Minor Histocompatibility Antigens Research Articles

T Cells Redirected to a Minor Histocompatibility Antigen Instruct Intratumoral TNFα Expression and Empower Adoptive Cell Therapy for Solid Tumors.

Donor-derived allogeneic T cells evoke potent graft versus tumor (GVT) effects likely due to the simultaneous recognition of tumor-specific and host-restricted minor histocompatibility (H) antigens. Here we investigated whether such effects could be reproduced in autologous settings by TCR gene-engineered lymphocytes. We report that T cells redirected either to a broadly expressed Y-encoded minor H antigen or to a tumor-associated antigen, although poorly effective if individually transferred, when simultaneously administered enabled acute autochthonous tumor debulking and resulted in durable clinical remission. Y-redirected T cells proved hyporesponsive in peripheral lymphoid organs, whereas they retained effector function at the tumor site, where in synergy with tumor-redirected lymphocytes, they instructed TNFα expression, endothelial cell activation, and intratumoral T-cell infiltration. While neutralizing TNFα hindered GVT effects by the combined T-cell infusion, a single injection of picogram amounts of NGR-TNF, a tumor vessel-targeted TNFα derivative currently in phase III clinical trials, substituted for Y-redirected cells and enabled tumor debulking by tumor-redirected lymphocytes. Together, our results provide new mechanistic insights into allogeneic GVT, validate the importance of targeting the tumor and its associated stroma, and prove the potency of a novel combined approach suitable for immediate clinical implementation. Cancer Res; 77(3); 658-71. ©2016 AACR.

Cellular Immunotherapies for Leukemia Patients

Cellular Immunotherapies for Leukemia Patients

Selective graft-versus-leukemia depends on magnitude and diversity of the alloreactive T cell response.

Patients with leukemia who receive a T cell-depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.

Dendritic Cell Cross Presentation of RBC Antigens in-Vivo Is Not Affected By RBC Storage Duration and Requires Red Pulp Macrophage "Help" in-Vitro

