512 Terminally exhausted CD8+ T cells potentiate the tolerogenic tumor microenvironment as functional suppressors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A548-A548
Author(s):  
Paolo Vignali ◽  
Kristin DePeaux ◽  
McLane Watson ◽  
Ashley Menk ◽  
Nicole Scharping ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Oxidative Metabolism ◽  
Advanced Melanoma ◽  
Effector T Cells ◽  
Checkpoint Blockade ◽  
Tumor Control ◽  
List Type ◽  
Murine Melanoma

BackgroundBlockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors; yet only in a subset of patients.1–3 Limited success with checkpoint blockade therapy suggests other cell extrinsic or intrinsic mechanisms may be dampening an effective immune response. Cytotoxic CD8+ T cells (CTL) encountering chronic antigen and metabolic restriction can differentiate to a terminally exhausted state (Texh), marked by hyporesponsiveness and metabolic, epigenetic, and transcriptional dysfunction.4–8 While enrichment of this population in tumor is a negative prognostic factor,9–10 it remains unclear whether Texh are simply non-functional or instead possess tolerogenic or suppressive properties. Transcriptional profiling of tumor-infiltrating PD-1int (progenitor exhausted) CTL versus PD-1hiTIM-3+ (terminally exhausted; Texh), reveals that exhausted cells express a pattern of genes associated with immune suppression. We hypothesize that Texh potentiate the suppressive microenvironment of solid tumor by autoregulation and inhibition of local immune responses.MethodsT cell populations were isolated from murine melanoma–B16-F10 or a lab-generated melanoma clone of the spontaneous BREF/PTEN model–by expression of inhibitory receptors and assayed in tandem in microsuppression assays. Murine melanoma clones with inhibited oxidative metabolism were generated by CRISPR-Cas9 deletion and validated for ablated mitochondrial respiration by extracellular flux analysis. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery.ResultsWhen sorted directly from tumor, PD-1hiTim3+ Texh, but not progenitor exhausted PD-1int CTL, induce marked suppression of T cell effector responses, comparable to Foxp3+ Treg from the same environment. Expression of the ectonucleotidase, CD39, is uniquely expressed in Texh and increases as T cells differentiate towards exhaustion. Genetic deletion of CD39 in Texh eliminates the regulatory phenotype of tumor-infiltrating Texh and enforced CD39 expression on effector T cells can inhibit T cell receptor signaling and downstream function. CD39 expression correlates with exposure to hypoxia and Texh sorted from tumors engineered to be less hypoxic displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, contributes to generation of immunosuppressive adenosine, and has been previously associated with terminal exhaustion.11–13ConclusionsOur data support a model that as CTL progress to terminal exhaustion, hypoxic exposure enforces the upregulation of CD39, providing Texh a mechanism to suppress proinflammatory processes. These findings suggest Texh are not solely dysfunctional but rather are deleterious to anti-tumor immunity and may need to be drastically reprogrammed or deleted in order to alleviate immunosuppressive functions.ReferencesWolchok JD. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med 2017; 377, 1345–1356.Hellmann MD, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an open-label, phase 1, multicohort study. Lancet Oncol 2017; 18, 31–41.Robert C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015; 372, 2521–2532.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Blackburn SD, Shin H, Freeman GJ & Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc. Natl. Acad. Sci 2008;105:15016–15021.Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165.Najjar YG, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019; 4.Loo K, et al. Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy. JCI Insight 2017; 2.Daud AI, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J. Clin. Invest 2016;126:3447–3452.. Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun 2018;9:2724.Canale FP, et al. CD39 Expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11, e1005177.

scholarly journals 234 Distinct efficacy and immunological responses to αPD-1, αPD-L1 and αPD-L2 immunotherapy in aged versus young hosts

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A249-A250
Author(s):  
Yilun Deng ◽  
Harshita Gupta ◽  
Myrna Garcia ◽  
Aravind Kancharla ◽  
Ryan Reyes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Checkpoint Blockade ◽  
List Type ◽  
Aged Mice ◽  
Melanoma Model ◽  
Ifn Γ

