Minor Histocompatibility Antigens Research Articles

Hypoxia- and Postirradiation reoxygenation-induced HMHA1/ARHGAP45 expression contributes to cancer cell invasion in a HIF-dependent manner.

Cancer cells in severely hypoxic regions have been reported to invade towards tumour blood vessels after surviving radiotherapy in a postirradiation reoxygenation- and hypoxia-inducible factor (HIF)-dependent manner and cause recurrence. However, how HIF induces invasiveness of irradiated and reoxygenated cancer cells remains unclear. Here, we identified human minor histocompatibility antigen 1 (HMHA1), which has been suggested to function in cytoskeleton dynamics and cellular motility, as a responsible factor and elucidated its mechanism of action using molecular and cellular biology techniques. HMHA1 expression was found to be induced at the transcription initiation level in a HIF-dependent manner under hypoxia. Boyden chamber invasion assay revealed that the induction of HMHA1 expression is required for the increase in invasion of hypoxic cancer cells. Reoxygenation treatment after ionising radiation in vitro that mimics dynamic changes of a microenvironment in hypoxic regions of tumour tissues after radiation therapy further enhanced HMHA1 expression and invasive potential of HMHA1 wildtype cancer cells in ROS- and HIF-dependent manners, but not of HMHA1 knockout cells. These results together provide insights into a potential molecular mechanism of the acquisition of invasiveness by hypoxic cancer cells after radiotherapy via the activation of the ROS/HIF/HMHA1 axis.

Abstract CT151: Trial in progress:A phase 1 trial of TSC-100 and TSC-101, engineered T cell therapies that target minor histocompatibility antigens to eliminate residual disease after hematopoietic cell transplantation

Abstract Engineered T cell therapies such as CAR-T cell therapies have transformed the treatment of B-cell but not non-B cell hematologic malignancies. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for hematologic malignancies but ~40% of patients relapse post-HCT with up to 90% mortality due to residual disease post-HCT. A key challenge in non-B cell malignancies is identifying the right antigens for T cell targeting. Cancer-associated antigens are heterogenous, enabling rapid escape of malignant cells with low antigens, while targeting lineage-specific antigens without distinct expression in malignant versus normal myeloid cells can cause prolonged neutropenia. A potential solution is targeting minor histocompatibility antigens (MiHAs) that are homogenously expressed on all hematopoietic cells and are genetically mismatched between donors and patients undergoing HCT. These mismatches enable T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, leaving donor cells untouched. TScan has developed allogeneic donor derived T-cell products TSC-100 and TSC-101, targeting MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive (>98% of whom are either HA-1 or HA-2 positive) and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 can potentially eliminate all residual patient-derived hematopoietic cells after HCT, to prevent disease relapse.Study NCT05473910 is a multi-center, multi-arm, non-randomized controlled Phase 1 umbrella study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Inclusion criteria (Figure 1) include adults with AML, MDS or ALL eligible for reduced intensity conditioning-based haploidentical donor transplantation from HLA or MiHA mismatched donors. HLA-A*02:01-positive patients undergo HA-1/HA-2 testing and are assigned to either TSC-100 or TSC-101 treatment arms in addition to HCT. HLA-A*02:01-negative patients in the control arm receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive either TSC-100 or TSC-101, administered as single or two doses. Primary endpoints include adverse event profiles and dose limiting toxicities. Secondary endpoints include relapse rates, disease-free survival and overall survival. Exploratory endpoints include surrogates of efficacy such as minimal residual disease (MRD) rates and donor chimerism rates and kinetics. MRD is measured before and after HCT using flow cytometry, NGS and ddPCR. Donor chimerism is measured by standard STR-based and novel high-sensitivity NGS-based assays to quantify residual patient-derived hematopoietic cells. Together, these assays measure elimination of residual patient hematopoietic cells, malignant or normal, and could provide early evidence of biological activity. Citation Format: Monzr Al Malki, Alla Keyzner, Hyung Suh, Uday Popat, Chen Yi-Bin, Saar Gill, Melhem Solh, Joseph Uberti, Iris Isufi, Erica Buonomo, Yun Wang, Jim Murray, Gavin MacBeath, Debora Barton, Shrikanta Chattopadhyay, Ran Reshef. Trial in progress:A phase 1 trial of TSC-100 and TSC-101, engineered T cell therapies that target minor histocompatibility antigens to eliminate residual disease after hematopoietic cell transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT151.

Abstract 3855: Kinetics of neoantigen-specific T cells in post-transplant relapse

Abstract Introduction: Graft-versus-leukemia effect (GVL) underpins the curative mechanism of allogeneic stem cell transplantation (alloSCT) for acute leukemias. Donor-derived T cells can recognize various antigens, including human leukocyte antigen (HLA), minor-histocompatibility antigens (miHA), over-expressed antigens, or neoantigens derived from somatic mutations of leukemia. However, the role of neoantigens has not been fully investigated in GVL. Here, we aimed to predict neoantigens and identify neoantigen-reactive T cells using samples after alloSCT. Materials: Leukemia blasts were purified from relapsed samples. Recipient saliva or T cells from pre-transplant peripheral blood (PB) were used as a recipient germline control, while donor monocyte or CD34 cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) paired with RNAseq. Somatic mutations were called using blasts and recipient germline controls by mutect and mpileup and annotation with SnpEff. Somatic mutations with variant allele frequency >20% were screened for neoantigen prediction bound to recipient HLA (IC50 < 500nM) by pVAC-seq. We also genotyped 73 SNPs associated with publicly known miHAs. Serial PB were stained for HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65. T cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: We screened neoantigens in 8 patients relapsing after alloSCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their donors (4 matched siblings, 1 matched unrelated, 3 haploidentical). In 7 subjects, WES/RNAseq showed an average of 273 somatic mutations per patient (range 108- 609). In one subject, targeted next-generation sequencing was used to detect 2 somatic mutations. Among these mutations, seven out of eight subjects (87.5%) had potential neoantigen candidates, with a median number of 9 antigens per subject. In contrast, only 2 subjects (25%) had potential miHA. UPN8, a responder to donor lymphocyte infusion (DLI), showed dominant CMVpp65-specific T cells at the time of relapse and emergence of TP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse. HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these miHA-specific T cells during GVHD. In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells specific to neoantigens were detectable by IFN-γ/CD107a production in reaction to mutant peptides (SDE2.pK123X or TP53.pR248W) from post-relapse samples. Discussion: Our study showed the feasibility of in-silico neoantigens prediction and neoantigen-specific T-cell detection from a subject who developed relapse after alloSCT. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with transplant outcomes. Citation Format: Biswas Neupane, Jui-En Ray Lee, Kedwin Ventura, Kayla Parr, Kevin Quann, Sawa Ito. Kinetics of neoantigen-specific T cells in post-transplant relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3855.

Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens

AbstractAllogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

Evidence of GVHD/GVL in allogeneic hematopoietic stem cell transplantation from sex-mismatched donors

Hematopoietic cell transplantation (HCT) is considered a curative treatment for hematological malignancies. However, HCT recipients often face complications such as graft-versus-host disease (GVHD) and disease relapse. Clinical factors like age and HLA disparity are recognized as risks for GVHD. Notably, sex-mismatched HCT, particularly with female donors and male recipients (F→M), is reported to increase the risk of chronic GVHD. This adverse effect of F→M HCT is thought to result from allogeneic immune response against minor histocompatibility antigens encoded on the Y-chromosome of a male recipient (HY-antigens). Indeed, antibodies against HY-antigens (HY-Abs) were detected three months after F→M HCT, and the cumulative number of HY-Abs was significantly associated with increased risks of chronic GVHD and non-relapse mortality. This review focuses on F→M HCT, shedding light on its impact in several clinical settings and presenting clinical evidence of its allogeneic response, encompassing GVHD and graft-versus-leukemia (GVL) effects. Additionally, potential clinical options to mitigate adverse effects in F→M HCT will be discussed. Further investigation is required to improve clinical outcomes and understand allogenic immunological reconstitution after F→M HCT.

Initial Results of a Phase 1 Trial of TSC-100 and TSC-101, Engineered T Cell Therapies That Target Minor Histocompatibility Antigens to Prevent Relapse after Allogeneic Hematopoietic Cell Transplantation

Introduction: To date, engineered T cell therapies have not proven effective for non-B cell hematologic malignancies due to a lack of antigens that spare healthy myeloid cells. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for these malignancies, yet ~40% of patients relapse post-HCT with high mortality after relapse. A potential solution to prevent relapse is to target hematopoietic lineage-specific minor histocompatibility antigens (MiHAs) that are genetically mismatched between HCT patients and donors. These mismatches enable engineered T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, and spare donor cells. TSC-100 and TSC-101 are allogeneic, donor derived T-cell receptor-engineered T (TCR-T) cells that target MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 are designed to eliminate residual patient hematopoietic cells post-HCT and thus prevent relapse (figure). Methods: Study TSCAN-001 (NCT05473910) is a multi-center, multi-arm non-randomized, controlled Phase 1 study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Adults with AML, MDS or ALL eligible for reduced intensity conditioning (RIC)-based haploidentical donor transplantation from HLA or MiHA mismatched donors are enrolled. HLA-A*02:01 and HA-1 or HA-2-positive patients receive either TSC-100 or TSC-101 after HCT. HLA-A*02:01-negative control arm patients receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive a single dose of TSC-100 or TSC-101 at Dose Level 1 or repeat doses at Dose Levels 2 and 3. Dose escalation follows interval 3+3 rules. Primary endpoints include adverse event profiles and dose-limiting toxicities (DLTs). Secondary endpoints are relapse rates, disease-free and overall survival. Exploratory endpoints include biomarkers of efficacy such as minimal residual disease (MRD) by deep next-generation sequencing (NGS), and donor chimerism using novel high-sensitivity and standard assays. Results: At submission time, 8 patients were enrolled, 5 in treatment arms (3 TSC-101, 2 TSC-100) and 3 in the control arm, with a median of 111 days post-HCT follow-up (range 33-266 days). No DLTs occurred, and patients were enrolled at Dose Level 2 in both TSC-100 and TSC-101 arms. Safety analysis found expected post-HCT adverse events similar in treatment and control arms. Incidence of graft-versus-host disease (GvHD) was similar in control (3 events) and treatment arms (4 events). Serious adverse events of grade 3 GvHD and infections were observed in TSC-101 and control arms (1 event each). No cytokine release syndrome or neurotoxicity occurred after TSC-100/101 and minimal changes in CRP/ ferritin occurred, consistent with general safety of TCR-Ts with low target cell burden. No clinical relapses occurred to date. Translational analysis found peak TSC-100/101 expansion and activation 7-14 days post dosing, with ongoing persistence at longest follow-up of 138 days. In repeat dose cohorts, substantial increases of early expansion were noted after dose 2 of TSC-101/100, supporting the benefit of repeat dosing. Chimerism analysis (table) using a novel high-sensitivity NGS-based AlloHeme assay (limit of detection (LOD) 0.13%) in whole blood, CD33+ or CD3+ subsets found mixed chimerism in 2 out of 2 control patients but complete donor chimerism in 4 out of 4 TSC-100/101 patients after Day 42. Both control patients had declining donor chimerism after Day 100 by AlloHeme and standard STR-based chimerism assays (LOD 1-2%) prompting early withdrawal of immunosuppression in one patient. Marrow MRD analysis by NGS (LOD <0.05%) found 1 control patient was MRD positive pre-HCT and remained MRD positive post-HCT. In contrast, 2 TSC-100/101 patients who were MRD positive pre-HCT turned MRD negative after HCT and TSC-100/101 therapy including a TP53 mutated MDS patient confirmed MRD negative by droplet digital PCR (LOD 0.01%). Conclusions: Targeting MiHAs HA-1 or HA-2 with TSC-100/ 101 following HCT shows early safety and biomarker evidence of efficacy by completing elimination of all detectable patient hematopoietic cells, normal or malignant, thereby reducing relapse risk. Updated results will be presented at the meeting.