BACKGROUND: Chronic transfusion is associated with an increased risk of bone marrow graft rejection. In prior studies with mouse models, minor histocompatibility antigens in red blood cell (RBC) and platelet products were presented in the context of recipient MHC class I to prime recipient cytotoxic T cells; these represent cross presentation and cross priming, respectively. Once primed in the recipient, these T cells may attack allogeneic bone marrow grafts. Thus, understanding the mechanism of transfusion-induced T cell cross priming may lead to new methods to reduce bone marrow graft rejection in chronically transfused patients. Cross presentation is performed by a specialized subset of antigen presenting cells (APCs) - CD11b lo, XCR1+ dendritic cells (XCR DCs). Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Fluorescent tracking of transfused RBCs showed that splenic red pulp macrophages (RPMs) ingest the majority of damaged RBCs, but RPMs are weak APCs. Splenic dendritic cells, including XCR DCs, also display increased uptake of stored vs. fresh RBCs. These data suggested that refrigerated storage may increase cross presentation of RBC antigens, thereby enhancing T cell cross priming.AIMS: To compare T cell cross priming after transfusion of fresh and refrigerator-stored RBCs, a mouse model was used with transgenic OVA-carrying RBCs as the antigen source and transgenic naive MHC class I-restricted (H-2Kb) OVA-specific T cells (OT-1) as responders. In parallel, an in-vitro system was established to determine the cellular elements required for cross presentation of RBC antigens.METHODS: Purified CD8 T cells from OT-1 mice were loaded with the cell proliferation tracking dye Cell Trace Far Red (CTFR) and adoptively transferred (4 x 10e6 per mouse) into cohorts of 8-12 week old C57BL/6 GFP+ mice (n=3 per group). The next day mice were transfused with 400 uL of fresh (<24 hours old), or stored RBCs (14 days old) from transgenic HOD mice (RBCs express surface HEL, OVA, and Duffy antigens). After 96 hours, flow cytometry was used to assess OT-1 cell proliferation by dye dilution and the expression of activation markers CD44, CD122 and CD62L. Control transfusions with non-HOD GFP+ RBCs confirmed the antigen specificity of the response, and transfusions of HOD blood that had undergone RBC lysis ruled out direct antigen presentation by MHC identical white cells in the transfusate. For the in-vitro cross presentation assay, 1x10e5 CTFR-loaded OT-1 cells were plated in U-bottom 96 well plates with all possible combinations of fresh or stored HOD RBCs (50 x 10e6 per well), bone marrow Flt3L-derived C57BL/6 murine dendritic cells (Flt3L-DCs, 5 X 10e4 per well), and magnetically selected BALB/c (H-2Kd) RPMs (5 x 10e4 per well). MHC mismatch rules out antigen presentation by BALB/c RPMs (H-2Kd) to OT-I T cells (H-2Kb).RESULTS: Transfusion of both fresh and stored HOD RBCs induced vigorous proliferation and activation of OT-1 cells. After 4 days, no differences were seen in the proliferation and activation profiles of OT-1 cells in mice receiving fresh (78±4% CD44hi, CTFR diluted) vs. stored (79±5%) HOD RBCs. The in-vitro cross presentation assay showed weak to absent OVA-specific OT-1 proliferation with co-cultures including fresh or stored HOD RBCs alone, and fresh or stored HOD RBCs plus Flt3L-DCs. In contrast, cultures including RPMs and Flt3L-DCs showed enhanced OVA-specific OT-1 proliferation with fresh (29±10% CTFR diluted) and stored (25±2%) HOD RBCs.CONCLUSION: Cross priming by fresh RBCs was stronger than expected based on prior studies showing minimal RBC uptake by XCR DCs after fresh GFP + RBC transfusion. Cross priming in this system may be more sensitive due to the high frequency of antigen specific T cells and, thus, is saturated by the small antigen load delivered by fresh transfusion. It is also possible that direct uptake of RBCs by DCs harms the cross-presentation machinery and offsets the effect of increased RBC antigen delivery. Optimal cross-presentation of RBC antigen may require cooperation with cells functionally specialized for metabolizing RBC by-products. Indeed, the in-vitro study shows RPMs promote DC-mediated cross presentation of antigens on fresh and stored RBCs. Thus, RPMs may share ingested RBC antigen with adjacent DCs and increase DC activation by secreting inflammatory cytokines.. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Integrated In Silico Prediction of Minor Histocompatibility Antigen Peptides in HLA-Matched Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Background and Rationale: T-cell responses to minor histocompatibility antigens (mHA) are important drivers of the beneficial graft versus leukemia (GvL) effect and harmful graft versus host disease (GvHD) pathology following HLA-matched allogeneic stem cell transplantation (alloSCT). Despite their importance, Genome Wide Association Studies (GWAS) have failed to elicit a prognostic set of individual mHA associated with the clinically observed GvL and GvH effects of alloSCT [Sato-Otsubo et al. Blood 2015]. This is likely due to a lack of public mHA shared across the global set of donor/recipient pairs (DRPs). Even an optimally frequent single nucleotide polymorphism (SNP) would result in a recipient restricted genetic variant in only 24% of DRPs with a matched unrelated donor (MUD) or 17% of DRPs with a matched related donor (MRD) [Armistead et al. PLoS One 2011]. Moreover, even when DRPs do share a recipient restricted genetic variant, any resulting peptide would have to bind a MHC molecule within the recipient for presentation to T-cells.Methods: To evaluate the role of mHA in alloSCT without requiring public mHA, we developed a bioinformatics pipeline to predict mHA peptides based on SNP differences taking into account peptide/MHC binding estimated by netMHCpan [Nielson et al. Genome Med 2016] and expected GvL vs GvH tissue expression derived from mRNA sequencing data from acute myeloid leukemia (AML) and normal hematopoietic, hepatobiliary, skin, and gastrointestinal tract tissues. In order to understand the distribution of mHA in HLA-matched alloSCT drawn from a diverse pool of DRPs, we applied this analysis to putative DRPs drawn from healthy individuals who underwent whole-exome sequencing as part of the 1000 Genomes Project (n = 1,916) [1000 Genomes Project Consortium et al. Nature 2015]. To evaluate the association of mHA with AML relapse and GvHD incidence following alloSCT, we predicted mHA from SNP data using clinical information from an earlier study [Armistead et al. PLoS One 2011].Results: To determine a baseline for the number of predicted mHA in an alloSCT, we considered every possible pair of samples in the 1000 Genomes data set as a theoretical alloSCT (n = 3,669,140). We determined each sample’s HLA type using PHLAT [Bai et al. BMC Genomics 2014] and then performed mHA predictions for all theoretical transplants with a 10 out of 10 HLA match (n = 10). Within each ethnicity represented in the 1000 Genomes data, the degree of HLA matching was greater than that of all ethnicities pooled, with the lowest HLA diversity in the Finnish, Chinese, and Japanese populations. The number of predicted mHA binding MHC with Kd < 500nM ranged from 6,217 to 13,545 in the 1000 Genomes theoretical HLA-matched DRPs, with mHA contained in genes selectively expressed in AML versus those selectively expressed in GvH target organs numbering 213 to 610 and 367 to 1,135, respectively. HLA-A*02:01 restricted mHA were predicted for 37 actual DRPs in the context of MUD alloSCT and 97 DRPs from MRD alloSCT [Armistead et al. PLoS One 2011]. There were significantly more predicted mHA in MUD transplants (Figure 1). Taking into account both predicted peptide/MHC binding and tissue expression, the number of predicted GvL mHA was significantly associated with remission and the aggregate number of predicted GvH mHA was significantly associated with grade 2-4 GvHD incidence in MUD transplants (Figure 2).Conclusions: Prediction of mHA based on whole-exome sequencing data is feasible and can be used to discover associations of mHA distribution features with clinical outcomes including AML remission and GvHD incidence. Future work in larger datasets will be required to validate these predicted associations and guide development of mHA-directed therapeutics. [Display omitted] [Display omitted] DisclosuresMolldrem:Astellas Pharma: Patents & Royalties.