BackgroundAging is the biggest risk factor for cancer, yet there are limited pre-clinical/clinical data regarding aging effects on immune checkpoint (IC) inhibition (ICI) outcomes. αPD-1 can potentially block PD-L1 and PD-L2 while αPD-L1 can block PD-1 and CD80. Melanoma response to αPD-1/αPD-L1 correlates with CD8+TCF-1+ T cell stem cell (TCSC) generation.1 Lack of host IL-17 can lead to increased IFN-γ production.2 3MethodsWe tested αPD-1 (200 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18–33 months) and young (3–8 months) mice challenged orthotopically with B16 (WT or PD-L1ko) or TPN61R melanoma (NRAS mutation melanoma model)4 (αPD-L2 only) (SQ). Tumors were analyzed by flow. We tested αPD-L2 (20 μg/ml) effects by co-culturing young or aged T cells ± young or aged myeloid cells.ResultsWe reported that αPD-1 treats young and aged with B16 whereas αPD-L1 treats young not aged.5 αPD-L2 treated B16 and TPN61R melanoma in aged but, remarkably, not young, the first single agent anti-cancer immunotherapy exhibiting this property (figure 1). B16 tumors from aged had differential IC content (PD-1, PD-L1, CD80, PD-L2) versus tumors from young (e.g., more PD-L2+ tumor and stroma cells in aged mice; figure 2). Efficacy in young (αPD-1, αPD-L1) and aged (αPD-L2) correlated with increased tumor TCSC content (figure 3). αPD-L2 efficacy against B16 in aged mice required host IFN-γ and IL-17 (figure 4). αPD-1 efficacy against B16 in aged appeared to be host and tumor PD-L1 independent (figure 5). PD-L1KO B16 response to αPD-1 in aged also correlated with increased tumor TCSC content. Myeloid cell PD-L2 signaling inhibited aged but not young CD8+ T cell IL-2 production in vitro (figure 6).Abstract 234 Figure 1Abstract 234 Figure 2Abstract 234 Figure 3Abstract 234 Figure 4Abstract 234 Figure 5Abstract 234 Figure 6ConclusionsTreatment differences in aged versus young could depend on IC, TCSC and/or host cytokine differences (IL-17/IFN-γ). αPD-1 efficacy in aged PD-L1KO mice challenged with PD-L1KO B16 suggests that PD-L2 block is sufficient for αPD-1 efficacy in aged. PD-L2 expression differences in the tumor microenvironment could also contribute to treatment efficacy differences. PD-L2 inhibitory signaling on aged but not young CD8+ T cells is a likely mechanism for αPD-L2 efficacy in aged but not young. We are now testing the role of IL-17 in αPD-L2 efficacy as it could be upstream of IFN-γ effects, and TCSC effects in aged versus young. Our work can improve cancer immunotherapy in aged hosts and provides insights into treatment failure, including in young hosts.AcknowledgementsSouth Texas MSTP training grant (NIH T32GM113896), TL1TR002647, NIH T32AI138944, R01 CA231325, Waxman Grant, UL1 TR001120ReferencesMiller BC, Sen DR, Al Abosy R, Bi K, Virkud YV, LaFleur MW, Yates KB, Lako A, Felt K, Naik GS, et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20(3):326–336.Moroda M, Takamoto M, Iwakura Y, Nakayama J, Aosai F. Interleukin-17A-deficient mice are highly susceptible to toxoplasma gondii infection due to excessively induced T. gondii HSP70 and interferon gamma production. Infection and immunity 2017;85(12):e00399–00317.Yi T, Zhao D, Lin C-L, Zhang C, Chen Y, Todorov I, LeBon T, Kandeel F, Forman S, Zeng D. Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease. Blood, The Journal of the American Society of Hematology 2008;112(5):2101–2110.Burd CE, Liu W, Huynh MV, Waqas MA, Gillahan JE, Clark KS, Fu K, Martin BL, Jeck WR, Souroullas GP. Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma. Cancer discovery 2014;4(12):1418–1429.Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, Svatek RS, Turk MJ, Drerup JM, Li R, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 2018;105:146–154.Ethics ApprovalAll animal studies are approved by UTHSA IACUC.

scholarly journals Reprogramming responsiveness to checkpoint blockade in dysfunctional CD8 T cells