Kinetics of Neoantigen Specific T Cells in Post-Transplant Relapse

Introduction: In allogeneic stem cell transplantation (alloSCT), donor-derived T cells mediate the graft-versus-leukemia effect (GVL) by recognizing antigens expressed by leukemia cells. These antigens may include either major- or minor-histocompatibility alloantigens, leukemia over-expressed antigens (leukemia-associated antigens), or neoantigens derived from leukemic somatic mutations. In solid tumors, higher somatic mutation burdens are associated with better response to checkpoint inhibitors, which underscores the importance of neoantigen-specific T-cells in cancer immunotherapy. However, mutation burdens are relatively low in acute leukemia, and the role of neoantigens in GVL remains unknown. Here, we screened potential neoantigens and identified neoantigen-reactive T cells in the patients who developed post-transplant relapse. Materials and methods: Leukemic blasts from patients in relapse were FACS-isolated from bone marrow aspirate or peripheral blood samples. Recipient T cells or saliva were isolated from pre-transplant peripheral blood as a germline control, and donor monocyte or CD34-positive cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) at 100X coverage, paired with RNAseq at 40M reads per sample. Somatic mutations were called between recipient blasts and germline controls with mutect and mpileup, followed by annotation with SnpEff. Somatic mutations with variant allele frequency >20% were subjected to pVAC-seq for prediction of neoantigen likely presented on recipient MHC (IC50 < 500nM). 73 SNPs loci associated with publicly known minor histocompatibility antigens (miHAs) were also genotyped to find the miHA disparities between donors and recipients. Serial peripheral blood mononuclear cell samples (PBMCs) were obtained before relapse if available, at the time of relapse, and after treatment for relapse. For HLA-A*02:01-restricted neoantigens, PBMCs were stained with HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65 (control). Purified CD3 cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: Eight patients with relapsed acute leukemia after allo-SCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their transplant donors (4 matched siblings, 1 matched unrelated, 3 haploidentical) were screened for neoantigens. We analyzed samples from 7 subjects with available WES/RNAseq and detected an average of 273 somatic mutations per patient (range 108- 609) on average. Targeted next-generation sequencing was used to detect 2 somatic mutations in one subject. Among these mutations, potential neoantigen candidates were identified in seven out of eight subjects (87.5%), with a median number of 9 candidate neoantigens per subject. In contrast, only 2 subjects (25%) were found with donor:receipient miHAs at tested loci. CD8 T cells derived from UPN8, a responder to DLI, showed dominant CMVpp65-specific T cells at the time of relapse and persistent low frequencyTP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse by HLA-A*02:01 tetramer staining (Figure A). HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these T cell clones during GVHD (Figure B). In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells reactive to neoantigens were detectable by IFN-γ/CD107a production specific to mutant peptides ( SDE2.pK123X and TP53.pR248W respectively). Discussion: Our in-silico analysis suggested the possibility of somatic mutations in acute leukemia to serve as putative neoantigens in alloSCT. We were able to detect neoantigen-specific T cells by tetramer staining and cytokine production assays from a subject who achieved a durable remission after DLI. Although the sample size is very small, we demonstrated the feasibility of studying neoantigen-specific T cells in post-transplant specimens. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with post-relapse outcomes, especially for response to DLI.

An Analytic Framework for the Prediction of Gvl Minor Histocompatibility Antigens to Prevent or Treat Post-Transplant Relapse

The therapeutic effect of allo-HSCT relies on T cell alloreactivity driven by genetic nonidentity between donor and recipient (D-R) pairs. In the setting of HLA-matched transplants, endogenous proteins in recipient cells differing from those of the donor due to genetic polymorphisms, can provide distinct HLA-binding peptides, serving as minor histocompatibility antigens (mHAgs). Hematopoietically-restricted mHAgs represent ideal targets to separate graft-versus-leukemia (GvL) from graft-versus-host-disease (GvHD) effects. To date,however, the quest for optimal GvL mHAgs has resulted in the identification of only a handful of targets, raising concerns on the clinical feasibility of their targeting. We hypothesized that systematic mHAg prediction from whole-exome sequencing (WES) of D-R pairs could help identifying novel candidate mHAgs with an acceptable safety profile for therapeutical targeting. We focused on AML/MDS, the most common indications for allo-HSCT in adults. To capture the transcriptional heterogeneity of malignant myeloid cells, we applied a single-cell based classifier to define genes expressed by leukemic cells in the Beat AML cohort, and then removed those expressed in non-hemopoietic tissues, per the GTEx RNA and protein database. Similarly, a broader set of pan-hematopoietic genes was defined starting from the expression profiles of 18 mature hematopoietic lineages and hematopoietic stem and precursor cells. Through this process, we identified 259 genes with preferential expression in AML and 615 broadly expressed in the hematopoietic compartment. We applied this analytical pipeline to WES from 220 HLA-matched D-R pairs transplanted at DFCI. To discover mHAgs with optimal GvL potential, we focused on the subgroup of 45 patients with long-term survival in the absence of both relapse and GvHD requiring systemic treatment (i.e. GvHD-free/relapse-free survival [GRFS]). For these patients experiencing purely GvL, we analyzed the individual repertoire of predicted GvL mHAgs, and found 86 SNPs recurring in ≥5 GRFS patients. These GvL SNPs were enriched in the subgroup of GRFS patients versus those without GRFS, suggesting that their overrepresentation was not dictated by high allelic frequency in the overall cohort. Using the median number of recurring GvL mHAgs in the GRFS subgroup (‘GRFS mHAgs‘) as a cutoff to stratify the entire study cohort, we found that a higher GRFS mHAg load was associated with a protective effect against relapse ( p=0.009) and conferred a benefit in 2-year overall survival ( p=0.03). Furthermore, HLA class I immunopeptidome analysis of 5 AML cell lines revealed evidence of presentation for 10 epitopes predicted from genes in the overall GvL filter, two of which were also part of the GRFS mHAg set. Given the limited size and lack of ethnic diversity of our discovery cohort (composed mainly of patients of European ancestry), we used genomic data from the 1000 Genomes project to estimate the feasibility of targeting our GRFS mHAg set in a broader population via in silico modeling of allo-HSCT. We simulated 2270 D-R pairs from 844 individuals across 5 populations of disparate ancestry (AFR, EAS, EUR, SAS, and AMR), mirroring a typical URD search in the NMDP registry. To gauge the feasibility of generating personalized mHAg-specific immunotherapy (i.e. cancer vaccines, adoptive cellular therapy) based on the GRFS mHAg set, we estimated that a 10-SNP design (i.e., the number of GRFS SNPs used for correlative analyses in our discovery cohort) would be possible for ~90% of the 1000 Genomes simulated cohort (EUR: 99%, EAS: 72%, SAS: 91%, AFR: 69%, AMR: 74%). Through a similar process, we calculated population coverage simulating a T cell-based approach targeting ≥1 epitope from the GRFS mHAg set. First, we identified 18 HLA alleles that could ensure ~99% coverage across diverse ethnic backgrounds. Second, for each HLA allele, the 3 most frequently predicted (and with highest agretopicity) epitopes from the GRFS SNP set were selected, defining a pool of 54 epitopes. We thus calculated that ≥1 epitopes could be potentially targeted for 81% of the simulated D-R pairs (EUR: 91%, EAS: 78%, SAS: 71%, AFR: 62%, and AMR:85%). Overall, we developed a robust approach to identify novel GvL mHAgs, which could form the basis for systematic personalized immunotherapy following allo-HSCT.

