Single-Cell Analysis of the Classical Hodgkin Lymphoma Immune Environment Reveals a Clonally-Expanded CD8+ T Cell Population with a Cytotoxic Phenotype

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a robust and complex immune cell infiltrate and the rare presence of malignant Hodgkin-Reed-Sternberg (HRS) cells. At the genetic level, HRS cells recurrently acquire alterations that lead to defective antigen presentation (β2 microglobulin mutations) and mediate T cell dysfunction (PD-L1 copy gains/amplifications) in order to subvert host immune surveillance. The clinical relevance of PD-L1 protein over-expression in cHL is clear, as response rates to PD-1 blockade therapy are extremely high among patients with relapsed/refractory (r/r) disease. Despite its remarkable efficacy, the cells that mediate response to anti-PD-1 therapy in cHL remain undefined. Recent analyses have highlighted a possible role for CD4+ T cells in mediating the clinical activity of anti-PD-1 therapy in cHL. CD4+ T cells significantly outnumber CD8+ T cells in cHL lesions and are more frequently juxtaposed to HRS cells in situ. Furthermore, HLA class II expression on HRS cells predicted higher complete response rates to PD-1 blockade therapy in r/r cHL patients. However, a candidate T cell population capable of specific reactivity to antigens expressed by HRS cells has yet to be identified. This information is critical as such T cells might be functionally reinvigorated to mediate HRS cell elimination following PD-1 blockade therapy. In order to address this key knowledge gap, we analyzed data at single cell (sc) resolution using paired RNA and T cell receptor (TCR) sequencing in 9 diagnostic cHL and 5 reactive lymph node (RLN) specimens. Methods: Sequencing was performed using the 10x Genomics Chromium Single Cell 5' Gene Expression and V(D)J workflows. B-cell depletion of each sample was achieved using CD19 microbeads and negative selection to enrich T cell populations. Reads were analyzed and aligned with CellRanger (v3.1.0) and Seurat (v3.2.0) was used to conduct clustering by a shared nearest neighbor (SNN) graph on scRNA data. TCR sequencing data was integrated using scRepertoire (v1.0.0). Results: A detailed map of the immune cell states in cHL was created using scRNA-seq (10X) data on 79,085 cells from 9 cHL (52,602 cells) and 5 RLN samples (26,484 cells) expressing a total of 21,421 genes (mean 5649 cells/sample; mean 2849 mRNA reads/cell). Dimensionality reduction and unsupervised graph-based clustering revealed 21 distinct cell type and activation state clusters, including T cells, NK cells, macrophages, and dendritic cells (Fig 1A-B). A cluster identifying HRS cells was not observed, consistent with a recently published report. Ten T cell clusters were identified (47,573 cells), including naive- and memory-like T cells, effector/cytotoxic CD8+ T cells, regulatory T cells, and T follicular helper cells. Unexpectedly, a putative exhausted T cell cluster was not clearly observed. The relative contributions of cHL and RLNs cases to these clusters are shown in Fig 1C. Paired TCR sequencing was available for 23,943 cells. Overall TCR diversity was lower among cHL samples compared to RLN specimens (Fig 1D). In cHL samples, modest clonal expansion within regulatory T cell and memory CD4+ T cell clusters was observed, but the most striking clonal expansion occurred among cells assigned to effector/cytotoxic CD8+ T cell clusters - a finding not observed in most RLN specimens (Fig 1E). Clonally-expanded effector/cytotoxic CD8+ T cells displayed high expression of granzymes (GZMA, GZMH, GZMK), cytokines (TNF, IFNG) and chemokines (CCL4/CCL5), and modest expression of exhaustion markers (PDCD1, ENTPD1, HAVCR2, ITGAE), contrasting with data from single-cell analyses of solid tumors. Clonal expansion of effector/cytotoxic CD8+ T cells was particularly robust in EBV-positive cHLs, likely due to recognition of viral-derived epitopes displayed on HRS cells (Fig 1F). Phenotypic and functional validation of key immune cell clusters in cHL specimens using spectral cytometry is underway and will be reported at the meeting. Conclusions: For the first time, our data have unveiled the nature of the T cell repertoire in cHL at single cell resolution. Our results reveal a recurrent pattern of clonal expansion within effector CD8+ cells, which may be the HRS antigen-specific T cells that mediate response to PD-1 blockade. This hypothesis requires confirmation through similar analyses of pre- and on-treatment biopsies of cHL patients receiving anti-PD-1 therapy. Disclosures Godfrey: Gilead: Research Funding; Merck: Research Funding; Verastem: Research Funding. Venkataraman:EUSA Pharma: Speakers Bureau. Smith:Janssen: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding. Kline:Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Single-Cell Transcriptional Analysis of Tumour Cells and the Immune Microenvironment during Disease Evolution in a Transgenic Mouse Model of Myeloma

