Integrative Immunogenomic Characterization of Diffuse Large B-Cell Lymphoma (DLBCL) Identifies Four Molecular Subtypes with Distinct Immune Landscapes

Background: Effective treatment of relapsed/refractory (r/r) DLBCL remains a major unmet need. Checkpoint blockade therapy (CBT) leads to durable responses in a small subset of r/r DLBCL patients, but limited understanding of predictive biomarkers and characteristics of host immune responses have slowed development of DLBCL immunotherapy. We previously described a subset of "T-cell inflamed" DLBCLs marked by PD-L1 gene alterations and increased likelihood of response to CBT. In this study, we aimed to identify and group gene expression patterns associated with immune features in a large number of DLBCL cases available in published datasets. We investigate the immunogenomic features of each group and corroborate findings in primary DLBCLs. Methods: Gene sets reflecting a broad array of activation states or subtypes of tumor-infiltrating immune cells were selected from previous studies (n = 143). Expression by case was scored for each set by applying gene set variation analysis (GSVA) to previously published DLBCL bulk RNAseq profiles (n = 1189). The resulting score matrix was reduced with principal component analysis (PCA); the first 10 components were used to hierarchically cluster each case into related groups. Immune cell fractions were estimated from RNAseq counts via deconvolution analysis. Differentially expressed genes (DEG) for each cluster were identified by false discovery rate (FDR) < 0.05 and log2 fold change of > 1.5. The cytolytic gene expression (CYT) score, associated with T-cell immunity against solid tumors, was computed for each case. Protein-coding mutations found in ≥ 5% of cases by whole exome sequencing analysis were filtered for driver mutations (MutSig2CV q-value < 0.1). PD-L1 gene amplification status was determined where copy number array data were available (n = 471). RNAseq (Illumina HiSeq 2000 platform) was performed on 24 fixed and embedded treatment-naive DLBCL tumors from an institutional biorepository. GSVA scores were projected onto the PCA and clusters assigned. CD4+ and CD8+ T-cells per high-power field (HPF) were assessed by immunohistochemistry (IHC). Results: Four clusters of immune gene set expression were identified in existing DLBCL datasets, termed "inflamed", "intermediate-M", "intermediate-T" and "cold". There is no association of any cluster with a difference in overall survival or enrichment in a cell of origin subtype. The inflamed cluster has the highest mean CYT score (Fig 1A, p < 0.001) and highest deconvolution-estimated fraction of CD8+ T-cells, M1 and M2 macrophages, dendritic cells, T-helper 1, and T-helper 2 cells (Fig 1C-E, p < 0.001). Significantly upregulated DEGs include CXCL9, CXCL10, CCL8, and CXCR6, which have been associated with a T-cell inflamed phenotype in solid tumors. Immune escape mechanisms in the inflamed cluster are suggested by upregulated DEGs of VSIG4 and IDO1, and significant enrichment for PD-L1 gene amplifications compared to the cold cluster (Fig 1B, 8.6% vs 1%, p = 0.01). The cold cluster has the lowest mean CYT score (p < 0.001), and lowest deconvolution scores for CD8+ T-cells, M1 and M2 macrophages, and dendritic cells (p < 0.001). The cold cluster harbors more mutations in MYD88 (27%),TMSB4X (11.6%), and FOXO1 (8%) and fewer SOCS1 (7%) mutations than other clusters (FDR < 0.25). Intermediate-T and intermediate-M clusters share mid-range values of estimated CD8+ T cells and CYT scores, but intermediate-M contained more frequent PD-L1 amplifications (Fig 1B, 9% vs 0.9%, p = 0.02), lower estimated Th1 fraction (p < 0.001), and higher estimated total macrophages (p < 0.001) than intermediate-T. Representatives of each cluster were identified in primary DLBCL tumors (n = 24). Mean CD4+ T-cell count per HPF was higher in inflamed cluster DLBCLs compared to cold (45.1 vs 10.1, p = 0.032). A non-significant increase in CD8+ cells was also seen (21.3 vs 14.2 per HPF). Conclusion: In this first comprehensive immunogenomic study of DLBCL, we define differences in host immune response by gene set expression, associate oncogenic mutations with immune exclusion, and discover expression of a number of immune escape genes in inflamed cases. Primary samples analyzed to date support the immune response patterns found by computational analysis. A greater understanding of heterogeneity in host response to DLBCL may help identify subsets of DLBCLs with inherent vulnerability to CBT and other immunotherapies. Disclosures Smith: Portola Pharmaceuticals: Research Funding. Kline:Merck: Honoraria; Merck: Research Funding.