Whole Transcriptome Sequencing (RNAseq) As a Comprehensive, Cost-Efficient Diagnostic Tool for Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is caused by cooperating oncogenic driver mutations that induce uncontrolled proliferation in combination with maturation arrest in myeloid precursor cells. The majority of oncogenic drivers are somatically acquired. Recurrent genetic lesions are used for molecular classification and prognosis of AML. In addition, expression levels of selected genes contribute to AML subclassification (CD34, KIT) and prognostication (e.g. EVI1). Actionable aberrations and pathways are molecular targets for personalized pharmacotherapy. Structures, incidences, and mutual associations of recurrent genetic aberrations have been elucidated by large whole genome/exome sequencing efforts. However, 10-20% of AML appear to be caused by hitherto unrecognized or individual driver lesions. Current AML diagnostics are poorly standardized and comprise a combination of morphology, flow cytometry, cytogenetics, targeted gene amplification with electrophoresis, sequence analysis, and qRT-PCR. We here test the hypothesis that whole transcriptome sequencing (RNAseq) without parallel germ-line sequencing could potentially be used as a single and cost-efficient platform for AML diagnosis and prognostication. Methods Poly(A)+ RNA was isolated from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%). These samples were obtained from 97 patients (2 diagnosis-relapse pairs, 1 de novo AML with subsequent tAML). RNA was sequenced at a depth of 59x106 paired-end reads per sample with median read length of 126 bp through a ISO17025-accredited Illumina HiSeq 2500 pipeline. After alignment against the GRCh38 reference genome, single nucleotide variants (SNV) and small indels were called by VarScan with a threshold of 20% aberrant reads. Variants were filtered for known polymorphisms occurring at &gt;5% in defined ethnic subpopulations of the 1000 Genomes and Genome of the Netherlands Projects. Internal tandem duplications (ITD) were identified by frequency and distribution of soft clipped reads. Detection of fusion genes was performed on raw reads using STAR-Fusion and FusionCatcher. Geneexpression levels were measured relative to HBMS as a housekeeping gene by counting the number of reads aligned to gene exons normalized to the sum of exon lengths. HAMLET was developed and implemented as an integrated RNAseq pipeline to measure relevant recurrent AML-associated aberrations, i.e. SNV and small indels in recurrently mutated genes, the FLT3-ITD, AML-associated gene fusions, and EVI1 overexpression. Results A total of 221 SNV/indels (15 homozygous) were detected at expected or higher frequencies in NPM1 (33%), FLT3 (ITD+TKD: 37%), DNMT3A (30%), TET2 (27%), IDH1 (14%), IDH2 (14%), RUNX1 (23%), CEBPA (4%), KIT (7%), WT1 (8%), ASXL1 (11%), and TP53 (3%). Sensitivities to detect SNV/small indels and FLT3-ITD were 96% and 94%, respectively, with no apparent relation to the blast count. Fusion genes relevant to the WHO AML classification were found with a sensitivity of 100% when compared to metaphase cytogenetics and FISH. Specifically, 10 cases of CBFB-MYH11, 3 RUNX1-RUNX1T1, 2 PML-RARA, 1 FUS-ERG, 1 KMT2A-MLLT3, and 1 DEK-NUP214 fusion events were correctly detected. In addition, 3 variant KMT2A translocations with MLLT1, MLLT4, and MLLT6 as fusion partners were correctly called. The specificity of HAMLET for recurrent SNV, indels, and fusion genes was 100%. Finally, RNAseq quantitatively detected variability in gene expression as exemplified for EVI1 overexpression and confirmed by qRT-PCR (r=0.85). Conclusions In conclusion, RNAseq analysis of AML samples without concomitant germ-line sequencing detects molecular information with relevance for classification, prognosis, and targeted therapy with 96% overall sensitivity and 100% specificity. HAMLET is currently optimized to further improve the sensitivity to detect SNV/small indel and ITD events. With a cost price of €980,- per case and full accreditation for diagnostic application of the RNAseq raw data, RNAseq facilitates comprehensive and cost-effective AML diagnostics in a single assay. Additional predicted benefits of this RNAseq approach to be explored include identification of non-recurrent individual drivers, neoantigens, and minor histocompatibility antigens, a potential to replace conventional HLA typing, and as a potential alternative for flow cytometry. Disclosures Janssen: GenomeScan BV: Employment.

Mother Donors Improve Outcomes after HLA Haploidentical Hematopoietic Transplantation: A Retrospective Study By the Cell Therapy and Immunobiology Working Party of the EBMT