Blood ◽

10.1182/blood-2018-99-118691 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 56-56 ◽

Cited By ~ 1

Author(s):

Danielle C Croucher ◽

Marta Chesi ◽

Zhihua Li ◽

Victoria Marie Garbitt ◽

Meaghen E Sharik ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Immune Cell ◽

Advisory Committees ◽

Disease Evolution ◽

Cell Exhaustion

Abstract Introduction: Multiple Myeloma (MM) is consistently preceded by pre-malignant asymptomatic monoclonal gammopathies (AMG). To date, our understanding of the pathogenesis of progression to MM remains incomplete. Genetic analyses of AMG cells compared to MM-derived plasma cells (PCs) have found few differences, suggesting that progression may be mediated in part by tumour-extrinsic mechanisms. To comprehensively examine the cellular and molecular complexities of MM pathogenesis, we performed an unbiased single cell RNA-sequencing (scRNA-seq) analysis of tumour cells as well as immune cells from the tumour microenvironment (TME) derived from transgenic mice transitioning from AMG to MM. Methods: We employed the Vk*MYC immune-competent mouse model of MM (C57BL/6/KaLwRij), which is a clinically and biologically faithful model of untreated disease that similarly progresses from AMG to MM with age. We established an age-based cohort of Vk*MYC mice to recapitulate a range of MM disease stages, generated single-cell suspensions from flushed bone marrow and subjected these cells to scRNA-seq profiling (10x Genomics). Results: Across 12 samples profiled to date, our scRNA-seq dataset contains 82,853 high-quality cells, expressing 17,922 genes. We employed dimensionality reduction and unsupervised graph-based clustering to visualize and group transcriptionally-similar cell populations, which revealed 42 clusters. Expression of known marker genes and computed correlation scores with bulk gene expression reference datasets enabled annotation of cell types, revealing both malignant cells and non-malignant immune cell populations. We first focused on single cell T/NK profiles in our data given the emerging utility of immune checkpoint inhibitors that target these populations. Although we did not observe numerical differences in the proportion of CD8+ T cells across disease stages, analysis of immune checkpoint receptor genes revealed increased expression of Pdcd1 (PD-1) and Lag3 in CD8+ T cells from mice with disease. Co-expression of LAG3 and PD-1 proteins was also confirmed using a Vk*MYC transplantable model, with a positive correlation between disease burden (%CD138+/B220- cells) and %PD1+LAG3+ CD8+ T cells by flow cytometry. Consistent with reports of PD-1 and LAG3 co-expression on non-functional exhausted T cells, CD8+ T cells from diseased mice demonstrated elevated T cell exhaustion scores in our scRNA-seq dataset. These observations suggest that T cell exhaustion may be mediated by multiple immune checkpoint receptors during disease evolution. Although combinatorial treatment with PD-1 and LAG3 antibodies failed to induce tumour regression in mice with established disease, the addition of cyclophosphamide (Cy) to these antibodies resulted in marked improvement in survival of the mice compared to Cy alone, presumably by promoting immunogenic cell death. Studies exploring the combination of LAG3 and PD-1 antibodies as a strategy to inhibit transition from AMG to MM in the Vk*MYC mice are ongoing and will be reported. We also performed subclustering analysis of 5,228 Sdc1+ (CD138) PCs in our scRNA-seq dataset revealing 11 distinct clusters, with evidence of inter- and intra-tumoural heterogeneity across all Vk*MYC mice. Differential gene expression analysis revealed a non-malignant PC (nPC) cluster as supported by lower Myc transgene and Ccnd2 expression. Moreover, this cluster was predominantly comprised of cells from age-matched control mice or mice with earlier disease. Single-cell chromosomal copy number analysis revealed loss of Chr5 in the majority of tumour cells from MM mice, but not in the nPC cluster. Loss of Chr5 was observed in tumor subclones from all AMG mice suggesting that it is an early and potentially unifying event in Vk*MYC mice during disease progression. Further, the data support the establishment of intratumoural heterogeneity early in disease evolution. Conclusions: Our approach of using scRNA-seq to characterize the pathogenesis of disease evolution in MM has enabled simultaneous measurement of intratumoural heterogeneity and immune cell phenotypes in the TME. In turn, this has provided insights into mechanisms that may contribute to transition from AMG to MM, including induction of T cell exhaustion and loss of mouse Chr5. Ongoing and future work aims to evaluate whether these mechanisms can be exploited therapeutically in pre-malignant AMG. Disclosures Sebag: Amgen Canada: Membership on an entity's Board of Directors or advisory committees; Janssen Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene Canada: Membership on an entity's Board of Directors or advisory committees; Takeda Canada: Membership on an entity's Board of Directors or advisory committees. Pugh:Prosigna: Honoraria; N/A: Patents & Royalties: Hybrid-capture sequencing for determining immune cell clonality; N/A: Patents & Royalties: Combined hybrid-capture DNA sequencing for disease detection; Boehringer Ingelheim: Research Funding; Chrysalis Biomedical Advisors: Honoraria; Merck: Honoraria; DynaCare: Consultancy.

