.c. FVIII and generated CD4+ T-cell hybridoma libraries that were tested for peptide specificities. For this purpose we used a FVIII peptide library containing 15 mers with an offset of 3 amino acids. Our results indicate that the pattern of FVIII-specific T-cell epitopes involved in the activation of FVIII-specific CD4+ T cells after i.v. and s.c. application of FVIII is almost identical and represents a small set of FVIII peptides distributed over the A1, A2, B, A3 and C1 domains. Based on our results we conclude that the new HLA-DRB1*1501 hemophilic mouse model represents an interesting opportunity to uncover the mechanisms that drive the decision of the immune system whether or not to develop antibodies against FVIII. Disclosures: Steinitz: Baxter BioScience: Employment. Binder:Baxter BioScience: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. de la Rosa:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment.

Immunosuppressive FVIII-Specific Human Cartregs in Hemophilia a

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 291-291 ◽  
Author(s):  
Jeong Heon Yoon ◽  
Anja Schmidt ◽  
Yong Chan Kim ◽  
Christoph Koenigs ◽  
David William Scott
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hemophilia A ◽  
Cell Clone ◽  
Coagulation Factor ◽  
Specific Staining ◽  
Promising Alternative ◽  
Effector Cells ◽  
T Effector Cells ◽  
T Cell Clone

Abstract Hemophilia A is an X-linked disorder, in which mutations in the coagulation Factor VIII (FVIII) gene lead to a loss of FVIII function and serious bleeding episodes. These episodes can be treated with recombinant FVIII protein replacement. Unfortunately, ~25% of hemophilia A patients produce inhibitory anti-FVIII antibodies because of lack of tolerance. Thus, it is necessary to develop effective tolerogenic therapies to prevent, as well as reverse, inhibitor formation. Previously, we generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T cell clone. The resulting engineered T cells bind MHC tetramers, proliferate in response to a specific FVIII epitope, and suppress effector responses to FVIII. In this study, we engineered a FVIII-specific chimeric antigen receptor (ANS8CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Following initial experiments in naïve CD4 T cells, this CAR was introduced into human Tregs. Western blot and specific staining with FVIII verified CAR expression. Transduced ANS8CAR Tregs proliferated in response to FVIII and were able to suppress the proliferation of FVIII-specific T effector cells in vitro. Additionally, the proliferation of T effector cells with different FVIII domain specificity was suppressed as well when ANS8CAR-transduced Tregs were activated with FVIII. Thus, engineered cells are able to promote bystander suppression. Cytokine expression of ANS8CAR-transduced Tregs was comparable to expression of untransduced and TCR-transduced Tregs indicating that the regulatory phenotype of Tregs was not negatively influenced by ANS8CAR expression. In conclusion, CAR-transduced Tregs seem to be a promising alternative to TCR-transduced Tregs for a future tolerogenic treatment of hemophilia A patients with inhibitory FVIII-specific antibodies. Supported by NIH grants HL061883 and HL126727 (DWS), the Society of Thrombosis and Hemostasis Research, and the Günter Landbeck Excellence Award (AS). Disclosures Kim: Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.

scholarly journals MHC-restricted minimal regulatory circuit initiated by a class II-autoreactive T cell clone.

1987 ◽  
Vol 165 (5) ◽  
pp. 1284-1295 ◽  
Author(s):  
K Sano ◽  
I Fujisawa ◽  
R Abe ◽  
Y Asano ◽  
T Tada
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
Class Ii ◽  
Secondary Antibody ◽  
Th Cells ◽  
Mhc Restriction ◽  
Autoreactive T Cell ◽  
Regulatory Circuit ◽  
T Cell Clone

