Intratumoral T-cell Research Articles

Impact of Metabolic Activity in Hepatocellular Carcinoma: Association With Immune Status and Vascular Formation.

We evaluated the prognostic value of fluorine‐18 fluorodeoxyglucose (18F‐FDG) positron emission tomography/computed tomography (PET/CT) in hepatocellular carcinoma (HCC). Their association with programmed death ligand 1 (PD‐L1) expression and vascular formation was further investigated. In this retrospective study, using a database of 418 patients who had undergone 18F‐FDG PET/CT before hepatic resection for HCC, immunohistochemical staining of PD‐L1, clusters of differentiation (CD) 8, CD68, and CD34 was performed. Patients with a high maximum standardized uptake value (SUVmax) on 18F‐FDG PET/CT showed a significantly worse recurrence‐free survival (RFS) (hazard ratio [HR]: 1.500; 95% confidence interval [CI]: 1.088‐2.069; P = 0.0133) and overall survival (OS) (HR: 2.259; 95% CI: 1.276‐4.000; P = 0.0052) than patients with a low SUVmax. Logistic regression analysis showed that a high SUVmax in HCC was significantly associated with PD‐L1‐positive expression (odds ratio: 4.407; 95% CI: 2.265‐8.575; P < 0.0001). SUVmax values of HCC were associated with intratumoral CD8‐positive T‐cell counts (P = 0.0044) and CD68‐positive macrophage counts (P = 0.0061). Stratification based on SUVmax, PD‐L1 expression, and the vessels that encapsulate tumor clusters (VETC) status was also significantly associated with RFS and OS. SUVmax, VETC, and PDL1 expression were independently predictive of survival on multivariable analysis. Conclusion: Our large cohort study showed that a high SUVmax on 18F‐FDG PET/CT is associated with a poor clinical outcome and PD‐L1 expression in patients with HCC. Additionally, stratification of patients based on the combination of SUVmax, PD‐L1 expression, and the VETC status predicts poor clinical outcome.

Durable Suppression of Acquired MEK Inhibitor Resistance in Cancer by Sequestering MEK from ERK and Promoting Antitumor T-cell Immunity.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF non-V600, and BRAF V600 mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF V600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521.

Digital Immunophenotyping Predicts Disease Free and Overall Survival in Early Stage Melanoma Patients.

Background: the prognostic significance of tumor infiltrating lymphocytes (TILs) in intermediate/thick primary cutaneous melanoma (PCM) remains controversial, partially because conventional evaluation is not reliable, due to inter-observer variability and diverse scoring methods. We aimed to assess the prognostic impact of the density and spatial distribution of immune cells in early stage intermediate/thick PCM. Materials and Methods: digital image acquisition and quantitative analysis of tissue immune biomarkers (CD3, CD4, CD8, CD68, PD-L1, CD163, FOX-P3, and PD-1) was carried out in a training cohort, which included patients with primary PCM ≥ 2 mm diagnosed, treated, and followed-up prospectively in three Italian centers. Results were validated in an independent Italian cohort. Results: in the training cohort, 100 Stage II–III melanoma patients were valuable. At multivariable analysis, a longer disease free survival (DFS) was statistically associated with higher levels of CD4+ intratumoral T-cells (aHR [100 cell/mm2 increase] 0.98, 95%CI 0.95–1.00, p = 0.041) and CD163+ inner peritumoral (aHR [high vs. low] 0.56, 95%CI 0.32–0.99, p = 0.047). A statistically significant longer DFS (aHR [high-high vs. low-low] 0.52, 95%CI 0.28–0.99, p = 0.047) and overall survival (OS) (aHR [high-high vs. low-low] 0.39, 95%CI 0.18–0.85, p = 0.018) was found in patients with a high density of both intratumoral CD8+ T-cells and CD68+ macrophages as compared to those with low density of both intratumoral CD8+ T-cells and CD68+ macrophages. Consistently, in the validation cohort, patients with high density of both intratumoral CD8+ and CD3+ T-cells were associated to a statistically better DFS (aHR[high-high vs. low-low] 0.24, 95%CI 0.10–0.56, p < 0.001) and those with high density of both intratumoral CD8+ and CD68+ were associated to a statistically longer OS (aHR[high-high vs. low-low] 0.28, 95%CI 0.09–0.86, p = 0.025). Conclusion: our findings suggest that a specific preexisting profile of T cells and macrophages distribution in melanomas may predict the risk of recurrence and death with potential implications for the stratification of stage II–III melanoma patients.