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Mitochondrial DNA Mutations Distinguish Individual Donor- and Recipient-Derived Immune Cells Following Matched Unrelated Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood-2021-145304 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1689-1689

Author(s):

Livius Penter ◽

Jackson Southard ◽

Shuqiang Li ◽

Caleb A. Lareau ◽

Leif S. Ludwig ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Mixed Chimerism ◽

Advisory Committees ◽

Post Transplant ◽

Mtdna Mutations

Abstract Reconstitution of donor hematopoiesis after allogeneic hematopoietic stem cell transplantation (HSCT) forms the basis for effective graft-versus-leukemia responses, but mixed chimerism is not an infrequent outcome. How the donor and host hematopoietic system interact under conditions of mixed chimerism remains incompletely understood. Multi-modal single cell sequencing platforms are increasingly available and provide information regarding cell identities and interactions at high resolution. However, the analysis of post-transplant immune reconstitution requires consistent distinguishing of donor- and recipient-derived cells, which for sparse single cell sequencing data until now has remained a challenge. Recently, mitochondrial DNA (mtDNA) mutations have been recognized for their potential as personal genetic barcodes that can be detected with mitochondrial single cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq). We hypothesized that individual-specific mtDNA mutations could provide a sensitive and robust approach for distinguishing donor- from recipient-derived cells, and therefore tested this approach on bone marrow (BM) samples from patients with relapsed acute myeloid leukemia (AML) post-HSCT. We employed ATAC with select cell surface antigen profiling by sequencing (ASAP-seq), which enables the detection of mtDNA mutations within distinct surface marker-defined cell populations alongside chromatin accessibility. We selected serial samples collected from the ETCTN 10026 study, which tested combined decitabine (days 1-5, every 4 weeks, start cycle 0) and ipilimumab (day 1, every 4 weeks, start cycle 1) in relapsed AML post-HSCT. We focused on 13 samples (study entry, on treatment and disease progression) from 3 patients: AML1012 (HSCT from a matched related donor [MRD]), and AML1010 and AML1026 (matched unrelated donor [MUD]-HSCT). In total, we obtained 33,943 ASAP-seq profiles, including 3,283 single T cells. While clustering using single cell chromatin profiles alone only allowed identification of either CD4 + or CD8 + T cells, integration with surface marker expression enabled more detailed annotations of 8 T cell subpopulations and NK cells. Further, phenotypically distinct subpopulations such as CD57 + CD4 + and CD8 + T cells shared highly similar chromatin profiles, and 11.1% of CD4 + and 33.7% of CD8 + T cells would have been mislabeled based on clustering of chromatin profiles alone. Thus, ASAP-seq identified T cell subsets with markedly improved accuracy and resolution than scATAC-seq alone. Upon evaluation of mtDNA mutations to discriminate donor- and recipient-derived single T cells, we found that this was unreliable for MRD-HSCT (AML1012), but highly robust in the setting of MUD-HSCT (AML1010, AML1026), consistent with maternal inheritance of mitochondrial genomes. For the latter two patients, we identified 48 donor- and 26 recipient-specific mtDNA mutations, all with high heteroplasmy (range 82 - 99%). Presence of donor- and recipient-derived mtDNA mutations was mutually exclusive, and recipient-specific mtDNA mutations were also detectable in AML cells. Clinical bulk and mtDNA mutation-based single T cell chimerisms were highly correlated (r = 0.97). AML1010 had sustained complete T cell chimerism (>97%) during study treatment. In AML1026, the mtDNA mutation-based T cell chimerism rose from 55% to 71% after 1 cycle of decitabine and then remained stable until disease progression 3 months later. This was associated with increased percentage of donor-derived CD4 + T cells (45% [study entry] vs. 71% [after 1 cycle of decitabine], p < 0.01), while donor-derived CD8 + T cells remained unchanged at 76%. Across all studied timepoints in AML1026, donor versus recipient skewing was also highest in CD4 + T cell subsets, with fewer naïve (20% vs. 31%, p < 0.01) but more donor-derived CD57 + CD4 + T cells (13% vs. 3%, p < 0.01). We demonstrate that mtDNA mutations can discriminate between donor- and recipient-derived single cells, enabling detection and in-depth characterization of chimeric immune cell dynamics after MUD HSCT. This approach will allow to systematically dissect conditions of mixed chimerism in the post-transplant setting with larger studies. Disclosures DeAngelo: Abbvie: Research Funding; Takeda: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Incyte: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Sankaran: Cellarity: Consultancy; Forma: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy; Ensoma: Consultancy. Soiffer: Jasper: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Precision Biosciences, USA: Consultancy; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics, USA: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Garcia: Genentech: Research Funding; Prelude: Research Funding; Pfizer: Research Funding; AstraZeneca: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company. OffLabel Disclosure: ipilimumab to modulate anti-leukemia immunity in the post-transplant and transplant-naive context