2019 ◽  
Vol 116 (7) ◽  
pp. 2640-2645 ◽  
Author(s):  
Christine E. Nelson ◽  
Lauren J. Mills ◽  
Jennifer L. McCurtain ◽  
Emily A. Thompson ◽  
Davis M. Seelig ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Gene Signature ◽  
Checkpoint Blockade ◽  
Tumor Control ◽  
Transcriptional Signature ◽  
Cell Dysfunction ◽  
T Cell Dysfunction ◽  
Cognate Antigen

Established T cell dysfunction is a barrier to antitumor responses, and checkpoint blockade presumably reverses this. Many patients fail to respond to treatment and/or develop autoimmune adverse events. The underlying reason for T cell responsiveness remains elusive. Here, we show that susceptibility to checkpoint blockade is dependent on the activation status of T cells. Newly activated self-specific CD8 T cells respond to checkpoint blockade and cause autoimmunity, which is mitigated by inhibiting the mechanistic target of rapamycin. However, once tolerance is established, self-specific CD8 T cells display a gene signature comparable to tumor-specific CD8 T cells in a fixed state of dysfunction. Tolerant self-specific CD8 T cells do not respond to single or combinatorial dosing of anti-CTLA4, anti–PD-L1, anti–PD-1, anti–LAG-3, and/or anti–TIM-3. Despite this, T cell responsiveness can be induced by vaccination with cognate antigen, which alters the previously fixed transcriptional signature and increases antigen-sensing machinery. Antigenic reeducation of tolerant T cells synergizes with checkpoint blockade to generate functional CD8 T cells, which eliminate tumors without concomitant autoimmunity and are transcriptionally distinct from classic effector T cells. These data demonstrate that responses to checkpoint blockade are dependent on the activation state of a T cell and show that checkpoint blockade-insensitive CD8 T cells can be induced to respond to checkpoint blockade with robust antigenic stimulation to participate in tumor control.

scholarly journals 679 Tumor hypoxia drives suppressor function in exhausted T cells limiting antitumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A707-A707
Author(s):  
Paolo Vignali ◽  
Kristin DePeaux ◽  
McLane Watson ◽  
Nicole Scharping ◽  
Ashley Menk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Hypoxia ◽  
Cd8 T Cell ◽  
Treg Cells ◽  
Checkpoint Blockade ◽  
Tumor Control ◽  
Cell Exhaustion ◽  
Hypoxia Exposure

BackgroundWhile CD8+ cytotoxic T cells are clearly critical for identification and elimination of cancer cells, factors concentrated within the tumor microenvironment drive altered differentiation of these cells to a hypofunctional, short-lived state termed T cell exhaustion1 (figure 1a). Exhaustion is a progressive lineage, and it is now clear that terminally exhausted T (tTexh) cells are not the targets of checkpoint blockade immunotherapy but may serve as factors that limit immunotherapeutic efficacy.2–6 Compared directly, tumor-infiltrating CD8+ tTexh cells bear notable phenotypic similarity to CD4+Foxp3+ regulatory T (Treg) cells in expression of immunosuppressive molecules suggesting beyond loss of effector function, tTexh cells may be directly anti-functional and constrain tumor-specific immunity. Thus, we hypothesize that tTexh cells potentiate the suppressive microenvironment of solid tumor and that strategies to limit their generation or reprogram their immunosuppressive nature will improve control of tumor progression.MethodsT cell populations were isolated from murine tumor lines, B16-F10 melanoma, Ptenflox/floxBrafLSL.V600ETyr2Cre.ERT2–derived Clone 24 melanoma, MEER head and neck carcinoma, and MC38 adenocarcinoma. T cell-specific CD39 (Entpd1) deletion was accomplished by crossing Entpd1flox/flox mice to Cd4Cre or E8iGFP-Cre-ERT2. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery. Tumor hypoxia was alleviated by CRISPR-Cas9-directed deletion of mitochondrial genes in B16-F10 or by treatment with axitinib or metformin.ResultsWhen sorted directly from tumor, CD8+PD-1hiTim-3+ tTexh cells, but not progenitor PD-1intTim-3– pTexh cells, induce marked suppression of T cell effector responses, comparable to CD4+Foxp3+ Treg cells from the same environment (figure 1b-c). The ectonucleotidase, CD39, increases as cells progressively differentiate and is associated with terminal exhaustion.7 8 CD8+ T cell-restricted deletion of CD39 restricts regulatory functions of tTexh cells (figure 1b), improving tumor control and augmenting response to checkpoint blockade (figure 1d). CD39 expression correlates with hypoxia exposure and tTexh cells sorted from tumors engineered to be less hypoxic or treated with hypoxia-mitigating agents displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, supports adenosine generation, and limits therapeutic efficacy.ConclusionsOur data support a model that as CD8+ T cells progress to terminal exhaustion, hypoxia exposure enforces the upregulation of CD39, providing tTexh cells a mechanism to suppress proinflammatory processes and promote tumor progression. These findings suggest tTexh cells are not solely dysfunctional but rather are deleterious to antitumor immunity and may need to be drastically reprogrammed or depleted to improve patient outcomes.ReferencesBlank CU, et al. Defining “T cell exhaustion”. Nat Rev Immunol 2019;19:665–674.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20:326–336.Blackburn SD, et al. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad Sci USA 2008;105:15016–15021.Sade-Feldman M, et al. Defining T Cell states associated with response to checkpoint immunotherapy in Melanoma. Cell 2018;175:998–1013.e20.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Siddiqui I, et al. Intratumoral Tcf1+PD-1+CD8+ T Cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 2019;50:195–211.e10.Canale FP, et al. CD39 expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11:e1005177.Abstract 679 Figure 1(a) Schematic depicting differentiation of CD8+ T cells to terminal exhaustion in cancer and subsequent suppression of local immune responses by expression of ectonucleosidase, CD39; (b) When assayed directly ex vivo, CD8+ terminally exhausted T (tTexh) cells, but not progenitor exhausted T (pTexh) cells, suppress effector functions as effectively as CD4+Foxp3+ Treg isolated from the same environment. Deletion of CD39 alleviates tTexh-mediated suppression; (c) CD8+ T cell suppression correlates with expression of CD39 on tTexh from various tumor models. (d) CD8+ T cell-specific deletion of CD39 slows tumor growth and improves immune response to checkpoint blockade-resistant tumors. Data are pooled from ≥3 experiments. Statistics are two-way ANOVA with multiple comparisons or Pearson correlation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