Download Full-text

Single-Cell Profiling Reveals Contribution of Tumor Extrinsic and Intrinsic Factors to BCMA-Targeted CAR-T Cell Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2021-150923 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 326-326

Author(s):

David T. Melnekoff ◽

Yogita Ghodke-Puranik ◽

Oliver Van Oekelen ◽

Adolfo Aleman ◽

Bhaskar Upadhyaya ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Cd8 T Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Poor Responders ◽

Car T Cell ◽

Car T

Abstract Background: BCMA CAR-T cell therapy has shown great promise in relapsed/refractory multiple myeloma (RRMM) patients, even though there is unpredictable variability in the duration and depth of response. The mechanisms behind these divergent outcomes and relapse are not well understood and heterogeneity of MM patients at the level of both tumor genomics and tumor microenvironment (TME) likely contributes to this important knowledge gap. To explore this question, we performed a longitudinal high resolution single cell genomic and proteomic analysis of bone marrow (BM) and peripheral blood (PB) samples in MM patients treated with BCMA CAR-T. Methods: Longitudinal comprehensive immune phenotyping of 3.5 million peripheral blood mononuclear cells (PBMC, CD45+CD66b-) from 11 BCMA CAR-T (idecabtagene vicleucel, ide-cel) patients was achieved via mass cytometry (CyTOF) with a panel of 39 markers. In addition, a total of 45,161 bone marrow mononuclear cells (BMMC) were analyzed from 6 patients before initiation of ide-cel therapy and at relapse by unbiased mRNA profiling via single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Downstream analysis was performed using the CATALYST and Seurat R packages, respectively. Immune cell populations are reported as % of PBMC and CD138- BMMC respectively, unless noted otherwise. Reported p values correspond to non-parametric tests or paired t test where applicable. Results: We compared baseline immune cell populations in the PB and the TME (BM) with regards to depth of CAR-T response. In PB, good responders (≥VGPR) had a higher proportion of CD8+ T cells (37% in good vs 11% in poor responders (<VGPR), p=0.08) and a lower proportion of CD14+ monocytes (30% vs 61%, p=0.28) and NK cells (2% vs 6%, p=0.08). In the TME, a similar trend was confirmed for CD8+ T cells and CD14+ monocytes. (Fig. 1A) Longitudinal analysis of PBMCs revealed phenotypic changes coinciding with CAR-T expansion; CD14+ monocytes declined from week 0 to week 4 after CAR-T infusion (40% vs 13%, p=0.04), while (non-CAR) CD8+ T cells expanded from week 0 to week 4 (32% vs 43%, p=0.15). The non-CAR CD8+ T cell expansion is characterized by differentiation towards a CD8+ effector-memory phenotype (EM, CCR7-CD45RA-) (73% vs 92% of CD8+ T cells, p=0.005). (Fig. 1B) BM samples at CAR-T relapse showed reversal of this shift: CD14+ monocyte levels remain constant or are slightly elevated, while non-CAR CD8+ T cells decrease at relapse. scRNA-seq of BMMC revealed significant gene expression changes between screening and relapse tumor samples, suggesting tumor-intrinsic factors of CAR-T response. For example, when comparing the pre and post tumor samples of a patient with durable response (PFS 652 days), we observed a significant upregulation of gene expression of pro-inflammatory chemokines (CCL3, CCL4), anti-apoptotic genes (MCL-1, FOSB, JUND), and NF-kB signaling genes (NFKBIA) in post tumor. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes showed significant enrichment for TNFA signaling via NF-kB Hallmark Pathway (p.adj = 0.04). We observed similar statistically significant findings between other screening and relapse samples within our cohort, as well as upon comparison of baseline samples of poor vs good responders. (Fig. 1C, D) Thus, our data suggest that anti-apoptotic gene expression could be one of the tumor intrinsic mechanisms of CAR-T therapy resistance. Notably, we did not observe loss of BCMA expression in any tumor samples. Conclusion: Single cell immune profiling and transcriptomic sequencing highlights changes in the PB, TME and within the tumor, which in concert may influence CAR-T efficacy. Our integrated data analysis indicates general immune activation after CAR-T cell infusion that returns to baseline levels at relapse. Specifically, the expansion of non-CAR cytotoxic CD8+ EM T cells provides a rationale for co-administration of IMiDs to enhance CAR-T efficacy. Significant up-regulation of anti-apoptotic genes at baseline in poor responders, and at relapse in good responders, suggest a novel tumor-mediated escape mechanism. Targeting the MCL-1/BCL-2 axis may augment CAR-T efficacy by sensitizing tumor cells and enhancing the effect of CAR-T killing. We will confirm these findings in a longitudinal cohort of BMMC/PBMC CITE-seq patients (n=23) and will present results at the conference. Figure 1 Figure 1. Disclosures Sebra: Sema4: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Download Full-text