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Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽

10.1182/blood-2020-138601 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Prajish Iyer ◽

Lu Yang ◽

Zhi-Zhang Yang ◽

Charla R. Secreto ◽

Sutapa Sinha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Gene Signature ◽

T Cell Subsets ◽

Rna Seq ◽

Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

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CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Combinatorial Cytokine Secretion Signature of Donor-Derived T Cells Infused with the Graft: A New Potential Biomarker of Acute Graft-Versus-Host Disease in Αβt-Cell/CD19 B-Cell Depleted Hematopoietic Stem Cell Transplant Recipients

Blood ◽

10.1182/blood-2021-149948 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1684-1684

Author(s):

Raul Montiel-Esparza ◽

Giulia Barbarito ◽

Samantha Peck ◽

Magali Bazzano ◽

Rachana Patil ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cytokine Secretion ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Tnf Α

Abstract Background: Hematopoietic stem cell graft manipulation strategies, such as αβT-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT), address the lack of matched donors and reduce the incidence of severe acute graft-versus-host disease (aGvHD). However, grade II-IV aGvHD still occurs in 25-30% of αβhaplo-HSCT recipients . Studies aimed at understanding the pathogenesis underlying aGVHD in αβhaplo-HSCT are lacking. We hypothesized that αβT cells adoptively transferred with the HSCT (<1x10 5/Kg) have unique combinatorial cytokine secretion signatures that may predict the occurrence of aGvHD. Here we used the IsoPlexis single-cell proteomics for CD4 + and CD8 + T cells to identify those putative signatures . Methods: Six patients with hematologic malignancies receiving fully myeloablative αβhaplo-HSCT at Lucile Packard Children's Hospital, Stanford, between 08/2018 and 05/2020 were enrolled upon signing IRB approved informed consent. Three patients developed grade II-IV aGvHD, while three did not. Aliquot of the graft and of peripheral blood collected at the time of aGvHD onset or at corresponding time points for the patients who did not develop aGvHD, were analyzed. Single sorted CD4 + and CD8 + T cells were profiled by single-cell barcode chip assay from IsoPlexis system (IsoPlexis, Branford, CT) after stimulation with PMA (50 ng/mL) and Ionomycin (1mcg/mL). Following the Human Adaptive Immune Panel, cytokines from CD4 + and CD8 + single T cells were captured by fluorescence ELISA, which measured the numbers of cytokine-producing cells (secretion frequency) and numbers of cytokines produced by individual cells across five functional groups: effector, stimulatory, chemoattractive, regulatory and inflammatory (Table 1). Polyfunctionality was defined as the secretion of 2+ cytokines from each CD4 + and CD8 + T cell. The T cell polyfunctional strength Index (PSI) was defined as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells. Additional statistical analysis was performed using the Student's t test. Results: We compared the combinatorial cytokine secretion signature of individual CD4 + and CD8 + T cells isolated from grafts infused into patients, who eventually did or didn't develop aGvHD. We are comparing the signature of post-HSCT CD4 + and CD8 + T cells isolated from patients who did or did not develop aGvHD. Collectively, we considered three variables: cytokine secretion frequency, numbers of cytokines produced by individual cells and characteristics of the cytokines secreted (functional group) upon stimulation. Single-cell functional heterogeneity evaluated by t-Distributed Stochastic Neighbor Embedding (t-SNE), showed higher CD4 + and CD8 + T-cell polyfunctionality (up to 4+ cytokines) with effector and stimulatory dominant functions in the grafts of patients who developed aGvHD, compared to those who did not develop aGvHD (Fig1). The average PSI (driven by Granzyme B, TNF-α, IFN-γ, MIP-1β, IL2, and IL-8) was found to be higher in both CD4 + and CD8 + T cells from the grafts of patients who developed aGvHD (Fig 2). Combinatorial cytokine secretion analysis showed that T cells from grafts of patients who did not develop aGvHD had unique signatures with CD4 + T cells having the predominant cytokine secretion signature of IL2 and TNF-α, and CD8 + T cells having three predominant cytokine secretion signatures: IL2, IL8, TNF-α; MIP-1β, IL8; and MIP-1β, IFN-γ (Fig3). Conclusions: Preliminary data from αβhaplo-HSCT pediatric recipients obtained using IsoPlexis single-cell functional proteomics for CD4 + and CD8 + T cells showed that an increased donor T-cell polyfunctionality with a Th1 dominant functional phenotype may be predictive of an increased risk of aGvHD, while CD4 + and CD8 + T cells infused into patients who didn't develop aGvHD, had combinations with limited cytokine secretion signatures. Ongoing analysis suggest that polyfunctional CD8 + T cells present in the graft of patients who developed aGvHD, are present at the time of aGvHD initiation, while the polyfunctional CD4 + T cell are not present at the onset of aGvHD. Correlation with ongoing studies on circulating cytokines and clonotypic analysis of αβT cells infused with the graft will be crucial to elucidate the cross talking between the donor's immune system and recipient's inflammatory milieu. Figure 1 Figure 1. Disclosures Parkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