Abstract OT-32-02: Irene study: Phase 2 study of incmga00012 (retifanlimab)and the oncolytic virus pelareorep in metastatic triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive subtype accounting for 15% of all breast cancer cases. It is characterized by larger tumor size, higher grade, early peak of recurrence, and a worse 5-year overall survival rate compared to other breast cancer subtypes. Chemotherapy serves as the backbone for the treatment of metastatic TNBC. Treatment with immunotherapy in combination with Abraxane, a taxane-based chemotherapy, is of benefit only in PD-L1 positive tumors, which represents a minority of the patients. Pelareorep, a proprietary isolate of the unmodified, replication competent reovirus type 3 Dearing (T3D), has been shown to upregulate PD-L1 expression in tumor and inflammatory cells and downregulate intra-tumoral regulatory T-cells in the tumor microenvironment in pre-clinical and early clinical studies. Retifanlimab is a PD-1 inhibitor currently in development. The rationale for this clinical study is that the administration of pelareorep will prime the tumor microenvironment for enhanced tumor response to PD-1 inhibitor retifanlimab.Trial design: This is a phase II multi-site single-arm clinical trial to study the combination of PD-1 inhibitor retifanlimab and the oncolytic virus Pelareorep in metastatic triple negative breast cancer who have progressed on chemotherapy. Eligible patients will receive pelareorep 4.5x1010 TCID50 /day IV, on Days 1, 2, 15 and 16 and retifanlimab 500mg IV on day 3 of every 28-day cycle until disease progression or unacceptable toxicity. Patient will be monitored clinically and radiologically for response to treatment. Tumor tissue, stool and blood samples will be collected while on treatment to evaluate changes in PD-L1 expression, gut microbiome and inflammatory cells induced by the study drugs. (ClinicalTrials.gov Identifier: NCT04445844) Eligibility criteria: Eligible patients will include premenopausal/postmenopausal women with metastatic TNBC who have previously received 1-2 prior lines of chemotherapy in the metastatic setting. ECOG PFS 0-2. Specific aims: Primary endpoint will be objective response rate (ORR) and safety, determined by the number, frequency, duration, and severity of AEs using CTCAE v5.0. The secondary end-points will be progression free survival (PFS), overall survival (OS) and duration of response (DOR) and quality of life measures using EORTC QLQ-C30. Statistical methods: Simon’s optimal 2-stage design will be used to calculate sample size. In the first stage, 14 patients will be accrued. If there are 1 or fewer responses in these 14 patients, the study will be stopped. Otherwise, 11 additional patients will be accrued for a total of 25. The null hypothesis will be rejected if 4 or more responses are observed in 25 patients. The first 6 patients will be enrolled in a staggering interval for the safety run-in phase of the study. Accrual: The study will enroll up to 25 patients at Rutgers Cancer Institute of New Jersey and Ohio State University Comprehensive Cancer Center Contact information: Mridula George, MD Email: mridula@cinj.rutgers.edu Citation Format: Mridula George, Nicole Williams, Maryam Lustberg, Coral Omene, Nancy Chan, Nisha Ohri, Maria Kowzun, Lindsay Potdevin, Firas Eladoumikdachi, Shicha Kumar, Robert Wesolowski, Grey Wilkinson, Danielle Tang, Sinae Kim, Shridar Ganesan, Bruce Haffty, Deborah Toppmeyer. Irene study: Phase 2 study of incmga00012 (retifanlimab)and the oncolytic virus pelareorep in metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-32-02.

Cancer-specific type-I interferon receptor signaling promotes cancer stemness and effector CD8+ T-cell exhaustion

ABSTRACT Type-I interferon (IFN-I) signaling is critical to maintaining antigen-presenting cell function for anti-tumor immunity. However, recent studies have suggested that IFN-I signaling may also contribute to more aggressive phenotypes, raising the possibility that IFN-I downstream signaling in cancer and myeloid cells may exert dichotomous functions.We analyzed the clinicopathologic correlation of cancer-specific IFN-I activation in 195 head and neck squamous cell carcinoma patients. We also characterized the immune impact of IFN-I receptor (IFNAR1)-deficiency in syngeneic tumor models using biochemistry, flow cytometry, and single-cell RNA-Seq. We stained HNSCC tissue microarrays with a sensitive IFN-I downstream signaling activation marker, MX1, and quantitated cancer cell-specific MX1 staining. Kaplan-Meier analysis revealed that MX1-high tumors exhibited worse survival, a phenotype that depends on the number of CD8+ intratumoral T-cells. We found that cancer-specific IFNAR1 engagement promotes cancer stemness and higher expression levels of suppressive immune checkpoint receptor ligands in cancer-derived exosomes. Notably, mice bearing Ifnar1-deficient tumors exhibited lower tumor burden, increased T-cell infiltration, reduced exhausted CD4+PD1high T-cells, and increased effector population CD8+IFN-γ+ T-cells. Then, we performed single-cell RNA-sequencing and discovered that cancer-specific IFN-I signaling not only restricts effector cells expansion but also dampens their functional fitness.The beneficial role of IFN-I activation is largely dependent on the myeloid compartment. Cancer-specific IFN-I receptor engagement promotes cancer stemness and the release of cancer-derived exosomes with high expression levels of immune checkpoint receptor ligands. Cancer-specific IFN-I activation is associated with poor immunogenicity and worse clinical outcomes in HNSCC.