Systematic Prediction of Minor Histocompatibility Antigens to Inform GvHD Outcomes after Allogeneic Stem Cell Transplantation

T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms (SNPs, indels, frameshifts), is at the core of the therapeutic effect of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite the crucial role of mHAgs in graft-versus-leukemia and graft-versus-host (GvHD) reactions, it has not been possible thus far to consistently link patient-specific mHAg repertoires to clinical outcomes. As a result, only D-R HLA matching and the activity of GvHD prophylaxis strategies are currently available to help clinicians in this challenge. We hypothesized that post-transplant outcomes could be impacted by genome-wide mHAg load, delineated in an organ- and malignancy-specific fashion. We have therefore devised an analytic framework to systematically identify autosomal and Y-encoded mHAgs, based on the integration of polymorphism detection by whole-exome sequencing (WES) of germline DNA from D-R pairs together with organ-specific transcriptional- and proteome-level expression together with HLA class I epitope prediction. A critical component of the pipeline was building a reliable expression atlas for acute or chronic GvHD-targeted tissues. To capture less frequent but biologically relevant cell types consistently underestimated in bulk RNA expression profiles, we evaluated multiple external single-cell datasets of healthy human skin, liver, lung, GI, oral mucosa, and lacrimal gland. For each tissue, we merged the corresponding single-cell data sets and then clustered and annotated tissue-resident cell types, for which specific expressed genes were then identified, totaling 13,512 genes expressed in ≥1 GvHD target tissue. To determine how mHAg load related to GvHD risk, we applied our mHAg pipeline to paired WES (≥85% target base coverage at ≥20x depth) generated from 220 D-R pairs treated with a matched-related donor allo-HSCT for AML/MDS. We considered the impact of diagnosis, prognostic risk score, status at transplant, conditioning regimen, and graft source against median mHAg load on risk for acute and chronic GvHD. Across these patients, only total autosomal mHAg load > median was associated with increased risk of developing grade II-IV aGvHD (HR = 2.54, 95% confidence interval: 1.03 - 6.26, p = 0.043). While the total autosomal mHAg load was not associated with development of cGvHD, subgroup analysis of 55 FàM transplants revealed that a combined score of autosomal and Y-encoded mHAgs > median was associated with development of NIH moderate/severe cGvHD (p = 0.042). We next asked whether organ-specific mHAg load could predict the risk for organ-restricted acute or chronic GvHD. By evaluating organ-specific GvHD occurrence across deciles of mHAg load, we identified 2 distinct patterns of association. In the case of lung cGvHD, all 14 positive cases occurred in patients with a lung-specific autosomal mHAg load > median. By logistic regression modelling, lung mHAg load > median was the sole factor associated with increased risk of lung cGvHD (OR = 17.22, 95% CI: 3.18 - 321.63, p = 0.008). In contrast, for aGvHD of the liver, we observed an opposite pattern, with 12 of 13 affected patients having a liver mHAg load < median. We hypothesized that a limited set of immunodominant liver mHAgs might drive the clinical manifestations in these patients. By analyzing the mHAg landscape of patients affected by liver aGvHD, we observed an enriched representation of 7 polymorphisms in genes with preferential expression in liver. Notably, these SNPs were less frequent in patients developing aGvHD without liver involvement (p = 0.002) and co-occurred at a lower frequency in patients experiencing liver cGvHD (p = 0.0002). For one patient experiencing liver aGvHD, we could trace ex vivo T cells specific for 1 (of the 3 predicted) driver liver mHAgs. Overall, our findings indicate that molecular characterization of D-R pairs to define GvHD risk could facilitate the design of personalized post-HSCT treatments to minimize this highly morbid condition, including incorporation of additional immunosuppressive therapies, introduced early for high-risk patients, or reduced-intensity approaches in low-risk settings. This is particularly valuable for lung GvHD, which is associated with the highest morbidity and mortality.