▪IntroductionTrans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect transplantation outcomes when the mother acts as donor for the child. Indeed, survival after T cell-depleted HLA haploidentical haematopoietic transplantation was improved using the mother as donor (vs all other family members) (Stern et al., Blood 112:2990, 2008; Kruchen et al., BMT 50:1367 2015). However, maternal donors were associated with increased incidence of GvHD and decreased survival after un-manipulated HLA haploidentical blood and marrow grafts (Wang Y et al., Blood 124:843, 2014).Patients and MethodsA retrospective EBMT registry-based study was performed in a combined series of adult (n=333) and pediatric (n=105) patients with acute leukemia (AML=268, ALL=160, Mixed phenotype=10) who received transplant from one MHC mismatched family donor. Forty-four percent (233) of patients were in I or II complete remission (CR) at the time of transplant, 193 were in chemo-resistant relapse or in CR > 2. Median age was 32.7 (range: 0.67-70). Seventy-one percent of patients received ex-vivo T cell depleted transplants. Twenty-two patients were given bone marrow and peripheral blood stem cells, the others were given peripheral blood cells only. The mother was used as donor in 98 patients.ResultsEighty patients developed acute GvHD ≥ grade II. At a median follow up of 58.7 months, 130 patients are alive without disease, 146 relapsed and 152 died of non-relapse mortality (NRM). Univariate analyses of factors influencing relapse-free survival (RFS) showed age, disease status at transplant, Karnofsky score and ex-vivo T cell depletion impacted significantly. As transplantation outcomes from family members other than mothers did not differ from one another, such transplants were combined for analyses. When compared with transplantation from all other family members (n=330), transplantation from mother donors (n=98) was associated with lower relapse incidence (RI) (28% vs 39%, P=0.06) and non-relapse mortality (NRM) (30% vs 40%, P=0.09) and, consequently, better relapse-free survival (RFS) (43% vs 21%, P < 0.001). Multivariate analyses showed transplantation from mother donors was an independent factor predicting better RFS (other donors vs mothers: HR: 1.42; CI: 1.01-2.00; P=0.04) and lower RI (other donors vs mothers: HR: 1.93; CI: 1.16-3.22; P = 0.01). In addition, transplantation in relapse (vs remission) predicted worse RFS (HR: 2.33, CI: 1.80-3.00, P < 0.001) such as ≥18 (vs < 18) adversely impacted on RFS (HR: 1.40, CI: 1.00-1.96, P=0.04).DiscussionOur retrospective analyses in 438 HLA haploidentical hematopoietic transplants for acute leukemia patients (pediatric and adult) show that transplantation from mother donors, when compared with transplantation from any other family member, is an independent factor predicting better outcomes, i.e., better RFS and lower RI. Mothers should therefore be preferred when selecting an HLA haploidentical family donor. Further clinical and preclinical studies are needed to unveil the mysteries underlying mother-to-child immune interaction during pregnancy and its bearing on the reproductive success of the human species. DisclosuresCiceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

NaïVe T Cell Depletion of PBSC Grafts Results in Very Low Rates of Chronic Gvhd and High Survival

BackgroundGraft-versus-host disease (GVHD) frequently causes morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) as a result of organ damage and infections. In HLA-identical HCT, GVHD results from recognition by donor T cells of minor histocompatibility (H) antigens on recipient tissues. Complete T cell depletion (TCD) of donor hematopoietic cell products is more effective than pharmacologic immunosuppression for preventing GVHD, but is complicated by delayed immune reconstitution and consequent life-threatening infections.Approaches to HCT which preferentially deplete the T cells that primarily cause GVHD and preserve pathogen-specific T cells may improve HCT outcomes.Mature CD3+ CD8+ and CD3+ CD4+ T cells can be classified into CD45RA+ CD62L+ naïve (TN) and CD45RO+ memory (TM) subsets, the latter of which includes effector memory (TEM) and central memory (TCM) cells. Murine studies in which allogeneic TCD bone marrow (BM) is transplanted with purified T cells from individual T cell subsets to irradiated minor H antigen disparate recipients have demonstrated that the most severe GVHD results from transplanting T cells of the TN subset. Purified TCM causes mild GVHD and TEM do not cause detectable GVHD and can transfer immunity to pathogens.In vitro studies have similarly demonstrated that human donor CD8+ T cells specific for recipient minor H antigens are found predominantly within the TN cell subset, suggesting selective TN cell depletion may alter the GVHD incidence and/or severity in human HCT.Methods and resultsWe developed an effective process for engineering human peripheral blood stem cell (PBSC) grafts that depletes CD45RA+ TN cells and retains CD34+ stem cells and functional CD45RO+ TM cells specific for a broad range of opportunistic pathogens (Bleakley BBMT 2014). We are conducting clinical trials to evaluate the selective depletion of TN cells from HLA-matched allogeneic PBSC grafts for the prevention of GVHD in patients with acute leukemia, the first of which has been published (Bleakley JCI 2015, N=35). Seventy patients have now been treated on three consecutive phase II trials. The median age was 34 years (1-56 years), 56% of patients had a diagnosis of ALL, 46% had previously relapsed or had detectable disease (MRD or relapse) at the time of HCT, and 23% had unrelated donor (URD) grafts. Intensive myeloablative, TBI-containing (13.2Gy) conditioning was used for 63 patients, whilst 7 patients received a medium intensity ’midi’ preparative regimen, including 4Gy of TBI. The TN-depletion procedure was successfully performed on URD PBSC products shipped overnight from donor centers throughout the US, as well as on MRD PBSC collected at our centers. Reliable engraftment with high-level donor chimerism was observed in recipients of ’midi’ as well as intensive myeloablative conditioning. The 2-year estimates of overall survival, disease-free survival, survival free of relapse and chronic GVHD (CRFS) and survival free of relapse, grade II-IV acute GVHD, and chronic GVHD (GRFS) are 79%, 73%, 69% and 63% respectively. Median follow-up among survivors is 26 months. The frequency and severity of chronic GVHD is remarkably low (5%) compared to historical rates of 40-60% chronic GVHD in HLA-matched PBSC transplantation with conventional calcineurin inhibitor-based immunosuppression. Relapse and non-relapse mortality (NRM) are acceptably low at 19% and 8%, respectively. No NRM occurred in patients <40 years. Updated results will be presented.ConclusionsThe outcomes of recipients of TN-depleted PBSC grafts compare very favorably to published results of HCT for patients with acute leukemia. For example, the 69% incidence of CRFS at 2 years in TN-depleted recipients compares with reported 2-year GRFS rates of 37% and 17% in recipients of allogeneic PBSC from HLA-matched related donors with or without ATG (Kroger et al. NEJM 2016). Our results suggest that TN-depletion of PBSC grafts may reduce the risk of chronic GVHD without negatively impacting other important HCT outcomes. DisclosuresRiddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria.