Acute Myeloid Leukemia (AML) Blasts Influence the Gene Expression Signature and Co-Signaling Receptor Expression of CD8+ T Cells

Blood ◽

10.1182/blood.v128.22.1700.1700 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1700-1700

Author(s):

Hanna A. Knaus ◽

Sofia Berglund ◽

Hubert Hackl ◽

Raúl Montiel-Esparza ◽

Mark J. Levis ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Induction Chemotherapy ◽

Cd8 T Cells ◽

Research Funding ◽

Receptor Expression ◽

Inhibitory Receptor ◽

Canonical Pathways

Abstract Background: T cell dysfunction in AML remains poorly understood. Our previous studies of AML-associated T cell dysfunction (Knaus, ASH 2015) have focused on expression of multiple inhibitory receptors by T cells in AML patients. Transcriptional signatures, however, remain relatively unexplored, as does the role of Blast/T cell interactions on T cell function. Deciphering those could be crucial for integration of future immunotherapies into clinical practice. Therefore, we aimed to characterize CD8+ T cell gene expression signatures in newly diagnosed AML patients before and after treatment, and to decipher the effects of AML blasts on the expression of co-signaling molecules by CD8+ T cells in co-culture experiments. Methods: Serial peripheral blood (PB) samples (at diagnosis and at the recovery after induction chemotherapy) were collected. To study transcriptional signatures, RNA isolated from FACS-purified PB CD8+ T cells from 6 patients [3 responders (R) and 3 non-responders (NR)] and 4 healthy controls (HC) was analyzed with the Human Prime View Gene Expression Array (Affymetrix). The data were normalized and log transformed. Expression fold change (FC), p values and false discovery rate were determined. Enrichment of canonical pathways was determined using Ingenuity Pathway Analysis (IPA, QIAGEN). To study AML blast-T cell interactions, we FACS-purified T cells and primary AML blasts at diagnosis (n=13) and T cells from HC (n=12). T cells were cultured in vitro for 3 days in the presence or absence of blasts (T cell:blast ratio 1:10) and analyzed by flow cytometry. Results: The transcriptional profile of CD8+ T cells at AML diagnosis significantly differed from that of HC. Genes were selected based on >2 FC between patient and HC, and p< 0.01. We identified a total of 453 dysregulated genes, of which 237 were up- and 216 down-regulated. Upregulated genes included immune inhibitory receptors LILRB1, 2B4, KLRG1, CD160, the transcription factors EOMES, TBET, TIGIT and cytokines (granzyme-A/B/K). In contrast, co-stimulatory receptor genes were downregulated, including CD40LG, CD28, CD30LG and CD28H. Canonical pathways analysis with IPA revealed that the NFAT pathway (involved in T cell differentiation and self-tolerance) was highly upregulated, while co-stimulatory CD28, ICOS and OX40 signaling pathways were downregulated in CD8+ T cells at AML diagnosis. Next, we compared R to NR after induction chemotherapy. There were a total of 351 dysregulated genes; 108/243 genes were up-/down-regulated, respectively. R patients upregulated immune stimulatory receptor genes like ICOS, whereas the top expressed genes for NR patients included the co-inhibitory receptor TIM3; several members of the inhibitory LIR receptor family; LST1 (involved in inhibition of lymphocyte proliferation); TWEAK-APRIL (associated with T cell apoptosis); and CD39 (terminally exhausted CD8+ T cells). In line with these findings, IPA showed that the co-stimulatory ICOS and OX40 signaling pathways were enriched in R patients. In contrast, the NFAT pathway, which had been highly upregulated at diagnosis, remained enriched in NR, but not in R patients. Results were confirmed by qPCR. The culture assay showed that the presence of primary AML blasts significantly reduced the viability of both AML and HC T cells (p <0.005 in both cases). The presence of AML blasts also significantly decreased the frequency of primary AML T cells expressing co-stimulatory receptors 41BB, ICOS and OX40, while it increased the frequency of HC T cells expressing co-inhibitory receptor 2B4 and the senescence/exhaustion marker CD57 compared to their counterparts cultured without blasts. Conclusions: Our study provides insight into the genomic CD8+ T cell signatures of AML patients at diagnosis and following chemotherapy. At diagnosis, T cells overexpressed genes that negatively regulate T cell immune responses, while genes that positively regulate immune responses were downregulated. Interestingly, after induction chemotherapy these changes persisted in NR only. Additionally, a pattern of decreased viability and co-stimulatory receptor expression was seen after in vitro co-culture of T cells with AML blasts, whereas immune inhibitory receptor expression was increased. Our data suggests that the blasts themselves influence the T cell phenotype and genotype in AML patients and that remission is associated with reversion to HC pattern. Disclosures Levis: Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Millennium: Consultancy, Research Funding.