Tumor cell-derived IL-10 promotes cell-autonomous growth and immune escape in diffuse large B-cell lymphoma

ABSTRACT Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy arising from germinal center or post-germinal center B-cells that retain many of the properties of normal B-cells. Here we show that a subset of DLBCL express the cytokine IL-10 and its receptor. The genetic ablation of IL-10 receptor signaling abrogates the autocrine STAT3 phosphorylation triggered by tumor cell-intrinsic IL-10 expression and impairs growth of DLBCL cell lines in subcutaneous and orthotopic xenotransplantation models. Furthermore, we demonstrate using an immunocompetent Myc-driven model of DLBCL that neutralization of IL-10 signaling reduces tumor growth, which can be attributed to reduced Treg infiltration, stronger intratumoral effector T-cell responses, and restored tumor-specific MHCII expression. The effects of IL-10R neutralization were phenocopied by the genetic ablation of IL-10 signaling in the Treg compartment and could be reversed by MHCII blockade. The BTK inhibitor ibrutinib effectively blocked tumor cell-intrinsic IL-10 expression and tumor growth in this Myc-driven model. Tumors from patients with high IL-10RA expression are infiltrated by higher numbers of Tregs than IL-10RAlow patients. Finally, we show in 16 cases of DLBCL derived from transplant patients on immunosuppressive therapy that IL-10RA expression is less common in this cohort, and Treg infiltration is not observed.

Intratumoral T-cell receptor repertoire is predictive of interim PET scan results in patients with diffuse large B-cell lymphoma treated with rituximab/cyclophosphamide/doxorubicin/prednisolone/vincristine (R-CHOP) chemoimmunotherapy.

ObjectivesA diverse intratumoral T‐cell receptor (TCR) repertoire is associated with improved survival in diffuse large B‐cell lymphoma (DLBCL) treated with rituximab/cyclophosphamide/doxorubicin/prednisolone/vincristine (R‐CHOP) chemoimmunotherapy. We explored the impact of intratumoral TCR repertoire on interim PET (iPET) done after four cycles of R‐CHOP, the relationships between intratumoral and circulating repertoire, and the phenotypes of expanded clonotypes.MethodsWe sequenced the third complementarity‐determining region of TCRβ in tumor samples, blood at pre‐therapy and after four cycles of R‐CHOP in 35 patients enrolled in ALLGNHL21 trial in high‐risk DLBCL. We correlated the TCR diversity metrics with iPET status, gene expression profiles and HLA‐class I genotypes. We then sequenced the FACS‐sorted peripheral blood T cells in six patients, and pentamer‐sorted EBV‐specific CD8+ T cells in one patient from this cohort.ResultsCompared with iPET− patients, the intratumoral TCR repertoire in iPET+ patients was characterised by higher cumulative frequency of abundant clonotypes and higher productive clonality. There was a variable overlap between circulating and intratumoral repertoires, with the dominant intratumoral clonotypes more likely to be detected in the blood. The majority of shared clonotypes were CD8+ PD‐1HI T cells, and CD8+ T cells had the largest clonal expansions in tumor and blood. In a patient with EBV+ DLBCL, EBV‐specific intratumoral clonotypes were trackable in the blood.ConclusionThis study demonstrates that clonally expanded intratumoral TCR repertoires are associated with iPET+ and that the blood can be used to track tumor‐associated antigen‐specific clonotypes. These findings assist the rationale design and therapeutic monitoring of immunotherapeutic strategies in DLBCL.