Dual Functions of Mitochondrial Calcium Uniporter in T Cell Alloimmunity

Despite pharmacological prophylaxis using calcineurin inhibitors, graft-versus-host disease (GVHD) still occurs in patients undergoing allogeneic hematopoietic stem cell transplantation. However, the mechanism that underlies the breakthrough of alloreactive T cell responses following calcium signaling inhibition remains poorly defined. The mitochondrial calcium uniporter (MCU), which is located in the inner mitochondrial membrane, controls Ca2+ uptake into the mitochondria matrix. Past dogma suggested that lack of mitochondria calcium uptake would compromise T cell immune response due to suboptimal NFAT activation upon TCR ligation. However, the exact role of MCU in the regulation of alloreactive T cell responses has not been previously determined. Here, we demonstrate dual functions of MCU in alloreactive T cell response and GVHD maintenance, highlighting MCU as a potential novel target to manipulate T cell alloreactivity. Using T cell-specific Mcu conditional knockout C57/BL6 (B6) mice (Mcu-cKO), we found that compared to WT T cells, B6 Mcu-cKO T cells induced less severe GVHD in Balb/c mice, with prolonged survival and significantly lower number of donor effector cells in the spleen and liver early after transplantation. Surprisingly, these Mcu-cKO T cell recipients developed severe liver GVHD by 60 days after transplantation, as evidenced by extensive lymphocytic infiltration in the liver, while to a lesser extent in the skin and intestine in these GVHD mice. Flow cytometric analysis of donor T cells, which were harvested between 5 and 8 weeks after transplantation, showed that Mcu-cKO T cells retained the IFN-g- and GM-CSF-producing capacity, but acquired stronger capacity of producing the cytotoxic molecule granzyme B. To comprehensively delineate the role of MCU in allogeneic T cell response, we used MHC-identical but minor histocompatibility antigen (miHA)-mismatched B6-to-Balb/b GVHD model, in which alloreactive CD8+ T cells can be identified with H60 peptide/MHC class-I dimer staining. Similarly, loss of Mcu in T cells caused less severe GVHD, attributed to the decreased number of H60+CD8+ effector T cells that showed impaired proliferation capacity. RNA-seq analysis of liver-infiltrating H60+CD8+ T cells revealed that Mcu ablation resulted in decreased expression of cell proliferation genes (Pclaf, Cdc45, Top2a, Cdca8, etc), increased expression of genes (Cd7, Bcl2, CD96, Eomes, Ezh1, etc) associated with memory T cells. These results demonstrated that Mcu is required for the expansion of alloreactive T cells, but restricts their memory-associated transcriptional programs during GVHD maintenance. Further characterization showed these alloreactive Mcu-cKO T cells produced higher levels of tissue migration molecules (CCR9, CCR5, etc) and lower death molecule (Lpin1) than their WT counterparts. Using gain-of-function strategy of retroviral Mcu overexpression in T cells (McuOE), we found McuOE CD8+ T cells displayed enhanced effector differentiation represented by higher expression of effector molecules (Ifng), decreased expression of stemness and memory associated molecules (Tcf7, Id3), and reduced expression of migration molecules (CCR9, CXCR3, etc). The cultured McuOE CD8+ T cells contracted earlier than WT. These results were further validated in vivo using the B6-to-Balb/c GVHD model. These data demonstrate a negative regulatory role of MCU in the tissue migration and persistence of alloreactive T cells. Notably, human T cells from patients with severe GVHD produced lower levels of MCU protein compared to that of non-GVHD patients. Our findings identified for the first time the dual functions of MCU in the regulation of T cell alloimmunity. MCU-mediated mitochondria calcium uptake promotes early expansion of alloreactive T cells but restricts the expression of memory-associated gene programs and tissue migration molecules. Strategies of enhancing MCU functionality in alloreactive T cells could potentially reduce their memory potential and tissue infiltration capacity, thereby reducing GVHD.

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

Prediction of Graft-Versus-Host Disease (GVHD) in Recipients of Hematopoietic Cell Transplant(alloHCT) from a Single Mismatched Unrelated Donor Using a Highly Multiplexed Proteomics Assay: MHC-Pepseq