Download Full-text

Immunodominant Cytomegalovirus Epitopes Suppress Subdominant Epitopes in the Generation of High-Avidity CD8 T Cells

Pathogens ◽

10.3390/pathogens10080956 ◽

2021 ◽

Vol 10 (8) ◽

pp. 956

Author(s):

Kirsten Freitag ◽

Sara Hamdan ◽

Matthias J. Reddehase ◽

Rafaela Holtappels

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Escape ◽

Cd8 T Cell ◽

Antigenic Drift ◽

Murine Cytomegalovirus ◽

High Avidity ◽

Infected Cells ◽

Cd8 T Cell Memory

CD8+ T-cell responses to pathogens are directed against infected cells that present pathogen-encoded peptides on MHC class-I molecules. Although natural responses are polyclonal, the spectrum of peptides that qualify for epitopes is remarkably small even for pathogens with high coding capacity. Among those few that are successful at all, a hierarchy exists in the magnitude of the response that they elicit in terms of numbers of CD8+ T cells generated. This led to a classification into immunodominant and non-immunodominant or subordinate epitopes, IDEs and non-IDEs, respectively. IDEs are favored in the design of vaccines and are chosen for CD8+ T-cell immunotherapy. Using murine cytomegalovirus as a model, we provide evidence to conclude that epitope hierarchy reflects competition on the level of antigen recognition. Notably, high-avidity cells specific for non-IDEs were found to expand only when IDEs were deleted. This may be a host’s back-up strategy to avoid viral immune escape through antigenic drift caused by IDE mutations. Importantly, our results are relevant for the design of vaccines based on cytomegaloviruses as vectors to generate high-avidity CD8+ T-cell memory specific for unrelated pathogens or tumors. We propose the deletion of vector-encoded IDEs to avoid the suppression of epitopes of the vaccine target.

Download Full-text

Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor

Nature Medicine ◽

10.1038/nm.1779 ◽

2008 ◽

Vol 14 (12) ◽

pp. 1390-1395 ◽

Cited By ~ 180

Author(s):

Angel Varela-Rohena ◽

Peter E Molloy ◽

Steven M Dunn ◽

Yi Li ◽

Megan M Suhoski ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Immune Escape ◽

Cell Receptor ◽

Hiv 1

Download Full-text

644 Tumor-mediated suppressive signaling and hypoxia supports altered chromatin landscapes that limit the transcriptional and functional potential of terminal T cell exhaustion

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.644 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A673-A673

Author(s):

Rhodes Ford ◽

Natalie Rittenhouse ◽

Nicole Scharping ◽

Paolo Vignali ◽

Greg Delgoffe ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Histone Modifications ◽