T-cell receptor repertoire analysis reveals positive correlation between intra-tumoral clonotype diversity and overall survival in patients with pancreatic ductal adenocarcinoma

Presenter: Vikram Pothuri BA | Washington University, St. Louis Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune therapy. Nevertheless, it is well established that intra-tumoral T-cell infiltration correlates with improved overall survival (OS). This highlights the importance of characterizing the global T-cell receptor (TCR) repertoire. We hypothesized that a more clonal TCR repertoire, suggestive of a primed tumor-specific T-cell response, would correlate with increased survival. Methods: Our study included 162 patients diagnosed with PDAC that underwent surgical resection between 2011-2019. Matched peripheral blood mononuclear cells (PBMC) and tumor tissue were banked from 123 patients and PBMC alone from 39 patients. TCR CDR3 regions were sequenced, enabling identification and quantification of both intra-tumoral and circulating T-cell clonotypes. We analyzed two metrics of TCR repertoire diversity: richness (number of unique clonotypes) and evenness (distribution of clonotypes). Results: In 114 matched samples, TCR repertoires in PBMC were significantly more diverse (Wilcoxon p < 0.003) than in tumor. When controlling for age, stage (I vs. II vs. III), neoadjuvant treatment (none vs. chemotherapy vs. chemotherapy plus radiation), and diversity in PBMC, fraction T-cells of nucleated cells positively correlated with increased OS (Multivariate Cox, HR (95% CI) = 0.05 (0.01-0.49), p = 0.01). When controlling for age, stage (I vs. II vs. III), neoadjuvant treatment (none vs. chemotherapy vs. chemotherapy plus radiation), and fraction T-cells in tissue, diversity in the upper quartile of the cohort is associated with increased OS compared to diversity in the lower three quartiles of the cohort (Multivariate Cox, HR (95% CI) = 0.42 (0.22-0.82), p = 0.01). Conclusion: Frequency of circulating T-cells, but not circulating TCR diversity, positively correlated with OS. Contrastingly, in tumors, TCR repertoire diversity, but not T-cell frequency, significantly correlated with OS. The intra-tumoral data indicates that a broader repertoire of TCRs within the tumor may offer a survival advantage. This offers insight into responses of PDAC to immunotherapy and emphasizes the importance of evaluating the tissue specific TCR repertoire, which may offer insights into patient outcome.

Clonal dynamics of tumor-infiltrating T-cell receptor beta-chain repertoires in the peripheral blood in response to concurrent chemoradiotherapy for Epstein–Barr virus-associated nasopharyngeal carcinoma

ABSTRACT The nasopharyngeal epithelium is highly susceptible to pathogenic infection. More than 95% of nasopharyngeal carcinomas (NPCs) are Epstein–Barr virus (EBV)-associated epithelial cancers densely infiltrated with EBV-free lymphocytes. It remains unknown whether the immune modulating effects of concurrent chemoradiotherapy (CCRT) on the tumor-infiltrating T-cell priming against EBV, tumor-associated antigens, and/or neoantigens can elicit systemic anti-tumor immunity and decrease recurrence or distant metastasis. Using matched EBV-associated NPCs, nasopharyngeal mucosal tissues, and longitudinal serial peripheral blood samples, we explored the spatiotemporal and quantitative changes in expansion and contraction of intratumoral T-cell clonotypes (ITCs) in peripheral blood samples from before, during, and after CCRT. The pre-treatment nasopharyngeal ITC repertoire contained unique mucosa-resident and commonly system-shared T-cell receptors (TCRs), portraying an individualized tumor-associated and/or metagenomic landscape. We found that the long-term disease-free patients had significantly more robust unique mucosa-resident ITCs that migrated into and expanded in the peripheral blood after CCRT than in the patients with recurrence or distant metastasis (Mann–Whitney U test, p = .0110). However, the system-shared productive ITC TCRs specific to the common viruses, such as EBV, cytomegalovirus, and influenzaA, in all the patients with and without recurrence demonstrated almost no expansion after CCRT. Thus, these findings underline the importance of determining the impact of unique intratumoral immune responses, reflected in the peripheral blood, on disease prognosis after treatment and challenge of mechanistically understanding the common systemic immune evasion of EBV in NPC patients.

Intrapleural interleukin-2–expressing oncolytic virotherapy enhances acute antitumor effects and T-cell receptor diversity in malignant pleural disease