Identifying Tissue-Resident Memory T Cells in Graft-Versus-Host Disease

Tissue-resident memory T cells (TRM) are a newly described subset of transcriptionally-distinct memory CD4 and CD8 cells that persist in barrier and non-barrier tissues. They are non-circulating, able to facilitate the recruitment of circulating effector cells and elicit rapid recall responses. The majority of TRM cells can be identified by the expression of CD69 and αE integrin, CD103. However, CD69+CD103- TRM cells have also been described.In graft-vs-host disease (GVHD), alloreactive effector T cells enter GVHD target tissues and mediate tissue damage through direct and indirect mechanisms.The recruitment of effector T cells into tissues is in general not dependent on the tissue expressing the target antigen; and even if a target antigen is available in a tissue, there could be niches free of presented alloantigen. We therefore hypothesized that even in GVHD where alloantigen is ubiquitous, TRM may develop.We first explored TRM formation in a CD4 T cell receptor (TCR) transgenic (Tg) GVHD system wherein donor BALB/c RAG2-/- TS1 TCR Tg T cells target the S1 peptide derived from HA, which is expressed ubiquitously in BALB/c RAG2-/- HA104 mice. In this model, GVHD is induced by <1000 TS1 cells and is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small bowel and colon. We harvested tissues from GVHD mice at days 21 and 28 post-transplant and quantitated TS1 cells with a TRM phenotype. At day 21, a fraction of TS1 cells expressed CD69+CD103+ (as % of total TS1 cells) in the epidermis (2.8% ± 1.2), dermis (11.3% ±7.9), colon (11.5% ±4.5) and small intestine (SI) intraepithelial (IEL) (33.4% ±5.8) and lamina propria (LP) (6.8% ±0.8). At day 28, CD69+CD103+ TS1 cells (% of total TS1) were present in the epidermis (13.6% ± 1.9), dermis (15.9% ± 7.7), colon (30.8% ± 6.7) and the SI IEL (69.1% ± 9.2) and SI LP (18.7 ±3.2). While there were no CD69+CD103+ TS1 in the spleen, bone marrow (BM) or liver (days 21 and 28), a small number (1.5% ±0.7, day 21; 1.7% ± 0.3, day 28) were found in the mesenteric lymph node (mLN). CD103- TRM have been described in the liver and secondary lymphoid organs. Consistent with this, CD69+CD103- TS1 cells (% of total TS1) were found in the liver (24.6% ±6.3, day21; 31.5% ±3.1, day 28), BM (23.1% ±3.1, day 21; 35.4% ±3.2, day 28), spleen (1.8% ±0.6, day 21; 9.1% ±0.7, day 28) and mLN (9.9% ±4.5, day 21; 23.5% ± 3.7, day 28). We are currently confirming the TRM identity of TS1 cells based on their transcriptional and migratory profiles and these data will be presented.Alloreactive TRM were also identified in the B6 (H-2b) into 129 (H-2b) MHC-matched, multiple minor histocompatibility antigen (miHA)-mismatched model in which GVHD is induced by a mix of CD4 and CD8 cells. A fraction of CD8 cells target the Kb-restricted miHA LTFNYRNL derived from H60, which can be tracked with tetramers (TetH60). At day 22 post-transplant, CD69+CD103+ CD4 cells (% of total CD4 T cells) were found in the epidermis (22.9% ±1.2), dermis (19.7% ±2.8), colon (9.4% ±3.1), SI IEL (26.5% ±2.6), SI LP (16.1% ± 3.7) and mLN (6.9% ±3.0). CD8+TetH60+ T cells (% of total CD8T cells) were detected in the epidermis (7.5% ± 3.5), dermis (10.6% ± 6.7), colon (6.9% ± 2.7), SI IEL (8.9% ±6.2), SI LP (10.2% ±3.8), mLN (3.0 ± 0.7) and spleen (9.0% ±2.9). A fraction of CD8+TetH60+ cells in the dermis (7.3% ±1.9), colon (18.1% ±12.2), SI IEL (50.1% ±4.3), SI LP (52.6% ±13.6), and mLN (6.3% ± 0.8) were CD69+CD103+, suggesting that alloreactive H60-directed CD8 T cells acquired a TRM phenotype.Using two different murine models, we found GVHD-inducing T cells with TRM phenotypes. Future experiments will confirm the TRM identity of these cells and will determine their importance in the maintenance of GVHD, perhaps by serving as a reservoir of cells that maintain GVHD locally. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Analysis of Activation and Proliferation Kinetics and Effector Functions of Human Lymphocyte Subsets in Xenogeneic GvHD Model

To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions.The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery.We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model.CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone.Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. DisclosuresKanda:Otsuka Pharmaceutical: Honoraria, Research Funding.