The in vivo administration of a self-class II-reactive Th clone MS202 derived from C3H into syngeneic mice resulted in the suppression of both primary and early secondary antibody responses against T cell-dependent antigens. The suppression was due to the generation of antigen-nonspecific Ts cells in the recipient, as the splenic T cells from the mice treated with MS202 were able to strongly suppress the in vitro secondary antibody response of primed syngeneic spleen cells. The dose-response curve of suppression indicated the generation of an effector type Ts that directly suppressed Th. The surface phenotype of Ts was Ly-1+,2-, L3T4+, I-J-. The presence of Ly-1+,2+ T cells was not required to induce the suppression. The suppression was strictly restricted to H-2k, as F1 Ts cells were able to suppress the response of C3H but not of B6 B cells helped by the same F1 Th cells. The experiments with chimeric mice indicated that the direct target of Ts is an MHC-restricted Th but not a B cell or APC. The results indicate the existence of a minimal regulatory circuit where an MHC-restricted Th induces a preprogrammed Ts that in turn directly suppresses Th with the same MHC-restriction specificity. The induction of and suppression by Ts appeared to be due to the direct recognition of MHC restriction sites of Th cells.

Humanized E17 Hemophilic Mice Are a Major Breakthrough in the Design of New Preclinical Models for Developing Factor VIII Products with Reduced Immunogenicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 782-782 ◽  
Author(s):  
Birgit M. Reipert ◽  
Christina Hausl ◽  
Maria Sasgary ◽  
Maria Schuster ◽  
Rafi U. Ahmad ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Factor Viii ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Hemophilia A ◽  
Class Ii ◽  
Mhc Class Ii Molecules

Abstract MHC class II molecules are crucial for regulating adaptive immune responses against self and foreign protein antigens. They determine the antigenic peptides that are presented to CD4+ T cells and are essential for shaping the CD4+ T-cell repertoire in the thymus. Thus, the structure of MHC class II molecules is a major determinant for protein antigen immunogenicity. Structural differences between murine and human MHC class II complexes fundamentally limit the use of conventional murine hemophilia A models for dissecting immune responses to human factor VIII and developing new factor VIII products with reduced immunogenicity. To overcome this limitation, we humanized the murine E17 model of hemophilia A by introducing the human MHC class II haplotype HLA-DRB1*1501 on the background of a complete knockout of all murine MHC class II genes. Any anti-FVIII antibody response in this new humanized hemophilia A model is driven by CD4+ T cells that recognize FVIII-derived peptides that are presented by human HLA-DRB1*1501. The MHC class II haplotype HLA-DRB1*1501 is particularly relevant for the situation in hemophilia A patients because it is found in about 25% of Caucasians and 32% of Africans and has been shown to be associated with an increased risk that patients with severe hemophilia A have for developing FVIII inhibitors. We validated the relevance of this new model by asking the question whether HLA-DRB1*1501 hemophilic E17 mice develop FVIII inhibitors that are similar to those observed in patients with hemophilia A. Furthermore, we wanted to show that anti-FVIII antibody responses in these mice depend on the expression of the human DRB1*1501 molecule. Mice were treated with 8 intravenous doses of human FVIII and tested for anti-FVIII antibodies, anti-FVIII antibody-producing plasma cells and FVIII-specific T cells. About 90% of all humanized hemophilic E17 mice tested developed anti-FVIII antibodies that were similar to FVIII inhibitors found in patients. These antibodies were not restricted isotypically and contained mainly IgG1, IgG2a and IgG2b antibodies. Detection of antibodies in the circulation correlated with the presence of anti-FVIII antibody-producing plasma cells in the spleen. Development of anti-FVIII antibodies depended on the activation of FVIII-specific T cells and strictly depended on the expression of the HLA-DRB1*1501 molecule. Mice that did not express any MHC class II molecules did not develop anti-FVIII antibodies. We conclude that this new humanized E17 model for hemophilia A is a major advance towards developing suitable animal models needed to design future immunomodulatory strategies for patients with FVIII inhibitors and develop new FVIII products with reduced immunogenicity. Furthermore, it provides a tool for identifying T-cell epitopes of human FVIII restricted by MHC class II molecules that can be used for monitoring FVIII-specific T cells in patients who receive replacement therapy with FVIII products.

Identification of Novel MHC Class II-Restricted Male-Specific mHAg Encoded bySMCY(JARID1D)..