ObjectiveThe mainstay of treatment for patients with malignant pleural disease is fluid drainage and systemic therapy. A tumor-specific oncolytic virus or T-cell–activating interleukin-2 immunotherapy may provide an opportunity for local control. We previously developed a vaccinia virus–expressing interleukin-2, an oncolytic virus that mediated tumor regression in preclinical peritoneal tumor models with expansion of tumor-infiltrating lymphocytes. We evaluated the antitumor efficacy and immune modulatory effects of vaccinia virus–expressing interleukin-2 in malignant pleural disease. MethodsA murine model of malignant pleural disease was established with percutaneous intrapleural deposition of the Lewis lung carcinoma cell line and monitored with bioluminescent imaging. After intrapleural or systemic administration of vaccinia viruses (vaccinia virus yellow fluorescent protein control, vaccinia virus–expressing interleukin-2), systemic anti–programmed cell death-1 antibody, or combination therapy (vaccinia virus–expressing interleukin-2 and anti–programmed cell death-1), tumor mass, immune cell infiltration, T-cell receptor diversity, and survival were assessed. ResultsIntrapleural vaccinia virus resulted in significant tumor regression compared with phosphate-buffered saline control (P < .05). Inclusion of the interleukin-2 transgene further increased intratumoral CD8+ T cells (P < .01) and programmed cell death-1 expression on CD8+ tumor-infiltrating lymphocytes (P < .001). Intrapleural vaccinia virus–expressing interleukin-2 was superior to systemic vaccinia virus–expressing interleukin-2, with reduced tumor burden (P < .0001) and improved survival (P < .05). Intrapleural vaccinia virus–expressing interleukin-2 alone or combined treatment with systemic anti–programmed cell death-1 reduced tumor burden (P < .01), improved survival (P < .01), and increased intratumoral αβ T-cell receptor diversity (P < .05) compared with systemic anti–programmed cell death-1 monotherapy. ConclusionsIntrapleural vaccinia virus–expressing interleukin-2 reduced tumor burden and enhanced survival in a murine malignant pleural disease model. Increased CD8+ tumor-infiltrating lymphocytes and αβ T-cell receptor diversity are associated with enhanced response. Clinical trials will enable assessment of intrapleural vaccinia virus–expressing interleukin-2 therapy in patients with malignant pleural disease.

Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity.

T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2–10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

IMMU-53. CHARACTERIZATION OF THE GENOMIC AND IMMUNOLOGICAL DIVERSITY OF MALIGNANT BRAIN TUMORS THROUGH MULTI-SECTOR ANALYSIS

Abstract While the degree of spatial genomic heterogeneity within primary glioblastoma (GBM) has been previously investigated, the extent of this heterogeneity in other malignant brain tumors and its connection to the local immune microenvironment remains unknown. To address this, we performed whole-exome, RNA, and TCR sequencing on multiple spatially distinct sectors from a cohort of malignant brain tumors comprised of both brain metastases and primary and recurrent GBMs. Our results suggest a striking difference in which a majority of mutations and predicted neoantigens are shared between spatially distinct regions of metastatic brain tumors in contrast to the spatial heterogeneity observed within both primary and recurrent GBMs. Additionally, despite substantial differences in immunotherapy responsiveness between brain metastases and GBM, we can detect significantly expanded T-cell clonotypes within both tumor types with some clonal frequencies exceeding 10% of intratumoral T-cells. Similar to the observed distribution of variants and neoantigens, the expanded clonotypes are more shared among spatially distinct sectors in metastatic brain tumors than in primary or recurrent GBMs. Interestingly, the frequencies of most of these enriched T-cell clones are significantly diminished within expanded tumor infiltrating lymphocyte (TIL) cultures, advising caution in the use of expanded TIL for the detection of tumor-specific responses. These results provide novel insight into the immunogenomic landscape of malignant brain tumors with implications for our understanding of tumor-immune interactions and the development of immunotherapies.

Regorafenib combined with PD1 blockade increases CD8 T-cell infiltration by inducing CXCL10 expression in hepatocellular carcinoma

Background and purposeCombining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms...