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Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation

Blood ◽

10.1182/blood-2021-152419 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4819-4819

Author(s):

Monzr M. Al Malki ◽

Sumithira Vasu ◽

Dipenkumar Modi ◽

Miguel-Angel Perales ◽

Lucy Y Ghoda ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Phase 1 ◽

Clinical Activity ◽

Advisory Committees ◽

Over Time

Abstract Patients who relapse after allogeneic HCT have a poor prognosis and few effective treatment options. Responses to salvage therapy with donor lymphocyte infusions (DLI) are driven by a graft versus leukemia (GvL) effect. However, relapses and moderate to severe graft versus host disease (GVHD) are common. Therapies that increase the GvL effect without inducing GVHD are needed. The NEXI-001 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunogenic, and antitumor activity of the NEXI-001 antigen specific T-cell product. This product is a donor-derived non-genetically engineered therapy that consists of populations of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, PRAME, and Cyclin A1 antigens. These T cells consist of populations with key memory phenotypes, including stem-like memory, central memory, and effector memory cells, with a low proportion (<5%) of potentially allogeneic-reactive T-naïve cells. Patients enrolled into the first cohort of the dose escalation phase received a single infusion of 50 million (M) to 100M cells of the NEXI-001 product. Bridging anti-AML treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2) that was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-001 product up to 72 hours later (Day1). Lymphocyte recovery to baseline levels occurred as early as three days after the NEXI-001 product infusion with robust CD4 and CD8 T cell reconstitution after LD chemotherapy. NEXI-001 antigen specific T cells were detectable in peripheral blood (PB) by multimer staining and were found to proliferate over time and to traffic to bone marrow. The phenotype composition of detectable antigen specific T cells at both sites was that of the infused product. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-001 product that were not detected in PB of patients tested at baseline. These unique clones subsequently expanded in PB and bone marrow (BM) and persisted over time. Neutrophil recovery, decreased transfusion burden of platelets and red blood cells, and increased donor chimerism were observed. Decreases in myeloblasts and reduction in the size of an extramedullary myeloid sarcoma were suggestive of clinical activity. One patient, a 23-year- old with MRD+ disease at baseline, received two doses of 200M NEXI-001 cells separated by approximately 2 months. Following the first infusion, antigen specific CD8+ T cells increased gradually in PB to 9% of the total CD3+ T cell population just prior to the second infusion and were found to have trafficked to bone marrow. By Day 2 following the second infusion, which was not preceded by LD chemotherapy, the antigen specific CD8+ T cells again increased to 9% of the total CD3+ T cell population in PB and remained at ≥5% until the end of study visit a month later. The absolute lymphocyte count increased by 50% highlighting continued expansion of the NEXI-001 T cells. These cells also maintained significant Tscm populations. Treatment related adverse events, including infusion reactions, GVHD, CRS, and neurotoxicity (ICANS), have not developed in these patients who have received 50M to 200M T cells of the NEXI-001 product either as single or repeat infusions. In conclusion, these results show that infusion of the NEXI-001 product is safe and capable of generating a cell-mediated immune response with early signs of clinical activity. A second infusion is associated with increasing the level of antigen specific CD8+ T cells and their persistence in PB and BM. TCR sequencing and RNA Seq transcriptional profiling of the CD8+ T cells are planned, and these data will be available for presentation during the ASH conference. At least two cycles of 200M NEXI-001 cells weekly x 3 weeks of a 4-week cycle is planned for the next dose-escalation cohort. Early data suggest that the NEXI-001 product has the potential to enhance a GvL effect with minimal GVHD-associated toxicities. Disclosures Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Neximmune: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Modi: MorphoSys: Membership on an entity's Board of Directors or advisory committees; Seagen: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding. Perales: Sellas Life Sciences: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Merck: Honoraria; Takeda: Honoraria; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; MorphoSys: Honoraria; Kite/Gilead: Honoraria, Other; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Medigene: Honoraria; NexImmune: Honoraria; Cidara: Honoraria; Nektar Therapeutics: Honoraria, Other; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment. Knight: Neximmune, Inc: Current Employment. Varela: Kite: Speakers Bureau; Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130197 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3202-3202