Cd8 T Cells ◽

Tumor Hypoxia ◽

T Cell Subsets ◽

T Cell Exhaustion ◽

Cell Exhaustion

BackgroundCD8+ T cells are a fundamental component of the anti-tumor response; however, tumor-infiltrating CD8+ T cells (TIL) are rendered dysfunctional by the tumor microenvironment. CD8+ TIL display an exhausted phenotype with decreased cytokine expression and increased expression of co-inhibitory receptors (IRs), such as PD-1 and Tim-3. The acquisition of IRs mark the progression of dysfunctional TIL from progenitors (PD-1Low) to terminally exhausted (PD-1+Tim-3+). How the chromatin landscape changes during this progression has not been described.MethodsUsing a low-input ChIP-based assay called Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we have profiled the histone modifications at the chromatin of tumor-infiltrating CD8+ T cell subsets to better understand the relationship between the epigenome and the transcriptome as TIL progress towards terminal exhaustion.ResultsWe have identified two epigenetic characteristics unique to terminally exhausted cells. First, we have identified a unique set of genes, characterized by active histone modifications that do not have correlated gene expression. These regions are enriched for AP-1 transcription factor motifs, yet most AP-1 family factors are actively downregulated in terminally exhausted cells, suggesting signals that promote downregulation of AP-1 expression negatively impacts gene expression. We have shown that inducing expression of AP-1 factors with a 41BB agonist correlates with increased expression of these anticorrelated genes. We have also found a substantial increase in the number of genes that exhibit bivalent chromatin marks, defined by the presence of both active (H3K4me3) and repressive (H3K27me3) chromatin modifications that inhibit gene expression. These bivalent genes in terminally exhausted T cells are not associated with plasticity and represent aberrant hypermethylation in response to tumor hypoxia, which is necessary and sufficient to promote downregulation of bivalent genes.ConclusionsOur study defines for the first time the roles of costimulation and the tumor microenvironment in driving epigenetic features of terminally exhausted tumor-infiltrating T cells. These results suggest that terminally exhausted T cells have genes that are primed for expression, given the right signals and are the basis for future work that will elucidate that factors that drive progression towards terminal T cell exhaustion at the epigenetic level and identify novel therapeutic targets to restore effector function of tumor T cells and mediate tumor clearance.

Download Full-text

Serum-derived exosomes promote CD8+ T cells to overexpress PD-1, affecting the prognosis of hypopharyngeal carcinoma

Cancer Cell International ◽

10.1186/s12935-021-02294-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Qian Gao ◽

Hui-Ting Liu ◽

Yu-Qin Xu ◽

Lin Zhang ◽

Yuan-Ru Liu ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Programmed Cell Death ◽

Cd8 T Cells ◽

Poor Prognosis ◽

Cell Function ◽

Immune Escape ◽

Cd8 T Cell ◽

Serum Exosomes

Abstract Background Hypopharyngeal cancer (HPC) is associated with a poor prognosis and a high recurrence rate. Immune escape is one of the reasons for the poor prognosis of malignant tumors. Programmed cell death ligand 1 (PD-L1) and programmed cell death-1 (PD-1) have been shown to play important roles in immune escape. However, the role of PD-1/PD-L1 in HPC remains unclear. In this experiment, we investigated the effect of exosomes from HPC patient serum on CD8+ T cell function and PD-1/PD-L1 expression and, thus, on prognosis. We hope to provide guidance for the identification of new targets for HPC immunotherapy. Methods PD-1 and CD8 expression in 71 HPC tissues and 16 paracarcinoma tissues was detected by immunohistochemistry. Concurrently, the clinicopathological data of the patients were obtained to conduct correlation analysis. Exosomes were isolated from serum and then identified by Western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Flow cytometry was used to assess the activity of CD8+ T cells after exosome stimulation. The effects of exosomes on the ability of CD8+ T cells to kill FaDu cells were assessed by CCK-8 assay. The expression of IL-10 and TGF-β1 was measured by enzyme-linked immunosorbent assay (ELISA). PD-L1 expression in HPC tissue samples was evaluated by immunohistochemistry, and the relationship between PD-1/PD-L1 expression and prognosis was investigated with patient specimens. Results PD-1 expression was significantly upregulated on CD8+ T cells in tumor tissues compared with those in normal tissues. The overall survival (OS) and disease-free survival (DFS) of PD-1-overexpressing patients were decreased. Serum exosomes from patients can elevate PD-1 expression on CD8+ T cells and suppress their killing capacity and secretory function. The rate of positive PD-L1 expression was increased in HPC tissues compared with paracancerous tissues. The DFS and OS of the PD-1(+)-PD-L1(+) group were significantly lower than those of the PD-1(−)-PD-L1(−) group. Conclusion Our findings indicate that serum exosomes from HPC patients can inhibit CD8+ T cell function and that the PD-1-PD-L1 pathway plays an important role in the immune escape of HPC. Exosomes combined with immunotherapy may guide the treatment of patients with advanced disease in the future.

