High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations

abstract: ...T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1 IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance.

... specifically recognizing target cells expressing the MAP4K1 IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of

... response and was obtained during the subsequent memory phase. The 8 cell lines tested showed a wide range of peptide sensitivity. Specifically, the cell lines specific for P218 MAP4K1 IMA and P46 HMHA1 VLH were capable of target recognition at low picomolar peptide concentrations, similar to the peptide concentration required for the HMHA-1-specific control T-cell clone. Cell lines specific for P91 FLT3 KVL , P309 HSPA6 SMC and P188 FLT3 ALA required low nanomolar peptide concentrations and those

... recognized by cell-lines P91 FLT3 KVL and P203 ATP2A3 KMN displayed a comparably high MHC affinity (as measured in Fig. 1 ) as those of the two highly sensitive cell lines P218 MAP4K1 IMA and P46 HMHA1 VLH . Thus, the low peptide sensitivity of many of the isolated T-cell lines formed a direct reflection of a low affinity TCR-pMHC interaction. 10.1371/journal.pone.0022523.g004 Figure 4 Analysis of peptide affinity of pMHC tetramer positive cell lines MHC tetramer positive T-cell lines demonstrated a wide

...), whereas 8 cell lines recognized self antigen variants. Interestingly, both high-avidity T-cell populations that were isolated (specific for P218 MAP4K1 IMA and P46 HMHA1 VLH ) recognized non-self antigen variants, whereas all cell lines that recognized self antigen variants were of intermediate or low-avidity. These results suggest a possible role for clonal deletion of high-avidity T-cells specific for these self epitopes when they are appropriately processed. Based on these results, we hypothesized

... P46 HMHA1 V L H D D L L E A A GG GG no non-self P89 ITGAM R L Q V P V E A V G GG GG no self P91 FLT3 K V L H E L F G M D I A AA AA no self P104 ZFP36L2 R L L P L W A A L P L G GG GG no self P109 FMNL1 S L W Q L G A A V M L G CC CG yes non-self P153 DOCK2 K L L Q I Q L R A G GG GG no self P203 ATP2A3 K M N V F D T N L A GG GG no non-self P218 MAP4K1 I M A I E L A E L A GG GG no non-self P235 (APM) PRKCB1 R I G Q R Q E T V G AA AA no non-self P235 (JMO) PRKCB1 R I G Q R Q E T V G AA AA no non-self

... demonstrated to be recognized by the HMHA-1 T-cell line as well as by the MAP4K1 IMA specific T-cell line ( Figure 6 ). These results indicate that when endogenous presentation of the predicted MAP4K1 IMA epitope is forced, recognition by P218 MAP4K1 IMA specific T-cells is strong. 10.1371/journal.pone.0022523.g006 Figure 6 Recognition of EBV target cells by high-avidity MiHA T-cells after minigene transduction. High-avidity cell lines P46 HMHA1 VLH (white) and P218 MAP4K1 IMA (grey) were stimulated with

... the Y-axis. Shown frequencies indicate pMHC tetramer positive T-cells out of total lymphocytes. ( c ) BDY3356 derived CD8 + cell line: P37 DOCK2 SIQ . ( d ) APM4461 derived CD8 + cell line: P46 HMHA1 VLH . Dot plots shown are representative for all generated cell lines. (PDF) Click here for additional data file. Table S1 Identified genes with a hematopoiesis-resricted expression pattern. (PDF) Click here for additional data file. Table S2 Total MiHA epitopes predicted by HLA-peptide binding

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Gene: HMHA1 - histocompatibility (minor) HA-1 - (NCBI)

Protein : 3ft3

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