Author(s):

Cameron S. Bader ◽

Henry Barreras ◽

Casey O. Lightbourn ◽

Sabrina N. Copsel ◽

Dietlinde Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Cd8 T Cell ◽

Advisory Committees ◽

Hematopoietic Stem

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

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Early Mycosis Fungoides Lesions with Increased CD3-CD4+ Histiocytes Are Associated with Dyslipidemia and Use of Statins

Blood ◽

10.1182/blood.v126.23.4609.4609 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4609-4609

Author(s):

Chee Won Oh ◽

Carlos Torres-Cabala ◽

Mikyoung Chang ◽

Madeleine Duvic

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Skin Lesions ◽

Medication History ◽

Advisory Committees ◽

History Of ◽

Skin Biopsies

Abstract Background The term "histiocyte" includes cells of the monocyte/macrophage series as antigen processing cells and the Langerhans cell/DC series as antigen-presenting cells. At least three DC subsets exist in skin: two expressing either CD1a or CD14 are dermal and Langerhans cells expressing CD1a are epidermal. Since the phenotype of histiocytic cells is typically CD3-CD4+, an estimation of the CD4+ histiocytic population can be made by comparing the numbers of CD3+ T cells with CD4+ cells. Programmed Cell Death 1 (PD-1) is an inhibitory receptor expressed on T cells, B cells, and some myeloid cells. During chronic antigen exposure, expression of PD-1 is sustained. Statins, inhibitors of cholesterol biosynthesis, are immunomodulatory agents acting on T cells and DCs, but their effects on skin immunology are unknown. Objectives To investigate whether infiltrates of CD3-CD4+histiocytes in early mycosis fungoides (MF) lesional skin biopsies are associated with any other factors, including history of medication and to reveal their histopathological pattern. Methods From Jan to Dec 2014, we identified cases of early MF from the clinic in which CD4+ cells exceeded CD3+ cells with biopsies to identify increased histiocytic population. Exclusion criteria included Sézary syndrome, granulomatous MF, T cell receptor beta monoclonality, abnormal T cell populations by flow cytometry, retinoid treatment, and progression of disease after treatment (n=12). Clinical and laboratory findings were retrospectively reviewed. Skin biopsies stained for H&E, CD3, CD4, CD7, and CD8 were reviewed. In 3 cases with paraffin blocks available, immunohistochemical stains for CD68, CD1a, CD163, PD-1, and PD-1 ligand PD-L1 were done. Results Clinical manifestations of early MF were pink scaly patches (9/12), capillaritis (2/12), and annular erythema - like patches (1/12). Eleven also had an increased monocytes in peripheral blood. All cases had a medication history of taking statins (atorvastatin 5/12; simvastatin 2/12; rosuvastatin 1/12) for dyslipidemia (hypercholesterolemia 7/12; both hypercholesterolemia and hypertriglyceridemia 3/12). In 9/12, symptoms persisted after MF treatment. A lichenoid or superficial perivascular lymphohistiocytic infiltration was observed in skin lesions. Focal basal vacuolization was found in all 12 patients. Upper dermal perivascular extravasation of RBCs suggesting vasculopathy was also found in 12/12 cases. All twelve cases showed predominant CD4+ T cells compared to CD8+ T cells in dermis and the CD4+ T cells were more prominent in dermis rather than in epidermis. CD7+ T cells were preserved (3/12) or partially lost (9/12). In all 3 cases, macrophage markers CD68 and CD163 were positive in dermal infiltrates. CD1a+ DCs were increased in both epidermis and dermis in all 3/3. Only one case of three showed PD1/PD-L1+ T cells in dermis. Discussion and Conclusion All our cases had a medication history of statins for dyslipidemia. Of interest, skin biopsies showed a vasculopathy previously reported during high-dose atorvastatin treatment (Tehrani et al, 2013) and infiltration of CD4/CD8+ T cells, CD1a+DCs and CD163/CD68+ macrophages. We hypothesize that statins or dyslipidemia in early MF were associated with cutaneous T cell immune reaction. In support of our hypothesis that dyslipidemia is associated with histiocytosis, we found a report of nine cases of granulomatous pigmented purpuric dermatosis with concurrent hyperlipidemia (Battle et al, 2015). Cholesterol induces monocytosis and M1 macrophages in mice. One study showed that predominant migration of mature CD1a+ DC is associated with release of IL-12p70 and efficient expansion of Th 1 cells and functional CD8+ T cells. On the contrary, IL-10 up-regulates migration of immature CD14+ DC, expression of the M2 macrophage marker CD163, poor expansion of CD4+ and CD8+ T cells, and skewing of Th responses conducive to expression of PD-L1. We cannot know whether skin lesions are secondary to hyperlipidemia or to treatment with statins. Although M1 and M2 macrophages can be distinguished by diverse markers, none of these antigens are suitable for single-marker identification by immunohistochemistry in paraffin embedded tissue blocks. Further study of the cutaneous effect and immunologic mechanisms leading to increased expression of DCs and T cell dysfunction after statin medication is necessary. Disclosures Duvic: Oncoceutics: Research Funding; Therakos: Research Funding, Speakers Bureau; Huya Bioscience Int'l: Consultancy; Tetralogics SHAPE: Research Funding; Innate Pharma: Research Funding; Cell Medica Ltd: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; MiRagen Therapeutics: Consultancy; Soligenics: Research Funding; Allos (spectrum): Research Funding; Array Biopharma: Consultancy; Spatz Foundation: Research Funding; Rhizen Pharma: Research Funding; Eisai: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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NME-2 Protein Functions as a Tumour Associated Antigen in HLA-A2+ Cells and Is Over Expressed in CML Via a Bcr/Abl Dependent Post Transcriptional Mechanism.