773 Adoptive Cell Therapy Response in Melanoma is Mediated by Stem-like CD8 T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A822-A822
Author(s):  
Sri Krishna ◽  
Frank Lowery ◽  
Amy Copeland ◽  
Stephanie Goff ◽  
Grégoire Altan-Bonnet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
High Dimensional ◽  
Tumor Control ◽  
Intrinsic Factors ◽  
Infiltrating Lymphocytes

BackgroundAdoptive T cell therapy (ACT) utilizing ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) can result in complete regression of human cancers.1 Successful immunotherapy is influenced by several tumor-intrinsic factors.2 3 Recently, T cell-intrinsic factors have been associated with immunotherapy response in murine and human studies.4 5 Analyses of tumor-reactive TILs have concluded that anti-tumor neoantigen-specific TILs are enriched in subsets defined by the expression of PD-1 or CD39.6 7 Thus, there is a lack of consensus regarding the tumor-reactive TIL subset that is directly responsible for successful immunotherapies such as ICB and ACT. In this study, we attempted to define the fitness landscape of TIL-enriched infusion products to specifically understand its phenotypic impact on human immunotherapy responses.MethodsWe compared the phenotypic differences that could distinguish bulk ACT infusion products (I.P.) administered to patients who had complete response to therapy (complete responders, CRs, N = 24) from those whose disease progressed following ACT (non-responders, NRs, N = 30) by high dimensional single cell protein and RNA analysis of the I.P. We further analyzed the phenotypic states of anti-tumor neoantigen specific TILs from patient I.P (N = 26) by flow cytometry and single cell transcriptomics.ResultsWe identified two CD8+ TIL populations associated with clinical outcomes: a memory-progenitor CD39-negative stem-like TIL (CD39-CD69-) in the I.P. associated with complete cancer regression (overall survival, P < 0.0001, HR = 0.217, 95% CI 0.101 to 0.463) and TIL persistence, and a terminally differentiated CD39-positive TIL (CD39+CD69+) population associated with poor TIL persistence post-treatment. Although the majority (>65%) of neoantigen-reactive TILs in both responders and non-responders to ACT were found in the differentiated CD39+ state, CR infusion products also contained a pool of CD39- stem-like neoantigen-specific TILs (median = 8.8%) that was lacking in NR infusion products (median = 23.6%, P = 1.86 x 10-5). Tumor-reactive stem-like T cells were capable of self-renewal, expansion, and persistence, and mediated superior anti-tumor response in vivo.ConclusionsOur results support the hypothesis that responders to ACT received infusion products containing a pool of stem-like neoantigen-specific TILs that are able to undergo prolific expansion, give rise to differentiated subsets, and mediate long-term tumor control and T cell persistence, in line with recent murine ICB studies mediated by TCF+ progenitor T cells.4 5 Our data also suggest that TIL subsets mediating ACT-response (stem-like CD39-) might be distinct from TIL subsets enriched for anti-tumor-reactivity (terminally differentiated CD39+) in human TIL.6 7AcknowledgementsWe thank Don White for curating the melanoma patient cohort, and J. Panopoulos (Flowjo) for helpful discussions on high-dimensional analysis, and NCI Surgery Branch members for helpful insights and suggestions. S. Krishna acknowledges funding support from NCI Director’s Innovation Award from the National Cancer Institute.Trial RegistrationNAEthics ApprovalThe study was approved by NCI’s IRB ethics board.ReferencesGoff SL, et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. J Clin Oncol 2016;34:2389–2397.Snyder A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014;371:2189–2199.McGranahan N, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 2019;176:404.Miller BC, et al. Subsets of exhausted CD8 T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Simoni Y, et al. Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Gros A, et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J Clin Invest 2014;124:2246–2259.