Vitamin A-Coupled Liposomes Carrying siRNA Against HSP47 Ameliorate Skin Fibrosis in Chronic Graft-Versus-Host Disease

Introduction: Chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is characterized by multiple-organ fibrosis and often refractory to conventional immunosuppressive therapies, profoundly affecting quality of life of SCT survivors. Heat shock protein 47 (HSP47), a collagen specific molecular chaperone, plays a critical role in collagen synthesis in myofibroblasts. Vitamin A-coupled liposomes carrying HSP47 siRNA (VA-Lip HSP47) have been shown to deliver HSP47 siRNA to pathogenic myofibroblasts and ameliorate mouse liver cirrhosis. We tested if VA-Lip HSP47 could inhibit collagen synthesis in myofibroblasts and ameliorate skin fibrosis in chronic GVHD, using a well-established murine model of cutaneous chronic GVHD.Methods: VA-Lip HSP47 was provided by Nitto Denko Corporation (Osaka, Japan). BALB/c mice were exposed to a single dose of 6 Gy total body irradiation, followed by intravenous injection with 8 x 106 bone marrow cells plus 2.5 x 107 splenocytes from minor histocompatibility antigen mismatched B10.D2 or syngeneic BALB/c donors on day 0. Recipient mice were intravenously injected with 4.5 mg/kg VA-Lip HSP47 or vehicle thrice a week from day +1 to +41 after bone marrow transplantation (BMT). To deplete macrophages, a group of recipients were intraperitoneally injected with 0.5 mg/body of anti-CSF1 receptor monoclonal antibodies (aCSF1R) thrice a week from day +7 after BMT. Skin samples were harvested on day +42. For in vitro analysis, primary fibroblasts isolated from mouse skin were stimulated by 5 ng/ml TGF-b in the presence or absence of 50 nM of VA-Lip HSP47 or VA-Lip containing scrambled siRNA. HSP47 expressions were measured by quantitative PCR.Results:Masson’s trichrome staining of the skin sections demonstrated skin thickening with massive fibrosis of the dermis 42 days after BMT. Quantitative collagen assay confirmed collagen volume was significantly increased in the skin of allogeneic recipients compared to syngeneic controls. Immunofluorescent study showed myofibroblasts expressing HSP47 were accumulated in the dermis of allogeneic animals, while these cells were hardly detected in the skin of syngeneic controls. Macrophage-depletion with repeated administration of aCSF1R dramatically reduced HSP47+ myofibroblasts, suggesting a critical role of macrophages in differentiation of HSP47+ myofibroblasts. In vitro culture showed VA-Lip HSP47 knocked down HSP47 in mouse skin myofibroblasts, while VA-Lip carrying scrambled siRNA did not alter HSP47 expression (Figure 1). To confirm in vivo distribution of VA-Lip HSP47 specifically to the fibrotic skin lesion, mice with bleomycin-induced localized skin fibrosis were injected with VA-Lip HSP47 labeled with fluorescent dye. We confirmed VA-Lip HSP47 was distributed specifically to fibrotic skin, whereas no VA-Lip HSP47 particle was found in non-fibrotic skin of the same mouse. Next, recipients of allogeneic BMT were treated with VA-Lip HSP47 from day +1 to +41. VA-Lip HSP47 knocked down HSP47 expression in the skin of allogeneic mice, resulting in significantly reduced collagen-deposition and skin thickness (Figure 2). In contrast, VA-Lip HSP47 had no effects on collagen volumes in the skin of naïve mice. Finally, allogeneic recipients were injected with VA-Lip HSP47 thrice a week from day +21 to test if VA-Lip HSP47 could promote regression of established skin fibrosis in chronic GVHD. Skin thickness and collagen volume on day +42 were again reduced in allogeneic mice treated with VA-Lip HSP47 compared to allogeneic controls, indicating that VA-Lip HSP47 is a promising therapeutic agent for established skin fibrosis. Because VA-Lip HSP47 did not affect donor T-cell expansion in the spleen or systemic GVHD scores after allogeneic BMT, VA-Lip HSP47 ameliorated skin fibrosis independently of systemic immunosuppression.Conclusion: VA-Lip HSP47 abrogates HSP47 expression in pathogenic myofibroblasts in the skin and ameliorates skin fibrosis in chronic GVHD without affecting collagen synthesis in the normal skin. VA-Lip HSP47 is a safe and promising anti-fibrotic agent for skin fibrosis in chronic GVHD without inducing systemic immunosuppression. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Does differential HLA-E and -F expression cause an aberrant immune tolerance in preeclampsia and HELLP?