Download Full-text

677 Reverse abscopal effect: intertumoral heterogeneity suppresses systemic CD8 T cell-mediated antitumor immunity and confers PD-1 inhibitor resistance in synchronous melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.677 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A705-A705

Author(s):

Shuyang Qin ◽

Booyeon Han ◽

Alexander Chacon ◽

Alexa Melucci ◽

Alyssa Williams ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Cd8 T Cells ◽

Antitumor Immunity ◽

Immune Escape ◽

Cd8 T Cell ◽

Abscopal Effect ◽

Immunogenic Tumor ◽

Ifn Γ

BackgroundDespite recent advancements in systemic therapy, only a minority of metastatic patients develop meaningful clinical responses to immune checkpoint inhibitors. Inherent genetic instability of melanoma generates genomically and microenvironmentally distinct metastases. These different tumor microenvironments (TMEs) contain numerous T cell suppression mechanisms, such as upregulation of the PD-1/PD-L1 exhaustion pathway. However, as synchronous metastases share one host immune system, intertumoral heterogeneity may result in increasing cross-talk between metastases that impairs systemic antitumor immunity and promotes PD-1 immunotherapy resistance.MethodsYUMM 1.7 (less immunogenic) and YUMMER 1.7 (more immunogenic cell line derived from YUMM following UVB irradiation) melanoma cell lines were simultaneously injected into opposite flanks of the same mice as a model of synchronous melanoma. We assessed tumor growth in wildtype, interferon-gamma (IFN-γ) knockout, and CD8-depleted mice as well as in response to PD-1 inhibitor. We characterized the TME with flow cytometry and performed TCR sequencing on tumor-infiltrating CD8 T cells.ResultsDistinct TMEs were observed for YUMM and YUMMER tumors simultaneously grown in the same mouse. The presence of the less immunogenic YUMM tumor allows the more immunogenic YUMMER tumors to escape IFN-γ and CD8 T cell-mediated rejection, despite abundant tumor-infiltrating, clonally expanded CD8 T cells. Identical immunodominant CD8 T cell clones were found in both YUMM and YUMMER tumors within the same mouse. Synchronous YUMMER-infiltrating CD8 T cells exhibit suppressed phenotypes, including increased persistence of surface PD-1 and decreased surface CD107a expressions. Simultaneously, these synchronous YUMMER tumors additionally upregulate macrophage surface PD-L1 expression, which potentially contributes to tumor immune escape. Lastly, synchronous YUMMER tumors become resistant to PD-1 inhibition, in direct contrast to control YUMMER tumors.ConclusionsIn a host with multiple melanoma lesions, immunogenicity of all tumors contribute to the systemic antitumor immune response. We show that two synchronous tumors with synonymous mutations (<40%), as is the case with metastatic patients, lead to skewed CD8 T cell expansion of the same clones in both tumors. The presence of a less immunogenic tumor prevents CD8 and IFN-γ mediated rejection of the more immunogenic tumor. Furthermore, CD8 T cells in the more immunogenic tumor exhibit decreased effector function and increased resistance to PD-1 blockade, as tumor-infiltrating macrophages concurrently become more immunosuppressive. These results are highly suggestive of a “reverse abscopal effect,” by which immunologically “cold” tumors generate systemic immunosuppression that facilitate PD-1 immunotherapy resistance and immune escape of all other tumors in synchronous metastatic melanoma patients.AcknowledgementsWe would like to thank Dr. Marcus Bosenberg from the Department of Dermatology at Yale University for kindly gifting us with the YUMMER 1.7 murine melanoma cell line.Ethics ApprovalAnimal experiments were approved by the University Committee on Animal Resources and performed in accordance with University of Rochester approved guidelines.