Introduction: Graft-versus-host disease (GVHD) remains a major barrier of allogenic hematopoietic stem cell transplantation (HCT). While direct recognition of mismatched HLA molecules is believed to be the major driver of alloimmune T cell response, indirect presentation of mismatched host class I or class II proteins may also contribute to CD4 T cell-mediated GVHD or graft-versus-leukemia (GVL). Using a novel highly multiplexed peptide-MHC binding assay (MHC-PepSeq), we developed a score of HLA-DRB1 restricted and binding allogeneic peptide sequences for each donor/recipient pair in a CIBMTR cohort of mismatched adult HCT recipients with hematological malignancies. We hypothesized that the risk score derived from the MHC-PepSeq assay is associated with the incidence and severity of acute and chronic GVHD. Methods: Using public population allele frequencies (allelefrequencies.net), we identified a set of alleles that covers >95% of HLA genotypes of 3 major US populations (European Caucasian, African American, Mexican Chicano) at 9 HLA-loci (-A, -B, -C, -DRA1, -DRB1, -DQA1, -DQB1, -DPA1, -DPB1) and converted their sequences to 7,744 unique, densely-tiled 15-mer peptides. A highly multiplexed synthesis protocol (“PepSeq”) was used to generate a library of DNA-barcoded peptides of the corresponding sequences, which we used to generate experimental binding measurements of each peptide against 12 common HLA-DRB1 molecules. The resulting data were then used to enumerate DR-binding peptides present in recipient HLA but not donor HLA genotype (“allopeptide score”), for each donor-recipient pair in a cohort of HLA-A, B, C and DRB1 (8/8) matched unrelated donors with a mismatch in HLA-DPB1 and a cohort of 7/8 Class I mismatched unrelated donors. Univariate analysis was used to compare clinical demographics and allopeptide association with clinical outcomes. In multivariable analysis, a logistic regression model was used to analyze aGVHD II-IV at day 100 (primary outcome), and Cox proportional hazards were applied for OS, relapse, chronic GVHD (cGVHD) and non-relapse mortality (NRM) (secondary outcomes). Results: In the 8/8 cohort, 6679 matched unrelated pairs were identified with allopeptide detected in 1563 (30.5%). In the 7/8 cohort, 1133 pairs were identified with allopeptide detected in 156 (15.9%). Patient characteristics were balanced in both cohorts and described in Table 1. In the 8/8 cohort, the cumulative incidence of aGVHD II-IV at day 100 post HCT was 42.3% (95% CI: 40.9-43.7%) in patients with a detectable allopeptide and 45.5% (95%CI: 43.0-48.0%) in patients with non-detectable allopeptide, respectively (p=.069). No significant differences were observed in univariate analysis for cGVHD, NRM, OS. In multivariate analysis there were no significant associations between presence of detectable allopeptide (Y/N) and aGVHD II-IV, cGVHD, OS, and NRM. However, a significant interaction between disease and allopeptide status was detected in the relapse model (P=0.0020), with allopeptide presence associated with a decreased risk of relapse in ALL (HR 0.71; 95%CI:0.54-0.94, p=0.0168) and an increased risk of relapse in AML (HR 1.23; 95%CI:1.05-1.43, p=0.0085). Allopeptide presence was not significantly associated with relapse in MDS (HR 0.89; 95% CI:0.74-1.06 p=0.1901). Adjusted cumulative incidence of relapse is shown in Figure 1. In the 7/8 cohort, cumulative incidence of aGVHD II-IV at day 100 post HCT was 43.4% (95% CI: 40.2-46.5%) and 41.8% (95% CI: 34.1-49.7%) (p=.77) in the allopeptide-detectable and non-detectable groups, respectively. In univariate analysis there were no statistically significant differences noted in cGVHD, NRM, OS. In multivariate analysis allopeptide presence was not significantly associated with any of the endpoints aGVHD II-IV, cGVHD, OS, relapse or NRM. Conclusion: In a large CIBMTR 8/8 and 7/8 matched unrelated donor cohort, allopeptide scores derived from MHC-pepseq were not predictive of aGVHD, possibly due to the limitation of the assay (i.e., low sensitivity, limited to class II presentation - HLA-DRB1 in this analysis) and/or low antigenicity in HLA molecules when indirectly presented as minor histocompatibility antigens. The observed reverse association of allopeptide presence with relapse in AML and ALL requires further investigation, and subgroup analyses for individual HLA-DRB1 presenting allele are currently underway.

Explorations of Post-DLI Low-Dose Cyclophosphamide for Preventing Severe aGVHD

Background Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a serious life-threatening complication. The granulocyte colony-stimulated factor mobilized donor lymphocyte infusions (gDLI) combined with chemotherapy is currently a commonly used treatment method. Nevertheless, gDLI may cause so severe acute graft-versus-host disease (aGVHD) as to impact prognosis. Posttransplant cyclophosphamide (PTCy) has been the backbone for GVHD prophylaxis by inducing tolerance to minor histocompatibility antigens in recipients, while the application of post-gDLI low-dose cyclophosphamide (PDCy) for GVHD prophylaxis has not yet been attempted. Methods To explore this possibility, a retrospective study was conducted. 20 patients relapsing after HSCT were administered 20 mg/kg/d cyclophosphamide（Cy）on day 3 (for matched related transplantation) or on days 3 and 4 (for haplo-identical or unrelated transplantation) after gDLI to prevent aGVHD (the PDCy group). Furthermore, through propensity score matching, 58 matched controls received other (for HID and URD) or no (for MSD) immunosuppressive therapy for GVHD prophylaxis (the Non-Cy group). Patients from the PDCy and the Non-Cy group were matched by transplantation type, CD3+ T cell counts, and age and gender of recipients and donors. An MRD complete response was undetectable MRD lower than 0.01% with qPCR, and a reduction of at least one-log fold in MRD load post gDLI was defined as an MRD response. Results The PDCy group comprised 9 and 11 men and women, respectively, with a median age of 38 (34-52, IQR) years. The Non-Cy group comprised 34 and 24 men and women, respectively, with a median age of 40 (29-47, IQR) years. The infused CD3+T cell dose in the PDCy and Non-Cy group was 3.0 *10^7/kg and 2.9 *10^7/kg, respectively (p=1.0). The interval between HSCT and gDLI was longer in the PDCy group when compared to the Non-Cy group (30.8 vs 6.5 months, p<0.01). And first recurrence accounted for a higher percentage in the Non-Cy group (93% vs 45%, p<0.01). Other transplant- and DLI-specific data did not exhibit any significant difference between the groups. With a median follow-up of 4.8 (0-37.1) months, the PDCy group had lower cumulative incidence of severe aGVHD (III-IV, 5% vs 31%, p=0.02; II-IV, 25% vs 52%, p=0.04, figure 1A, B), but no significant differences existed in 4-month OS (64% vs 59%, p=0.51, figure 1E) and NRM (20% vs 19%, p=0.5, figure 1D). Among 49 patients undergoing salvage treatment (PDCy group, n=16; Non-Cy group, n=33), there was no statistical difference in 4-month CIR (20% vs 47%, p=0.11, figure 1C) and rates of objective response (PR+CR, 68.8% vs 54.5%, p=0.6). Among 29 patients undergoing preemptive treatment (PDCy group, n=4; Non-Cy group, n=25), there was no statistical difference in the rates of MRD complete response (25% vs 42%, p=1) and MRD response (25% vs 50%, p=0.6) between the PDCy group and the Non-Cy group. The PDCy regimen didn't increase the incidence of severe bacterial infection (45% vs 33%, p=0.3), bacteremia (25% vs 16%, p=0.11), fungus infection (20% vs 29.3%, p=0.4), virus infection (5% vs 19%, p=0.1), hemorrhagic cystitis (n=1 vs n=1), and cardiac events (n=0 vs n=0). Conclusions In conclusion, on the premise of safety, the PDCy regimen could effectively protest against severe aGVHD after gDLI while preserving therapeutic response rates. However, the research results still require verification through longer follow-up and large prospective randomized studies.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