The non-classical HLA-groups (E, F, G), representing Minor Histocompatibility antigens (miHA) are essential for fetal-maternal immune tolerance and normal placental development. Reduced HLA-G expression on extravillous trophoblasts is linked to pregnancy disorders like preeclampsia (PE) due to weak trophoblast invasion in the maternal decidua.

The Value of Online Algorithms to Predict T-Cell Ligands Created by Genetic Variants

Allogeneic stem cell transplantation can be a curative treatment for hematological malignancies. After HLA-matched allogeneic stem cell transplantation, beneficial anti-tumor immunity as well as detrimental side-effects can develop due to donor-derived T-cells recognizing polymorphic peptides that are presented by HLA on patient cells. Polymorphic peptides on patient cells that are recognized by specific T-cells are called minor histocompatibility antigens (MiHA), while the respective peptides in donor cells are allelic variants. MiHA can be identified by reverse strategies in which large sets of peptides are screened for T-cell recognition. In these strategies, selection of peptides by prediction algorithms may be relevant to increase the efficiency of MiHA discovery. We investigated the value of online prediction algorithms for MiHA discovery and determined the in silico characteristics of 68 autosomal HLA class I-restricted MiHA that have been identified as natural ligands by forward strategies in which T-cells from in vivo immune responses after allogeneic stem cell transplantation are used to identify the antigen. Our analysis showed that HLA class I binding was accurately predicted for 87% of MiHA of which a relatively large proportion of peptides had strong binding affinity (56%). Weak binding affinity was also predicted for a considerable number of antigens (31%) and the remaining 13% of MiHA were not predicted as HLA class I binding peptides. Besides prediction for HLA class I binding, none of the other online algorithms significantly contributed to MiHA characterization. Furthermore, we demonstrated that the majority of MiHA do not differ from their allelic variants in in silico characteristics, suggesting that allelic variants can potentially be processed and presented on the cell surface. In conclusion, our analyses revealed the in silico characteristics of 68 HLA class I-restricted MiHA and explored the value of online algorithms to predict T-cell ligands that are created by genetic variants.

P078 The relationship between MiHAs compatibility and HLA matching degree of related donor and recipient pairs in allogeneic hematopoietic stem cell transplantation

Aim This study is aimed at investigating the relationship between minor histocompatibility antigens (MiHAs) compatibility and human leucocyte antigens (HLA) matching degree of related donor and recipient pairs in allogeneic hematopoietic stem cell transplantation (allo–HSCT). Methods The 1116 samples is consisted of 439 leukemia patients and 677 related potential donors including 229 HLA-matched, 245 HLA-semi-matched and 204 HLA-unmatched groups. PCR-SSP technique was used to genotype the 14 autosomal MiHAs coding genes. Typing data were analyzed for allelic, genotypic and phenotypic frequencies of the 14 MiHAs. The disparity rate of MiHAs in HLA-matched, HLA-semi-matched and HLA-unmatched groups were analyzed respectively. Linkage analysis was performed on PECAM-1A (codon 125) – PECAM-1A (codon 563)-PECAM-1A (codon 670) haplotypes. Results The disparity rates of HA-1,HA-2,HA-8,ACC-1, PECAM-1 (codon 125) V,PECAM-1 (codon 563) N,PECAM-1 (codon 670) G,CD62 (codon 206) and CD62 (codon 213) were observed significantly lower in HLA-matched group than those in HLA-semi-matched group ( P P P P P ≅ 1). Conclusion This is the first time to report the positive correlation between MiHAs matching degree and HLA compatibility of donor-recipient pairs, which provided important data to select best suitable donor for a recipient in allo-HSCT. Keywords: allo-HSCT; MiHAs; HLA; GVHD.

Integrated Whole Genome and Transcriptome Analysis Identified a Therapeutic Minor Histocompatibility Antigen in a Splice Variant of ITGB2.

In HLA-matched allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells recognizing minor histocompatibility antigens (MiHAs) can mediate desired antitumor immunity as well as undesired side effects. MiHAs with hematopoiesis-restricted expression are relevant targets to augment antitumor immunity after alloSCT without side effects. To identify therapeutic MiHAs, we analyzed the in vivo immune response in a patient with strong antitumor immunity after alloSCT. T-cell clones recognizing patient, but not donor, hematopoietic cells were selected for MiHA discovery by whole genome association scanning. RNA-sequence data from the GEUVADIS project were analyzed to investigate alternative transcripts, and expression patterns were determined by microarray analysis and qPCR. T-cell reactivity was measured by cytokine release and cytotoxicity. T-cell clones were isolated for two HLA-B*15:01-restricted MiHA. LB-GLE1-1V is encoded by a nonsynonymous SNP in exon 6 of GLE1 For the other MiHAs, an associating SNP in intron 3 of ITGB2 was found, but no SNP disparity was present in the normal gene transcript between patient and donor. RNA-sequence analysis identified an alternative ITGB2 transcript containing part of intron 3. qPCR demonstrated that this transcript is restricted to hematopoietic cells and SNP-positive individuals. In silico translation revealed LB-ITGB2-1 as HLA-B*15:01-binding peptide, which was validated as hematopoietic MiHA by T-cell experiments. Whole genome and transcriptome analysis identified LB-ITGB2-1 as MiHAs encoded by an alternative transcript. Our data support the therapeutic relevance of LB-ITGB2-1 and illustrate the value of RNA-sequence analysis for discovery of immune targets encoded by alternative transcripts. Clin Cancer Res; 22(16); 4185-96. ©2016 AACR.