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1344-1344
Author(s):  
Nobuharu Fujii ◽  
Kellie V Rosinski ◽  
Paulo V Campregher ◽  
Edus H Warren
Keyword(s):  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Cell Clone ◽  
T Cell Responses ◽  
Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
Cell Responses ◽  
T Cell Clone

Abstract Abstract 1344 Poster Board I-366 Male recipients of female hematopoietic cell grafts, when compared with all other donor/recipient gender combinations, have an increased risk for both acute and chronic GVHD, but also have a significantly decreased risk of posttransplant relapse. F→M HCT is also characterized at the cellular level by donor (female) T cell responses against male-specific minor histocompatibility (H-Y) antigens, which can contribute to both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) activity. SMCY is a Y-chromosome gene that has previously been shown to encode at least two distinct MHC class I-restricted H-Y antigens presented by HLA-A*0201 and HLA-B*0702, respectively. Also, association between CD8+ T cell responses specific for the SMCY311-319 FIDSYICQV epitope and GVHD or GVL has been reported. A CD8+ FIDSYICQV-specific T cell clone was also reported to induce histological signs of GVHD reaction in an in vitro skin-explant assay. To date, however, only two MHC class I-restricted, and no MHC class II-restricted, H-Y antigens encoded by SMCY have been characterized. Given the large size of the SMCY and the homologous SMCX proteins and the fact that they are only 85% identical at the amino acid sequence level, we hypothesized that SMCY encodes other MHC class I- and class II-restricted H-Y antigens, and that T cell responses against these epitopes may likewise contribute to GVHD and GVL activity after F→M HCT. Arrays of pentadecapeptides with eleven-residue overlap were designed to tile regions of the SMCY protein that are non-identical to the corresponding regions of its X chromosome-encoded homologue SMCX, and then used to generate SMCY-specific T cell lines recognizing novel SMCY-encoded MHC class I- and class II-restricted H-Y antigens. Peripheral blood mononuclear cells (PBMC) were obtained on posttransplant day +126 from a 46 year-old male patient with monosomy 7 AML who had received a hematopoietic cell graft from his MHC-identical sister, and were stimulated in vitro with dendritic cells derived from his pretransplant PBMC that had been pulsed with the SMCY pentadecapeptides. After three stimulations, a SMCY peptide-specific CD4+ T cell line as well as a SMCY311-319 (FIDSYICQV)-specific CD8+ T cell line were obtained. After cloning by limiting dilution, we further characterized the SMCY-specific CD4+ T cell clone, 13H3. The 13H3 T cell clone recognizes the SMCY232-246 15-mer peptide, ELKKLQIYGPGPKMM, presented by HLA-DRB1*1501, and has a CD3+, CD4+, CD8−, CD45RA−, CD45RO+ surface phenotype. The cytokine release profile of this clone when assessed with SMCY232-246-loaded donor-derived EBV-LCL, as measured by the Luminex assay, is characterized mainly by Th1 cytokines (IFN-g and IL-2), but the clone also produced low to moderate levels of the Th2 cytokines IL-4, IL-10, and TGF-β. A minigene encoding SMCY232-246 was recognized by the 13H3 clone in a HLA-DRB1*1501-dependent fashion when transfected into COS-7 cells, but a minigene encoding the homologous SMCX-derived ELKKLQIYGAGPKMM peptide was not recognized, demonstrating that the clone is SMCY-specific. The 13H3 clone recognized 3 of 5 HLA-DRB1*1501+ male primary leukemia cells, but did not recognize either of 2 HLA-DRB1*1501− male or either of 2 HLA-DRB1*1501+ female primary leukemia cells. These results suggest that CD4+ T cell responses against the SMCY232-246 epitope could potentially contribute to GVL activity after F→M HCT. A SMCY232-246/HLA-DRB1*1501 tetramer has been constructed which specifically marks the 13H3 T cell clone, and future studies will use this reagent to determine whether CD4+ T cells specific for this epitope can be detected directly ex vivo in posttransplant blood samples from HLA-DRB1*1501+ F→M HCT recipients. Disclosures No relevant conflicts of interest to declare.

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