Download Full-text

Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.815.815 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 815-815

Author(s):

Farhad Ravandi ◽

Naval Daver ◽

Guillermo Garcia-Manero ◽

Christopher B Benton ◽

Philip A Thompson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

T Cell Subsets ◽

Newly Diagnosed ◽

Effector Cells ◽

T Effector Cells ◽

Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p<0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

Download Full-text

CD4 T cell-intrinsic role for the T helper 17 signature cytokine IL-17: Effector resistance to immune suppression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005010117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19408-19414 ◽

Cited By ~ 1

Author(s):

Michael P. Crawford ◽

Sushmita Sinha ◽

Pranav S. Renavikar ◽

Nicholas Borcherding ◽

Nitin J. Karandikar

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Suppression ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

T Helper ◽

T Helper 17 ◽

Immune Resistance ◽

Inflammatory Bowel

Untoward effector CD4+ T cell responses are kept in check by immune regulatory mechanisms mediated by CD4+ and CD8+ T cells. CD4+ T helper 17 (Th17) cells, characterized by IL-17 production, play important roles in the pathogenesis of autoimmune diseases (such as arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, among others) and in the host response to infection and cancer. Here, we demonstrate that human CD4+ T cells cells exposed to a Th17-differentiating milieu are significantly more resistant to immune suppression by CD8+ T cells compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F, and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T cells themselves, but not through their action on CD8+ T cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T cells to induce suppressive resistance, and this resistance can be reversed by blockade of IL-1β, IL-6, or STAT3. These studies reveal a role for IL-17 cytokines in mediating CD4-intrinsic immune resistance. The pathways induced in this process may serve as a critical target for future investigation and immunotherapeutic intervention.

Download Full-text

Inhibitory Effects of Lactic Acid on Human Antigen-Specific CD8+ T-Cells.

Blood ◽

10.1182/blood.v104.11.3844.3844 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3844-3844

Author(s):

Marina Kreutz ◽

Karin Fischer ◽

Petra Hoffmann ◽

Simon Volkl ◽

Matthias Edinger ◽

...

Keyword(s):

T Cells ◽

Lactic Acid ◽

T Cell ◽

Cd8 T Cells ◽

Acid Production ◽

Cell Function ◽

Immune Escape ◽

Lactic Acid Production ◽

Target Cells ◽

Thymidine Uptake

Abstract A characteristic feature of inflammatory lesions or tumor sites is local acidosis, which is attributed to the local increase in lactic acid production. We studied the effect of such an acidic environment on the immune functions of antigen-specific CD8+ T-cells by incubating the cells in the presence of various concentrations of lactic acid for up to 48h. CD8+ T-cells were isolated from healthy donors and expanded by weekly stimulation with autologous dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. The obtained T cell population consisted of at least 90% CD8+ and about 60% Melan-A specific T cells, as determined by Melan-A multimer staining. Incubation of CD8+ T cells with up to 20mM lactic acid for 24h did not cause T-cell apoptosis or cell death as determined by combined annexin/propidium iodide staining. However, the proliferative capacity of CD8+ T cells, as determined by 3H-thymidine uptake, was strongly inhibited. Similar results were obtained when we determined cytokine production and cytotoxic activity of the cells after a 24h culture period in 5-20 mM lactic acid. Production of both, IL-2 and IFN-gamma was strongly diminished in comparison to untreated cells, as determined by intracellular staining after stimulation with PMA/ionomycin for 5h in the presence of monensin. Analysis of the antigen-specific cytolytic capacity revealed that CD8+ T cells pre-cultured with lactic acid were less effective in killing antigen-loaded T2 target cells as compared to untreated CD8+ T cells. In parallel, the intracellular contents of the cytotoxic effector molecules granzyme-B and perforin was diminished. Re-adjusting the pH of the medium to a physiological value of pH7.4 could partially revert the effect of lactic acid. Treatment of CD8+ T cells with sodium lactate instead of lactic acid had no inhibitory effect. We conclude, that lactic acid is an important modulator of CD8+ T-cell function and may contribute, together with other factors, to immune escape mechanisms in the tumor environment.

Download Full-text