Blood ◽

10.1182/blood.v114.22.3266.3266 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3266-3266

Author(s):

Sabine Tschiedel ◽

Melanie Adler ◽

Karoline Schubert ◽

Annette Jilo ◽

Enrica Mueller ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cell Response ◽

Research Funding ◽

Kinase Activity ◽

T Cell Response ◽

Advisory Committees

Abstract Abstract 3266 Poster Board III-1 Introduction: NmE2 (Nm23-H2, NDP kinase B) is one of a family of proteins that catalyze the transfer of gamma-phosphate between nucleoside-triphosphates and diphosphates. The two major family members, NmE1 and NmE2 are strongly implicated in the control of differentiation, proliferation, migration and apoptosis via interactions which are often independent of their kinase activity, NmE2 being a transcriptional activator of the c-myc gene. We recently identified NmE2 as a tumour associated, HLA-A32+ restricted, antigen in a patient with CML and found the protein (but not the mRNA) to be generally over expressed in CML but not in other haematological malignancies. We also detected a specific T-cell response in peripheral blood cells of a patient 5 years after transplantation. This identifies NmE2 as a potential target for both molecular and immunotherapy of CML. However, the development of immunotherapeutic approaches will depend on the ability of NmE2 to function as a tumour antigen in common HLA backgrounds. The aims of this study were firstly to investigate the antigenicity of NmE2 in the HLA-A2 background (which accounts for more than 50% of the Caucasian population), and secondly to characterise the regulatory relationship between Bcr/Abl and NmE2 using a cell line model of CML. Materials and Methods: 5 nonameric NmE2 peptides with predicted anchor amino acids for HLA-A2 were loaded at concentrations of 10μM separately onto HLA-A2 expressing antigen presenting cells. Elispot Assays were carried out with CD8+ MLLCs (for the identification of antigenic peptides) or CD8+ cells isolated directly from a CML patient at different time points after HCT. Ba/F3 cells stably expressing wild type and mutant forms of Bcr/Abl were treated with imatinib and nilotinib (0 – 10 μM) for 48h. Bcr/Abl activity was assessed by FACS using antibodies specific for the phosphorylated forms of CrkL and Stat5. NmE2 and c-Myc protein were detected by immunocytochemistry and Western blotting with specific antibodies [Santa Cruz, clones L-16 and 9E10 respectively]. Levels of nme2 and c-myc mRNA were determined by quantitative real time PCR. Results: Full length NmE2 protein and 2 of 5 HLA-A2 anchor-containing peptides tested (NmE2132–140 and NmE2112–120) were specifically recognized by the HLA-A2+ CD8+ MLLC, demonstrating the antigenicity of NmE2 in the HLA-A2 background in vitro. Furthermore, while CD8+ T-cells from a transplanted HLA-A2+ CML patient showed little or no specific reactivity in the first 10 months after HCT, a distinct reactivity (up to 0.6 % NmE2 reactive CD8+ T cells) became apparent at later stages, consistent with the development of an immune response against NmE2-expressing cells in vivo. The patient remained negative for bcr/abl transcripts throughout this period. BA/F3 Bcr/Abl cells expressed increased levels of NmE2 protein (but not mRNA) compared to the parent BA/F3 line. Interestingly, treatment with imatinib or nilotinib reduced NmE2 protein expression in BA/F3 Bcr/Abl, but not in cells expressing Bcr/Abl mutants resistant to the respective inhibitors. Treatment of BA/F3 Bcr/Abl cells with the PI3K inhibitor Ly294002 resulted in reduced Bcr/Abl activity and a corresponding reduction in both c-Myc and NmE2 protein levels, without affecting mRNA levels. Conclusion: The over expression of NmE2 is closely linked to Bcr/Abl kinase activity, the predominant level of regulation being post-transcriptional and dependent on PI-3K activity. The NmE2 protein is restricted by HLA-A2 as well as by HLA-A32. The development of an NmE2-specific T-cell response in a CML patient after stem cell transplantation suggests that NmE2 functions as a tumour antigen in HLA-A2+ patients in vivo and may be relevant to the long term immune control of CML. NmE2 is therefore a promising candidate for the development of new immunotherapeutic strategies for the treatment of CML. Disclosures: Lange: BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Niederwieser:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