Abstract IA02: Immune exhaustion and suppression in lymphoma

Abstract The tumor microenvironment in lymphoproliferative disorders, including lymphoma, shows a prevalence of intratumoral T cells suggesting an effective immune response to malignant B cells. This supposes that antigen-presenting cells present tumor-associated peptides to the immune system, resulting in T cells that are primed to target the malignant clone. It is anticipated that activated T cells will target the malignant cells and eradicate the tumor. However, while T cells appear plentiful in the tumor microenvironment in B-cell lymphoma, they are not able to eradicate the malignancy. Rather, the T cells appear inhibited, either due to immune suppression or immune exhaustion, and the lymphoma steadily progresses. An effective antitumor response requires presentation of tumor antigens to T cells in the context of major histocompatibility complex (MHC) proteins. The cells then receive a second activating signal resulting in proliferation of the T cells, expansion of a tumor-specific T-cell clone, and generation of cytotoxic proteins that lyse the lymphoma cell. To control this process, however, inhibitory receptors are typically upregulated upon T-cell activation that allow the process to be suppressed. This prevents damage to healthy tissue from T cells that remain inappropriately activated for protracted periods of time. In lymphoid malignancies, however, malignant B cells commonly overexpress the ligands for PD-1, namely PD-L1 and PD-L2. This results in suppression of the activated T cells and prevents the malignant clone from being eradicated. This leads to a subsequent problem, in which effector T cells are restimulated by the persistent presence of malignant cells that expressed tumor antigens which they are targeting. This repeat stimulation results in activation-induced exhaustion and upregulation of additional receptors that make the cells susceptible to apoptosis. These receptors, including TIM3, TIGIT, and LAG3, are then expressed on T cells whose effector function is severely impaired. The effector T cells expressing TIM3 and LAG3 have very little ability to proliferate or generate cytokines. Additional mechanisms that inhibit T-cell activation are present in lymphoma and include cytokine- and cell-mediated suppression. Among cytokines that mediate T-cell suppression, TGF-β plays a pivotal role in regulating immune responses by inhibiting the proliferation and differentiation in both CD4+ and CD8+ T cells. In B-cell malignancies, both malignant B cells and intratumoral T cells can synthesize and secrete TGF-β, and TGF-β inhibits intratumoral T-cell proliferation, upregulates Foxp3 expression, and suppresses the development of TH1 and TH17 cells. Similarly, IL-10 also inhibits the proliferation, cytokine production, and migratory capacities of T cells. IL-10 downregulates expression of MHC class II as well as costimulatory molecules CD80 and CD86 on dendritic cells, resulting in suboptimal stimulation of T cells. Furthermore, IL-10-expressing dendritic cells promote T regulatory cell development. In B-cell NHL, it has been shown that serum IL-10 levels are elevated and correlate with a poor prognosis. IL-10 further affects monocytes by downregulating HLA-DR expression, and IL-10-induced CD14+HLA-DRlow/- monocytes inhibit T-cell activation and proliferation. In addition to regulatory cytokines, cell-mediated suppression plays a crucial role in T-cell suppression. Myeloid-derived suppressor cells (MDSCs) and CD4+ regulatory T cells are two major types of cells mediating T-cell suppression. In B-cell NHL, increased numbers of MDSCs may contribute to elevated absolute monocytosis at diagnosis that correlates with poor outcome in In DLBCL patients. Tumor-infiltrating MDSCs promote cross-tolerance in B-cell lymphoma by expanding or recruiting regulatory T cells, which supports tumor growth. Regulatory T cells also suppress immune function. In B-cell NHL, we have found that the frequency of regulatory T cells is increased in biopsy specimens. Regulatory T cells suppress proliferation and cytokine production of intratumoral CD4+ cells as well as granule secretion by CD8+ T cells, resulting in suppressed cytotoxicity of effector T cells. We found that both recruitment of naturally occurring regulatory T cells and induction of inducible regulatory T cells contribute to elevated frequency of these cells in the tumor microenvironment of B-cell NHL. Furthermore, malignant B cells play a role in the recruitment and induction of regulatory T cells as chemokine CCL22 secreted by lymphoma cells and CD70 expression by CD19+ lymphoma cells are involved in the process. All of these mechanisms that result in T-cell suppression and exhaustion prevent an effective antitumor immune response in lymphoma. Combination strategies that both activate T cells and prevent their inhibition and suppression are needed to rectify these issues. Citation Format: Stephen M. Ansell. Immune exhaustion and suppression in lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA02.

Overcoming primary and acquired resistance to anti-PD-L1 therapy by induction and activation of tumor-residing cDC1s

The ability of cancer cells to ensure T-cell exclusion from the tumor microenvironment is a significant mechanism of resistance to anti-PD-1/PD-L1 therapy. Evidence indicates crucial roles of Batf3-dependent conventional type-1 dendritic cells (cDC1s) for inducing antitumor T-cell immunity; however, strategies to maximize cDC1 engagement remain elusive. Here, using multiple orthotopic tumor mouse models resistant to anti-PD-L1-therapy, we are testing the hypothesis that in situ induction and activation of tumor-residing cDC1s overcomes poor T-cell infiltration. In situ immunomodulation with Flt3L, radiotherapy, and TLR3/CD40 stimulation induces an influx of stem-like Tcf1+ Slamf6+ CD8+ T cells, triggers regression not only of primary, but also untreated distant tumors, and renders tumors responsive to anti-PD-L1 therapy. Furthermore, serial in situ immunomodulation (ISIM) reshapes repertoires of intratumoral T cells, overcomes acquired resistance to anti-PD-L1 therapy, and establishes tumor-specific immunological memory. These findings provide new insights into cDC1 biology as a critical determinant to overcome mechanisms of intratumoral T-cell exclusion.

CDK4/6 inhibition enhances antitumor efficacy of chemotherapy and immune checkpoint inhibitor combinations in preclinical models and enhances T-cell activation in patients with SCLC receiving chemotherapy.

BackgroundCombination treatment with chemotherapy and immune checkpoint inhibitors (ICIs) has demonstrated meaningful clinical benefit to patients. However, chemotherapy-induced damage to the immune system can potentially diminish the efficacy of chemotherapy/ICI combinations. Trilaciclib, a highly potent, selective and reversible cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor in development to preserve hematopoietic stem and progenitor cells and immune system function during chemotherapy, has demonstrated proof of concept in recent clinical trials. Furthermore, CDK4/6 inhibition has been shown to augment T-cell activation and antitumor immunity in preclinical settings. Therefore, addition of trilaciclib has the potential to further enhance the efficacy of chemotherapy and ICI combinations.MethodsIn murine syngeneic tumor models, a schedule of 3 weekly doses of trilaciclib was combined with chemotherapy/ICI regimens to assess the effect of transient CDK4/6 inhibition on antitumor response and intratumor T-cell proliferation and function. Peripheral T-cell status was also analyzed in patients with small cell lung cancer (SCLC) treated with chemotherapy with or without trilaciclib to gain insights into the effect of transient exposure of trilaciclib on T-cell activation.ResultsPreclinically, the addition of trilaciclib to chemotherapy/ICI regimens enhanced antitumor response and overall survival compared with chemotherapy and ICI combinations alone. This effect is associated with the modulation of the proliferation and composition of T-cell subsets in the tumor microenvironment and increased effector function. Transient exposure of trilaciclib in patients with SCLC during chemotherapy treatment both preserved and increased peripheral lymphocyte counts and enhanced T-cell activation, suggesting that trilaciclib not only preserved but also enhanced immune system function.ConclusionsTransient CDK4/6 inhibition by trilaciclib was sufficient to enhance and prolong the duration of the antitumor response by chemotherapy/ICI combinations, suggesting a role for the transient cell cycle arrest of tumor immune infiltrates in remodeling the tumor microenvironment. These results provide a rationale for combining trilaciclib with chemotherapy/ICI regimens to improve antitumor efficacy in patients with cancer.

A Genetic Screen to Identify Gain- and Loss-of-Function Modifications that Enhance T-cell Infiltration into Tumors.

T-cell-mediated cancer immunotherapies, including anti-PD-1 and T cells expressing chimeric antigen receptors (CAR-T cells), are becoming standard treatments for many cancer types. CAR-T therapy, in particular, has been successful in treating circulating, but not solid, tumors. One challenge limiting immunotherapy success is that tumors lacking T-cell infiltration do not respond to treatment. Therefore, one potential strategy to overcome resistance is to enhance the ability of T cells to traffic into tumors. Here, we describe an unbiased in vivo genetic screen approach utilizing the Sleeping Beauty mutagenesis system to identify candidate genes in T cells that might be modified to drive intratumoral T-cell accumulation. This screen identified over 400 candidate genes in three tumor models. These results indicated substantial variation in gene candidate selection, depending on the tumor model and whether or not mice were treated with anti-PD-1, yet some candidate genes were identified in all tumor models and with anti-PD-1 therapy. Inhibition of the most frequently mutated gene, Aak1, affected chemokine receptor expression and enhanced T-cell trafficking in vitro and in vivo Screen candidates should be further validated as therapeutic targets, with particular relevance to enhancing infiltration of adoptively transferred T cells into solid tumors.