scholarly journals Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade

2019 ◽  
Vol 20 (3) ◽  
pp. 326-336 ◽  
Author(s):  
Brian C. Miller ◽  
Debattama R. Sen ◽  
Rose Al Abosy ◽  
Kevin Bi ◽  
Yamini V. Virkud ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Checkpoint Blockade ◽  
Tumor Control

303 Phase II trial of neoadjuvant nivolumab (Nivo) and intra-tumoral (IT) CMP-001 in high-risk resectable melanoma (Neo-C-Nivo): final results

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A330-A330
Author(s):  
Diwakar Davar ◽  
Arivarasan Karunamurthy ◽  
Douglas Hartman ◽  
Richelle DeBlasio ◽  
Joe-Marc Chauvin ◽  
...  
Keyword(s):  
T Cells ◽  
High Risk ◽  
Cd8 T Cells ◽  
Advanced Melanoma ◽  
Pathologic Response ◽  
Type I ◽  
List Type ◽  
Cell Lung Carcinoma ◽  
Radiographic Response ◽  
Pathologic Features

BackgroundNeoadjuvant PD-1 blockade produces major pathological responses (MPR) in ~30% of patients (pts) with high-risk resectable melanoma (MEL) with durable relapse-free benefit, and increased circulating activated CD8+ T cells.1 2 CMP-001 is a type A CpG packaged within a virus-like particle that activates tumor-associated plasmacytoid dendritic cells (pDC) via TLR9 inducing type I interferons and anti-tumor CD8+ T cells. CMP-001/pembrolizumab produces durable anti-tumor responses in PD-1 refractory melanoma.3 We previously reported preliminary evidence of efficacy of neoadjuvant IT CMP/Nivo in high-risk resectable MEL; and herein present final results on 30 evaluable patients.Methods30 pts with stage III B/C/D MEL were enrolled. Pre-operatively, CMP-001 was dosed at 5 mg subcutaneous (SC, 1st), then 10 mg IT (2nd-7th) weekly; Nivo was dosed 240 mg q2 weeks for 3 doses – both agents given for 7 weeks. Post-operatively, Nivo was dosed 480 mg q4 weeks with CMP-001 5 mg q4 weeks SC for 48 weeks. Primary endpoints included major pathologic response rate (MPR), and incidence of dose-limiting toxicities (DLT). Secondary endpoints were radiographic response, relapse-free survival (RFS) and overall survival (OS). Pathological response was scored blinded by pathologists based on residual volume of tumor (RVT) using prior specified cutoffs:4 60% (complete response, pCR); 0%<rvt<rvt50% (non-response, pNR). Radiographic response was assessed using RECIST v1.1. Sequential blood draws and tumor biopsies were collected and analyzed for CD8+ T cell infiltrate (TIL), multiparameter flow cytometry (MFC) and multiplex immunofluorescence (mIF).Results30 pts with regionally advanced MEL were enrolled, of stages IIIB (57%), IIIC (37%), IIID (7%). 29/30 (97%) of pts completed 7 weeks of neoadjuvant Nivo/CMP; while 1 pt had a delay in surgery related to a pre-operative infection unrelated to therapy. No DLTs were reported; grade 3/4 irAE were reported in 3 pts (11%) leading to CMP-001 discontinuation in 2 pts (7%). Radiographic responses were seen in 13 pts (43%), while 9 pts (30%) had stable disease and 8 pts (27%) had progressive disease. Pathological responses (RVT <50%) were seen in 70% of pts: pCR 15 (50%), pMR 3 (10%), 3 pPR (10%); only 9 (30%) had pNR. Pathological responders (pCR/pMR) had increased CD8+ TIL and CD303+ pDC intra-tumorally by mIF; and peripherally activated PD1+/Ki67+ CD8+ T cells by MFC.ConclusionsNeoadjuvant CMP/Nivo has acceptable toxicity and promising efficacy. MPR is 60% in 30 pts. 1-year RFS was 82% (all pts) and 89% (among those with pCR/pMR); median RFS is 9 months (among pNR/pPR) and not reached (among pCR/pMR). Response is associated with evidence of immune activation intra-tumorally and peripherally. IT CMP001 increases clinical efficacy of PD-1 blockade with minimal additional toxicity in pts with regionally advanced MEL. Further study of this combination in high-risk resectable MEL is planned.AcknowledgementsWe thank Dr. Jagjit Singh and the pathology grossing room staff for their assistance and Checkmate Pharmaceuticals for funding and CMP-001.Trial RegistrationClinical trial information: NCT03618641Ethics ApprovalThe study was approved by University of Pittsburgh’s Institutional Review Board, approval number MOD19040237-002.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.ReferencesAmaria RN, Reddy SM, Tawbi HA, et al. Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma. Nat Med 2018. Nov;24(11):1649–1654.Huang AC, Orlowski RJ, Xu X, et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nat Med 2019. Mar;25(3):454–461. doi: 10.1038/s41591-019-0357-y.Milhem M, Gonzales R, Medina T, et al. Abstract CT144: Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14–18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract CT144.Tetzlaff MT, Messina JL, Stein JE, et al. Pathological assessment of resection specimens after neoadjuvant therapy for metastatic melanoma. Ann Oncol 2018. Aug 1;29(8):1861–1868.Cottrell TR, Thompson ED, Forde PM, et al. Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC). Ann Oncol 2018 Aug 1;29(8):1853–1860. doi: 10.1093/annonc/mdy218.Stein JE, Soni A, Danilova L, et al. Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response. Ann Oncol 2019 Apr 1;30(4):589–596. doi: 10.1093/annonc/mdz019.

scholarly journals 622 Disrupted oxygen supply and tumor hyper- oxygen consumption contribute independently to prostate cancer immune privilege

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A652-A652
Author(s):  
Priyamvada Jayaprakash ◽  
Meghan Rice ◽  
Krithikaa Rajkumar Bhanu ◽  
Brittany Morrow ◽  
Joseph Marszalek ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
T Cells ◽  
T Cell ◽  
Oxidative Metabolism ◽  
Complex I ◽  
Tumor Regression ◽  
Tumor Hypoxia ◽  
Checkpoint Blockade ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex

BackgroundDespite the success of immunotherapy in immune-infiltrated ”hot” tumors like melanoma, ”cold” tumors like prostate cancer remain unresponsive [1,2,3]. We find that these tumors harbor regions of hypoxia that act as islands of immune privilege that exclude T cells, while retaining immunosuppressive myeloid cells. Targeting hypoxia using the hypoxia-activated prodrug, TH-302 (Evofosfamide) reduced hypoxic regions and co-operated with immune checkpoint blockade (anti-CTLA-4+anti PD-1) to drive tumor regression in transplantable and spontaneous murine prostate tumors [4]. In a Phase I clinical trial, the combination of Evofosfamide and anti CTLA-4 (Ipilimumab) elicited both objective responses and prolonged disease stabilization in late-stage ”cold” tumor patients. However, Evofosfamide reduces but does not eliminate hypoxia and patient tumors resistant to treatment with Evofosfamide and Ipilimumab were hyper-metabolic [5]. Heightened tumor oxidative metabolism has been shown to generate hypoxic zones that resist PD-1 blockade therapy [6] and treatment with Metformin, a mitochondrial complex I inhibitor may reduce hypoxia and improve responses [7]. We hypothesized that targeting tumor oxidative metabolism using mitochondrial complex I inhibitors might diminish tumor hypoxia and, in conjunction with Evofosfamide, sensitize unresponsive tumors to immunotherapy.MethodsWe investigated the capacity of two mitochondrial complex I inhibitors to reduce tumor oxidative metabolism, diminish myeloid suppressive capacity and improve anti-tumor T cell immunity, alone and in combination with Evofosfamide and checkpoint blockade. We assessed tumor burden and immune composition and characterized metabolic profiles using Seahorse XFe96 analyzer (Agilent).ResultsWhile Evofosfamide or inhibition of oxidative metabolism alone did not significantly impact tumor regression, dual combination and triple combination with checkpoint blockade led to a significant reduction in tumor burden. Assessment of the tumor immune microenvironment identified improvements in CD8 and CD4 effector T cell proliferation. In vitro metabolic and functional profiling of TRAMP-C2 prostate tumors, pre-activated T cells and myeloid derived suppressor cells revealed differential effects of complex I inhibition, with inhibition resulting in reduced tumor proliferation and myeloid suppressive function but increases in proliferation and cytotoxic function of pre-activated T cells.ConclusionsOur findings indicate that tumor hypoxia and associated immune suppressive programming can be reduced through both local tissue remodeling and limitation of tumor oxygen metabolism. Complex I inhibition selectively inhibits tumor and myeloid cell function, while sparing T cells. This provides opportunities to craft synergistic immuno-metabolic therapies with the potential to treat ”cold” tumor patients refractory to current FDA approved immunotherapeutics.ReferencesCurran MA, Montalvo W, Yagita H, and Allison JP. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A. 2010; 107(9): 4275–80.Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013; 369(2): 122–33.Kwon ED, Drake CG, Scher HI, Fizazi K, Bossi A, van den Eertwegh AJ, et al. Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, doubleblind, phase 3 trial. Lancet Oncol. 2014;15(7):700–12.Jayaprakash P, Ai M, Liu A, Budhani P, Bartkowiak T, Sheng J, et al. Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy. J Clin Invest. 2018; 128 (11): 5137–5149.Hegde A, Jayaprakash P, Couillault CA, Piha-Paul S, Karp D, Rodon J, et al. A Phase I Dose-Escalation Study to Evaluate the Safety and Tolerability of Evofosfamide in Combination with Ipilimumab in Advanced Solid Malignancies. Clin Cancer Res. 2021; 27(11): 3050–3060.Najjar YG, Menk AV, Sander C, Rao U, Karunamurthy A, Bhatia R, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019 4(5): e124989. A.Scharping NE, Menk AV, Whetstone RD, Zeng X, Delgoffe GM. Efficacy of PD-1 Blockade Is Potentiated by Metformin-Induced Reduction of Tumor Hypoxia. Cancer Immunol Res. 2017; 5(1):9–16.

scholarly journals 668 Lipid-instructed metabolic rewiring unleash the anti-tumor potential of CD8+ T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A696-A696
Author(s):  
Teresa Manzo ◽  
Carina Nava Lauveson ◽  
Teresa Maria Frasconi ◽  
Silvia Tiberti ◽  
Ignazio Caruana ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd8 T Cells ◽  
Mitochondrial Function ◽  
Cd8 T Cell ◽  
T Cell Differentiation ◽  
Metabolic Reprogramming ◽  
Tumor Control ◽  
Metabolic Fitness