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Characterization of T-Cell Exhaustion in Rapid Progressing Multiple Myeloma Using Cross Center Scrna-Seq Study

Blood ◽

10.1182/blood-2021-153863 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 401-401

Author(s):

William Pilcher ◽

Beena E Thomas ◽

Swati S Bhasin ◽

Reyka G Jayasinghe ◽

Adeeb H Rahman ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Surface Marker ◽

Cell Cluster ◽

Advisory Committees ◽

Surface Marker Expression

Abstract Introduction: Multiple myeloma (MM) is a complex hematological malignancy with the heterogenous immune bone marrow (BM) environment contributing to tumor growth, drug resistance, and immune escape. T-Cells play a critical role in the clearance of malignant plasma cells from the tumor environment. However, T-Cells in multiple myeloma demonstrate impaired cytotoxicity, proliferation, and cytokine production due to the activation of immune inhibitory receptors from ligands produced by the myeloma cells. In this study, we investigate the behavior of T-Cells in MM patients by using single-cell RNA-Seq (scRNA-Seq) to compare the transcriptomic profiles of BM T-Cells of patients with rapid progressing (FP; PFS < 18mo) and non-progressing (NP; PFS > 4yrs) disease. Methods: Newly diagnosed MM patients (n=18) from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) were identified as either rapid progressors or non-progressors based on their progression free survival since diagnosis. To capture transcriptomic data, scRNA-Seq was performed on 48 aliquots of frozen CD138-negative BM cells at three medical centers/universities (Beth Israel Deaconess Medical Center, Boston, Washington University in St. Louis, and Mount Sinai School of Medicine, NYC). Samples were collected at diagnosis prior to treatment. Surface marker expression for 29 proteins was captured for at least one sample per patient using CITE-Seq. After integration and batch correction, clustering was performed to identify cells of T or NK lineage. Uniform Manifold Approximation and Projection (UMAP) and differential expression were used to identify T-Lymphoid subtypes, and differences in NP and FP samples. Results: In this study, single cell transcriptomic profiles were identified for ~102,207 cells from 48 samples of 18 MM patients. 40,328 T (CD3+) and NK (CD3-, NKG7+) cells were isolated, and subclustered for further analysis (Fig 1A). Using differentially expressed markers for each cluster, the T-Lymphoid subset was refined into seven subtypes, consisting of various CD4+ T-Cells, CD8+ T-Cells, and NK cells (Fig 1B). The CD8+ cells were divided into three distinct phenotypes, namely a GZMK-, GZMB- CD8+ T-Cell cluster, a GZMK+ CD8+ Exhausted T-Cell cluster enriched in TIGIT and multiple chemokines (CCL3, CCL4, XCL2), and a GZMB+ NkT cluster enriched in cytolytic markers (PRF1, GNLY, NKG7) (Fig 1C). Differential expression between NP and FP samples in this CD8+ subset showed enrichment of the NkT cytotoxic markers in NP samples, while FP samples were enriched in the CD8+ Exhausted chemokine markers (Fig 1D). Furthermore, the proportion of CD8+ Exhausted T-Cells was enriched in FP samples (p.val < 0.05) (Fig 1E). Exhaustion markers were measured through both RNA and surface marker levels. In RNA, TIGIT was uniquely associated with the FP-enriched CD8+ Exhausted T-Cell cluster, and CD160 was uniquely expressed in FP samples (Fig 1F). CITE-Seq surface marker expression confirms enrichment of both TIGIT and PD1 in the CD8+ Exhausted T-Cell cluster, and along with more exhaustion in FP samples (p.val < 0.01). Conclusion: In this study, we have identified significant differences in T-Cell activity in patients with non-progressing and rapid-progressing multiple myeloma. T-Cells in rapid progressing patients appear to be in a suppressed state, with low cytolytic activity and enriched exhaustion markers. This GZMK+ T-Cell population shows strong similarities with an aging-associated subtype of effector memory T-Cells found to be enriched in older populations (Mogilenko et al, Immunity 54, 2021). These findings will be further validated in an expanded study, consisting both of a larger number of samples, and multiple samples at different timepoints from the same patient. Figure 1 Figure 1. Disclosures Jayasinghe: MMRF: Consultancy; WUGEN: Consultancy. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Kumar: Carsgen: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Roche-Genentech: Consultancy, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

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