A Novel High-Throughput Method for Identifying T-Cell Receptor: Minor Histocompatibility Antigen Interactions in Mouse Models of Graft-Vs-Host Disease

Graft-vs-host disease is a complication of allogeneic stem cell transplant (alloSCT) in which donor T cells target alloantigens expressed by healthy recipient tissues. In major-histocompatibility-matched alloSCT, CD8-mediated GVHD is caused by T cell receptor (TCR) allorecognition of class-I presented minor histocompatibility antigens (miHAs). The vast majority of miHAs remain unknown between any given donor-recipient pairing. However, determining the identities of these miHAs and their cognate TCRs would prove useful towards the development of allografts that minimize GVHD yet preserve the therapeutic benefits of alloSCT. Here, we implement a high-throughput TCR cloning method, TCXpress, to screen alloreactive TCRs from a mouse model of MHC-matched alloSCT against putative miHA targets determined informatically. 129 (H-2 b) mice, which express the immunodominant miHA H60, were lethally irradiated and reconstituted with bone marrow and CD4 + and CD8 + T cells from B6 (H-2 b) donors. At day +10, recipient spleens were harvested and CD44 +PD-1 + donor CD8 + cells were single-cell sorted according to H-2Kb:H60 tetramer (Tet H60) staining into unknown miHA-reactive (Tet H60-) and control H60-reactive (Tet H60+) cohorts for TCR cloning using the TCXpress platform (BlueSphere Bio). Clonotyping was performed by sequencing the CDR3 region of TCRs. Putative miHAs were identified informatically by filtering non-synonymous variants between B6 and 129 reference exomes on predicted class I binding affinities. To interrogate TCR target specificity, Jurkat reporter cells, which express GFP and CD69 upon TCR activation, were transduced with selected alloreactive TCRs and reacted in a multiplexed fashion against a panel of B6WT3 antigen-presenting cells (H-2 b) transduced with tandem minigenes (TMG) of predicted miHAs. To test TCR reactivity against hematopoietically-expressed miHAs independent of our calling strategy, Jurkat reporters were also reacted against bone-marrow derived dendritic cells (BMDCs) from control B6 and 129 mice. From 3 recipient mice, 966 alloreactive TCRs were cloned and sequenced (735 Tet H60- and 231 Tet H60+) corresponding to 553 unique clonotypes (437 Tet H60- and 116 Tet H60+). Sixty-seven percent (N=668) of TCRs sequenced had clonotype occurrences of ≥ 2, indicating clonal expansion post-alloSCT, whereas there were no repeated clonotypes among donor B6 CD8 + cells pre-transplant (N=173 TCRs). A total of 47 Tet H60- TCR clones and 3 Tet H60+ control TCR clones were selected for transduction into Jurkat reporters and screened against a limited TMG library of 158 putative B6→129 miHAs, as well as H60. Expectedly, all TCR clonotypes isolated from the Tet H60+ sort gate reacted against H60-expressing B6WT3 cells and 129 BMDCs. From the Tet H60- TCRs, one clone was validated against another documented 129 miHA, H4, while an additional 4 clones were found to react against novel B6→129 miHAs (Serpinb8 and Cdt1). Interestingly, 2 highly over-represented clones were autoreactive against B6 tissues (B6WT3 cells and BMDCs), but not 129 BMDCs. In summary, we demonstrate the capacity of a novel TCR cloning platform to validate informatically-predicted miHAs and their cognate TCRs in a mouse model of GVHD. This approach may be readily adapted to develop new TCR:miHA therapeutics and gain a deeper understanding of GVH responses targeting miHAs.

TCF-1is Required By CD8 + T Cells for the Maintenance of Alloimmune Responses in Graft-Vs-Host Disease

Graft-vs-host disease (GVHD) is a common complication of allogeneic stem cell transplant (alloSCT) wherein donor T cells target alloantigens on recipient tissues. It is unclear how alloimmune responses are maintained in GVHD despite abundant antigen, which causes T cell anergy, deletion and exhaustion. Previously, we identified alloreactive TCF-1 high T cells arising post-transplant that resemble exhausted progenitors (T EXP) capable of propagating immune responses in other chronic antigen models. Here, we sought to further characterize these cells in the B6→129 MHC-matched GVHD mouse model, in which 129 recipients express the immunodominant H-2K b-restricted minor histocompatibility antigen (miHA) H60. At day +7 post-transplant, alloreactive CD8 + cells specific to H60 (as determined by MHC-I-tetramer staining; Tet H60+) were nearly uniformly PD-1 hiTox hi whereas Tet H60- cells displayed a bimodal distribution into discrete PD-1 hiTox hi and PD-1 loTox lo populations, indicative of more diverse antigen experiences. Among these both Tet H60+ and Tet H60- cells were TCF-1 hi cells. TCF-1 hi Tet H60+ cells were uniformly CD39 loTox hiPD-1 hi, which is a canonical T EXP phenotype. In contrast, among activated Tet H60- cells there were TCF-1 hi cells that were CD39 loTox hiPD-1 hi and Tox loPD-1 lo. At later times in spleen and lymph node, and in GVHD target tissues, these populations of TCF-1 + Tet H60+ and Tet H60- were found. To test if these CD39 loTCF-1 hi T EXP had proliferative advantages in GVHD, we sorted congenic TCF-1 hiCD39 lo and TCF-1 loCD39 hi CD8 + cells from recipient spleens 14-days post-transplant and adoptively transferred them in competition in a 1:1 ratio (of Tet H60+ cells) into newly transplanted recipients. Among Tet H60+ cells in all tissues at day 14 post-transfer, TCF-1 hiCD39 lo-sorted progeny greatly outperformed TCF-1 loCD39 hi-sorted progeny. In line with their role as a source of GVHD effectors, progeny of TCF-1 hiCD39 lo cells were mostly TCF-1 loCD39 hi; however, a fraction remained TCF-1 hi consistent with their being able to undergo self-renewal. Conversely, we observed few if any TCF-1 + progeny of CD39 hi cells. We next tested whether TCF-1 was an important mediator of T cell fitness or whether it was only a marker for functionality. To do so we competed congenic wild-type (WT) and Tcf7 p45-/- (p45 -/-) donor CD8 cells, which lack the N-terminal β-catenin binding domain of TCF-1, in allogeneic (129) and syngeneic (B6) recipients. Strikingly, p45 -/- CD8 cells were greatly outcompeted by WT CD8 cells in 129 recipients in all tissues and at all times post-transplant, among both Tet H60+ and Tet H60- cells. In contrast, in B6 recipients, WT and p45 -/- cells remained evenly matched, suggesting that full-length TCF-1 isoforms are dispensable for lymphopenia-induced T cell expansion. Further, p45 -/- cells were also not disadvantaged when adoptively transferred into B6 mice and acutely challenged with H60 antigen by vaccination. Together these data suggest a model wherein TCF-1 hi progenitor like T cells are seeded in GVHD target organs where they may serve as a key local source for GVHD effectors, and moreover, full-length TCF-1 is itself critical for alloreactive T cell fitness in GVH responses.