Role of Toll-Like Receptor Signaling in the Pathogenesis of Graft-versus-Host Diseases.

Graft-versus-host disease (GVHD) and infection are major complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the leading causes of morbidity and mortality in HSCT patients. Recent work has demonstrated that the two complications are interdependent. GVHD occurs when allo-reactive donor T lymphocytes are activated by major histocompatibility antigens or minor histocompatibility antigens on host antigen-presenting cells (APCs), with the eventual attack of recipient tissues or organs. Activation of APCs is important for the priming of GVHD and is mediated by innate immune signaling pathways. Current evidence indicates that intestinal microbes and innate pattern-recognition receptors (PRRs) on host APCs, including both Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), are involved in the pathogenesis of GVHD. Patients undergoing chemotherapy and/or total body irradiation before allo-HSCT are susceptible to aggravated gastrointestinal epithelial cell damage and the subsequent translocation of bacterial components, followed by the release of endogenous dangerous molecules, termed pathogen-associated molecular patterns (PAMPs), which then activate the PRRs on host APCs to trigger local or systemic inflammatory responses that modulate T cell allo-reactivity against host tissues, which is equivalent to GVHD. In other words, infection can, to some extent, accelerate the progression of GVHD. Therefore, the intestinal flora’s PAMPs can interact with TLRs to activate and mature APCs, subsequently activate donor T cells with the release of pro-inflammatory cytokines, and eventually, induce GVHD. In the present article, we summarize the current perspectives on the understanding of different TLR signaling pathways and their involvement in the occurrence of GVHD.

Programming of donor T cells using allogeneic δ-like ligand 4–positive dendritic cells to reduce GVHD in mice

AbstractAlloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)–mismatched and MHC-identical but minor histocompatibility antigen–mismatched mouse models. We established a novel platform that produced δ-like ligand 4–positive dendritic cells (Dll4hiDCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4hiDC stimulation, CD4+ naïve T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4hiDC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4hiDC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4hiDC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4hiDC-induced CD4+ T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4hiDC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.

Incomplete clonal deletion as prerequisite for tissue-specific minor antigen tolerization.

Central clonal deletion has been considered the critical factor responsible for the robust state of tolerance achieved by chimerism-based experimental protocols, but split-tolerance models and the clinical experience are calling this assumption into question. Although clone-size reduction through deletion has been shown to be universally required for achieving allotolerance, it remains undetermined whether it is sufficient by itself. Therapeutic Treg treatment induces chimerism and tolerance in a stringent murine BM transplantation model devoid of myelosuppressive recipient treatment. In contrast to irradiation chimeras, chronic rejection (CR) of skin and heart allografts in Treg chimeras was permanently prevented, even in the absence of complete clonal deletion of donor MHC-reactive T cells. We show that minor histocompatibility antigen mismatches account for CR in irradiation chimeras without global T cell depletion. Furthermore, we show that Treg therapy-induced tolerance prevents CR in a linked suppression-like fashion, which is maintained by active regulatory mechanisms involving recruitment of thymus-derived Tregs to the graft. These data suggest that highly efficient intrathymic and peripheral deletion of donor-reactive T cells for specificities expressed on hematopoietic cells preclude the expansion of donor-specific Tregs and, hence, do not allow for spreading of tolerance to minor specificities that are not expressed by donor BM.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Comprehensive pipeline for bioinformatics prediction of immunogenic minor histocompatibility antigens

Abstract Hematopoietic stem cell transplantation (HSCT) is widely used for treatment of haematological malignancies. Both raft versus tumor (GVT) effect and potentially lethal graft versus host disease (GVHD) may develop after transplantation. Current approach to limit GVHD, is based on donor selection and immunosuppression. But as alloreactivity is not restricted to the HLA locus, up to 40% of fully HLA-matched transplantations are complicated by severe forms of GVHD. Non-synonymous single nucleotide polymorphisms (SNPs) in the genome coding regions can give rise to polymorphic peptides – minor histocompatibility antigens (MiHA) – recognized by the immune system as foreign. Limiting alloreactivity to SNPs in hematopoietic genes could allow to discriminate between GVHD and GVT. Thus information of which polymorphisms are immunogenic is of great importance. So far only a limited number of MiHA have been discovered. Methods for verification of SNP immunogenicity are slow and laborious so there is a need for algorithms predicting potentially immunogenic polymorphisms. We have developed TIE predict (TransImmuno Epitope predictor) which is a comprehensive pipeline for in silico prediction of MiHAs based on exome data of donors and patients. This pipeline utilizes several algorithms for peptide to MHC binding prediction, which increases the overall accuracy of prediction. TIE predict is linked to TransImmuno Epitope database holding the data on common genetic poymophisms genomic sequences and annotations. We have tested this approach on publically available genetic data and currently starting to use it on patients and donors undergoing HSCT in combination with immunogenicity assays.