BackgroundAdoptive cell therapy (ACT) harnesses the immune system to recognise tumor cells and carry out an anti-tumor function. However, metabolic constraints imposed by the tumour microenvironment (TME) suppress anti-tumor responses of CTL by reshaping their metabolism and epigenetic landscape. We have recently demonstrated that progressive accumulation of specific long-chain fatty acids (LCFAs) impair mitochondrial function and drives CD8+ T cell dysfunction. In this scenario, maintaining T cells in a less-differentiated state and with high metabolic plasticity during ex vivo T cell production and after infusion may have a strong therapeutic impact. Here, we propose a novel strategy to boost ACT efficacy by implementing T cell long-term functionality, metabolic fitness and preventing exhaustion through lipid-induced mitochondrial rewiring.MethodsWe screen different LCFAs and assess their ability to shape CD8+ T cell differentiation using multi-parametric flow cytometry, proliferation and cytotoxic assays, together with a complete transcriptomic and epigenomic profiling. Metabolic reprogramming of lipid-treated CD8+ T cell was examined by bioenergetic flux measurements paired with metabolomic and lipidomic analysis. Finally, the anti-tumor responses of lipid-instructed CD8 T cells was evaluated in a melanoma mouse model, known to poorly respond to immunotherapy.ResultsLCFAs-treated CD8+ T cells are endowed with highly effector and cytotoxic features but still retaining a memory-like phenotype with decreased PD1 protein levels. Consistently, analysis of the bioenergetic profile and mitochondrial activity has shown that LCFA-instructed CD8+ T cells display a greater mitochondrial fitness. Thus, in vitro LCFA-instructed CD8+ T cells are characterized by higher mitochondrial fitness, potent functionality, memory-like phenotype and PD-1 down-regulation, overall evoking the ideal T cell population associated with a productive anti-tumor response. The therapeutic potential of CD8 T cells lipid-induced metabolic rewiring was further confirmed in vivo. ACT performed with LCFA-reprogrammed CD8 T cells induces higher frequency of memory T cells, which show high polyfunctionality and mitochondrial function, decreased PD1 expression, ultimately resulting in improved tumor control. In addition, LCFA-induced metabolic rewiring during manufacturing of human CAR-redirected T cells, generated a CD8+ T cell memory-like population with higher mitochondrial fitness coupled with a much potent cytotoxic activity.ConclusionsThese results suggest that LCFAs dictate the fate of CD8+ T cell differentiation and could be considered as a molecular switch to fine-tune memory T cell formation and metabolic fitness maintenance, linking lipid metabolism to anti-tumor surveillance. This will be of fundamental importance for a new generation of adoptive T cell-based therapies.Ethics ApprovalThe experiments described were performed in accordance with the European Union Guideline on Animal Experiments and mouse protocols were approved by Italian Ministry of Health and the IEO Committee.

scholarly journals Single-Cell Transcriptome Analysis Reveals RGS1 as a New Marker and Promoting Factor for T-Cell Exhaustion in Multiple Cancers

2021 ◽  
Vol 12 ◽  
Author(s):  
Yunmeng Bai ◽  
Meiling Hu ◽  
Zixi Chen ◽  
Jinfen Wei ◽  
Hongli Du
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Poor Prognosis ◽  
Effector T Cells ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Cell Transcriptome ◽  
Cancer Tissues ◽  
Single Cell Transcriptome

T-cell exhaustion is one of the main reasons of tumor immune escape. Using single-cell transcriptome data of CD8+ T cells in multiple cancers, we identified different cell types, in which Pre_exhaust and exhausted T cells participated in negative regulation of immune system process. By analyzing the coexpression network patterns and differentially expressed genes of Pre_exhaust, exhausted, and effector T cells, we identified 35 genes related to T-cell exhaustion, whose high GSVA scores were associated with significantly poor prognosis in various cancers. In the differentially expressed genes, RGS1 showed the greatest fold change in Pre_exhaust and exhausted cells of three cancers compared with effector T cells, and high expression of RGS1 was also associated with poor prognosis in various cancers. Additionally, RGS1 protein was upregulated significantly in tumor tissues in the immunohistochemistry verification. Furthermore, RGS1 displayed positive correlation with the 35 genes, especially highly correlated with PDCD1, CTLA4, HAVCR2, and TNFRSF9 in CD8+ T cells and cancer tissues, indicating the important roles of RGS1 in CD8+ T-cell exhaustion. Considering the GTP-hydrolysis activity of RGS1 and significantly high mRNA and protein expression in cancer tissues, we speculated that RGS1 potentially mediate the T-cell retention to lead to the persistent antigen stimulation, resulting in T-cell exhaustion. In conclusion, our findings suggest that RGS1 is a new marker and promoting factor for CD8+ T-cell exhaustion and provide theoretical basis for research and immunotherapy of exhausted cells.

Sign in / Sign up

Export Citation Format

Share Document