Abstract 900: Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer

Abstract The clinical success of cyclophosphamide (Cy)-based haploidentical stem cell transplants in hematological malignancies indicates that this drug has the potential to reorchestrate the immune system against cancer cells. Along a similar way, Cy is administered before the infusion of CAR-T cells to improve their clinical efficacy. We have previously found that daily, low/medium-dose Cy (20mg/Kg), in association with vinorelbine (V) was able to improve the preclinical efficacy of checkpoint inhibitors (CIs) anti-PD-1 and anti-PD-L1 in preclinical models of breast cancer and lymphoma (Orecchioni et al, 2018). Randomized clinical trials have recently indicated that the addition of CIs to other chemotherapy drugs such as taxanes (T), doxorubicin (D), or platinum (P) might be beneficial in triple negative breast cancer (TNBC) patients. Thus, we designed the present study in two orthotopic, immunocompetent, local and metastatic models of TNBC (CI-resistant 4T1 and EMT6, moderately responsive to CIs) to investigate whether Cy and V, in different schedules and doses, were more effective than T, D, or P. In vivo studies in local and metastatic models using both 4T1 and EMT6 TNBC cells clearly indicated that intermittent (ie every 6 days), medium-dosage (140 mg/Kg) Cy, was more effective than any other combinatorial regimens including CIs plus daily low/medium-dose Cy, or T, D, or P at their regular dosages. The association of V further increased the preclinical efficacy of intermittent Cy plus CIs, and abrogated tumor growth in both 4T1 and EMT6 models. In vivo studies with neutralizing monoclonal antibodies targeting in separate experiments T, B, NK, and myeloid cells demonstrated that CD3+CD4+, CD3+CD8+ T cells as well as CD11b+ monocyte/macrophage dendritic cells were crucial to abrogate tumor growth in TNBC-bearing mice treated with intermittent Cy, V and CIs. Single-cell transcriptome analysis of more than 60,000 intratumoral immune cells and flow cytometry studies in circulating and intratumoral subsets of immune cells indicated that V promoted the generation and maturation of myeloid APC cells, and that intermittent Cy generated new clones of tumor-infiltrating CD3+CD4+ and CD3+CD8+ TCR alpha beta cells. After treatment with intermittent Cy, V and CIs, intratumoral immune cells showed a unique gene signature of 42 genes (CD3D, Ptprc, CD69, Lat, Lck, CD2, CD28, CD27, and CD3E among others) suggestive of a change in morphology and behavior resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, as well as APC-to-T cell adhesion. Taken together, our data support the hypothesis that APC priming and T cell clonal replacement can significantly improve CI efficacy in vivo in TNBC models. We are further investigating the Cy-related mechanisms inducing intratumoral T-cell clonal replacement and how these T cells cooperate with V-induced APCs. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, Francesco Bertolini. Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 900.

Tumor CD155 Expression Is Associated with Resistance to Anti-PD1 Immunotherapy in Metastatic Melanoma.

Resistance to anti-PD1-based immune checkpoint blockade (ICB) remains a problem for the treatment of metastatic melanoma. Tumor cells as well as host myeloid cells can express the immune checkpoint ligand CD155 to regulate immune cell function. However, the effect of tumor CD155 on the immune context of human melanoma has not been well described. This observational study characterizes tumor CD155 ligand expression by metastatic melanoma tumors and correlates results with differences in immune cell features and response to ICB. Pretreatment tumor specimens, from 155 patients with metastatic melanoma treated with ICB and from 50 patients treated with BRAF/MEK-directed targeted therapy, were assessed for CD155 expression by IHC. Intratumor T-cell features were analyzed using multiplex-immunohistofluorescence for CD8, PD1, and SOX10. Correlations were made between CD155 tumor level and bulk tumor RNA sequencing results, as well as clinical RECIST response and progression-free survival. High pretreatment CD155 tumor levels correlated with high parenchymal PD1+CD8+/CD8+ T-cell ratios (PD1tR) and poor response to anti-PD1 therapy. In PDL1 negative tumors, high CD155 tumor expression was associated with patients who had poor response to combination anti-PD1/CTLA4 therapy. Our findings are the first to suggest that tumor CD155 supports an increase in the fraction of PD1+CD8+ T cells in anti-PD1 refractory melanoma tumors and, further, that targeting the CD155 pathway might improve response to anti-PD1 therapy for patients with metastatic melanoma.

BRAF Inhibitors: Molecular Targeting and Immunomodulatory Actions.

The BRAF inhibitors vemurafenib, dabrafenib and encorafenib are used in the treatment of patients with BRAF-mutant melanoma. They selectively target BRAF kinase and thus interfere with the mitogen-activated protein kinase (MAPK) signalling pathway that regulates the proliferation and survival of melanoma cells. In addition to their molecularly targeted activity, BRAF inhibitors have immunomodulatory effects. The MAPK pathway is involved in T-cell receptor signalling, and interference in the pathway by BRAF inhibitors has beneficial effects on the tumour microenvironment and anti-tumour immune response in BRAF-mutant melanoma, including increased immune-stimulatory cytokine levels, decreased immunosuppressive cytokine levels, enhanced melanoma differentiation antigen expression and presentation of tumour antigens by HLA 1, and increased intra-tumoral T-cell infiltration and activity. These effects promote recognition of the tumour by the immune system and enhance anti-tumour T-cell responses. Combining BRAF inhibitors with MEK inhibitors provides more complete blockade of the MAPK pathway. The immunomodulatory effects of BRAF inhibition alone or in combination with MEK inhibition provide a rationale for combining these targeted therapies with immune checkpoint inhibitors. Available data support the synergy between these treatment approaches, indicating such combinations provide an additional beneficial effect on the tumour microenvironment and immune response in BRAF-mutant melanoma.