Impact of Multiple Minor Histocompatibility Antigen Differences on Graft-Versus-Host Disease and Relapse Risk in Pediatric Allogeneic Hematopoietic Cell Transplantation Recipients

BACKGROUND Success of allogeneic hematopoietic cell transplantation (alloHCT) is sharped by alloreactivity, i.e. the ability of donor T-cells to recognize particular recipient motifs as non-self and get activated. Donor T-cells recognizing recipient tissue can cause harmful graft-versus-host disease (GvHD) while activity against leukemic blasts can protect against relapse (GvL). In HLA-matched alloHCT, alloreacticity is driven by subtle nonpathogenic variants in natural proteins, socalled minor histocompatibility antigens (miHAgs), however, not all variants are immunogenic. METHODS To analyze the impact of mHAgs-differences on alloHCT outcomes, we selected a purely pediatric cohort of 102 HLA-matched allo HCTs transplanted between 2003 and 2017. Panel sequencing of 46 validated miHAgs was performed using Ilumina MiSeq, only variants in the graft-versus-host direction were considered. RESULTS In a logistic regression model for the whole cohort acute GvHD incidence was significantly dependent on the proportion of miHAg mismatches (all genes) with an 95%-CI 1.0263-1.1998, p=.01 and an odds ratio of 1.1066. From all variants tested, only mismatches in TRIM42 were significant as a predictor with a FDR <.05. Furthermore, proportion of mismatches was a predictor of all-cause mortality. Risk of relapse was evaluated by regression analysis for competing risks: proportion of miHAg mismatches failed to be predictive for relapse ( p=.37). However, looking at patients with acute lymphobastic leukemia (ALL) only (n=36), mismatches in hematopoiesis-restricted genes protected from relapse (95%-CI 1.0016-1.0712, p=.04, hazard ratio 1.0358). CONCLUSION Thus, in children GvHD and mortality seem to be related to the overall number of miHAg-differences whereas variants in hematopoiesis-restriced genes protect from ALL relapse. These data implicate that selection of TCRs with a specificity for hematopoietic miHAgs could be a way to improve GvL effect without increasing the risk of GvhD.

Immunosuppressive Activity of Exosomes from Granulocytic Myeloid-Derived Suppressor Cells in a Murine Model of Immune Bone Marrow Failure.

We previously reported that granulocytic myeloid-derived suppressor cells (G-MDSCs) suppressed T-cell activation and attenuated bone marrow failure (BMF) in a minor histocompatibility (minor-H) antigen mismatched murine aplastic anemia (AA) model. In the current study, we tested the hypothesis that exosomes, a subset of extracellular vesicles, are responsible at least partially for G-MDSCs' therapeutic efficacy. Indeed, exosomes isolated from GMDSCs (G-MDSC-exos) suppressed CD4+ and CD8+ T-cell proliferation in vitro and mildly attenuated immune BMF in the minor-H mismatched AA model. G-MDSC-exos treatment significantly increased red blood cells, hemoglobin, and total bone marrow (BM) cells, and moderately reduced BM CD8+ T cells. G-MDSC-exos' effects were associated with upregulations in an array of lymphocyte-suppression-related miRNAs such as hsa-miR-142-5p, miR-19a-3p, and miR-19b-3p in both BM CD4+ and CD8+ T cells. We concluded that G-MDSC-exos attenuate immune BMF via modulating the delivery of immunosuppressive miRNAs into activated T lymphocytes.

Minor histocompatibility antigens represented in HLA-A*02:01 and their search strategies

Minor histocompatibility antigens (MiHAs) are polymorphic peptides on the cell surface derived from self-proteins that are capable to induce an immune response during allogeneic hematopoietic stem cells transplantation. Their presentation occurs in the context of the certain major histocompatibility complex (HLA – human leucocyte antigen) alleles. One of the most common HLA alleles is HLA-A*02:01. Accordingly, for a significant number of donors and recipients pairs, it is possible to use the MiHAs presented in the HLA-A*02:01 as a target for relapsed leukemia therapy. This review discusses the main known MiHAs presented in the context of HLA-A*02:01, their characteristics and approaches used for identification. The described approaches may be used to search for new MiHAs for immunotherapy.

Novel T cell/organoid culture system allows ex vivo modeling of intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRβ+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches.