Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set.

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan-Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R2 = 0.73 and R2 = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer.

Advances in the development of cancer immunotherapies.

Manipulating the immune system in order to induce clinically relevant responses against cancer is a longstanding goal. Interventions to enhance tumor-specific immunity through vaccination, sustaining effector T cell activation, or increasing the numbers of tumor-specific T cells using ex vivo expansion, have all resulted in clinical successes. Here, we examine recent clinical advances and major ongoing studies in the field of cancer immunotherapy. Single agents have so far benefited a limited proportion of patients, and future studies combining different types of immunotherapies and other therapeutic modalities, such as drugs against specific signaling pathways driving cancer cell growth, are needed to pave the way for the development of effective anticancer treatments causing durable responses.

Broad cross-presentation of the hematopoietically derived PR1 antigen on solid tumors leads to susceptibility to PR1-targeted immunotherapy.

PR1 is a HLA-A2-restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived APCs, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific CTLs (PR1-CTL) and the anti-PR1/HLA-A2 Ab 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an Ag become susceptible to therapies that target cross-presented Ags and suggest PR1 as a broadly expressed tumor Ag.

2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

Randomized phase II trial of lymphodepletion plus adoptive cell transfer of tumor-infiltrating lymphocytes, with or without dendritic cell vaccination, in patients with metastatic melanoma.

BACKGROUND: The adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen-loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized that the combined transfer of TIL, containing a melanoma antigen recognized by T cells 1 (MART-1) specific population, with MART-1-pulsed DC will result in enhanced proliferation and prolonged survival of transferred MART-1 specific T cells in vivo ultimately leading to improved clinical responses. DESIGN: We tested the combination of TIL and DC in a phase II clinical trial of patients with advanced stage IV melanoma. HLA-A0201 patients whose early TIL cultures demonstrated reactivity to MART-1 peptide were randomly assigned to receive TIL alone or TIL +DC pulsed with MART-1 peptide. The primary endpoint was to evaluate the persistence of MART-1 TIL in the two arms. Secondary endpoints were to evaluate clinical response and survival. RESULTS: Ten patients were given TIL alone while eight patients received TIL+DC vaccine. Infused MART-1 reactive CD8+ TIL were tracked in the blood over time by flow cytometry and results show good persistence in both arms, with no difference in the persistence of MART-1 between the two arms. The objective response rate was 30% (3/10) in the TIL arm and 50% (4/8) in the TIL+DC arm. All treatments were well tolerated. CONCLUSIONS: The combination of TIL +DC showed no difference in the persistence of MART-1 TIL compared with TIL therapy alone. Although more patients showed a clinical response to TIL+DC therapy, this study was not powered to resolve differences between groups. TRIAL REGISTRATION NUMBER: NCT00338377.

COVID-19: a pandemic experience that illuminates potential reforms to health research.

COVID-19 has halted research around the globe and forced researchers out of their laboratories. Non-emergency medical appointments were canceled. Ongoing clinical trials were challenged to create new modes of operation while public pressure mounted to find therapeutic options against COVID-19. Yet, the inability to conduct research during COVID-19 was overcome with cooperation, resource sharing, and compassion, which provides important lessons on how to improve health related research as we enter a new normal.

A new approach for the large-scale generation of mature dendritic cells from adherent PBMC using roller bottle technology.

BACKGROUND: Human monocyte-derived DC (mDC) loaded with peptides, protein, tumor cell lysates, or tumor cell RNA, are being tested as vaccines against multiple human malignancies and viral infection with great promise. One of the factors that has limited more widespread use of these vaccines is the need to generate mDC in large scale. Current methods for the large-scale cultivation of mDC in static culture vessels are labor- and time- intensive, and also require many culture vessels. Here, we describe a new method for the large-scale generation of human mDC from human PBMC from leukopheresis or buffy coat products using roller bottles, never attempted before for mDC generation. We have tested this technology using 850 cm2 roller bottles compared to conventional T-175 flat-bottom static culture flasks. METHODS: DC were generated from adherent human PBMC from buffy coats or leukopherisis products using GM-CSF and IL-4 in T-175 static flasks or 850 cm2 roller bottles. The cells were matured over two days, harvested and analyzed for cell yield and mature DC phenotype by flow cytometry, and then functionally analyzed for their ability to activate allogeneic T-cell or recall antigen peptide-specific T-cell responses. RESULTS: Monocytes were found to adhere inside roller bottles to the same extent as in static culture flasks. The phenotype and function of the mDC harvested after maturation from both type of culture systems were similar. The yield of mDC from input PBMC in the roller bottle system was similar as in the static flask system. However, each 850 cm2 roller bottle could be seeded with 4-5 times more input PBMC and could yield 4-5 times as many mDC per culture vessel than the static flasks as a result. CONCLUSION: Our results indicate that the roller bottle technology can generate similar numbers of mDC from adherent PBMC as traditional static flask methods, but with having to use fewer culture vessels. Thus, this may be a more practical method to generate mDC in large-scale cutting down on the amount of laboratory manipulations, and can save both time and labor costs.

Utilizing T-cell Activation Signals 1, 2, and 3 for Tumor-infiltrating Lymphocytes (TIL) Expansion: The Advantage Over the Sole Use of Interleukin-2 in Cutaneous and Uveal Melanoma.

In this study, we address one of the major critiques for tumor-infiltrating lymphocyte (TIL) therapy-the time needed for proper expansion of a suitable product. We postulated that T-cell receptor activation in the first phase of expansion combined with an agonistic stimulation of CD137/4-1BB and interleukin-2 would favor preferential expansion of CD8 TIL. Indeed, this novel 3-signal approach for optimal T-cell activation resulted in faster and more consistent expansion of CD8CD3 TIL. This new method allowed for successful expansion of TIL from cutaneous and uveal melanoma tumors in 100% of the cultures in <3 weeks. Finally, providing the 3 signals attributed to optimal T-cell activation led to expansion of TIL capable of recognizing their tumor counterpart in cutaneous and uveal melanoma. This new methodology for the initial phase of TIL expansion brings a new opportunity for translation of TIL therapy in challenging malignancies such as uveal melanoma.

Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome.

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence.Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated.Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome.Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416-28. ©2018 AACR.

Improving antitumor immune responses by circumventing immunoregulatory cells and mechanisms.

Although numerous immunotherapeutic strategies have been studied in patients with cancer, consistent induction of clinical responses remains a formidable challenge. Cancer vaccines are often successful at generating elevated numbers of tumor-specific T lymphocytes in peripheral blood, however, despite this, tumors usually continue to grow unabated. Recent evidence suggests that endogenous regulatory cells, known to play a major role in the induction of immune tolerance to self and prevention of autoimmunity, as well as suppressive myeloid cells invoked in the tumor-bearing state, may be largely responsible for preventing effective antitumor immune responses. This review will focus on the major regulatory cell subtypes, including CD4(+)CD25(+) T-regulatory cells, type 1 regulatory T cells, natural killer T cells, and immature myeloid cells. Studies in humans and in animal models have shown a role for all of these cells in tumor progression, although the mechanisms by which they act to suppress immunity remain largely undefined. Elucidation of the dominant molecular mechanisms mediating immune suppression in vivo will allow more precise targeting of the relevant regulatory cell populations, as well as the development of novel strategies and clinical reagents that will directly block molecules that induce the suppression of antitumor immunity.

Immunosuppression in melanoma immunotherapy: potential opportunities for intervention.

Although melanomas are substantially more immunogenic than other tumors, current immunotherapeutic approaches for melanoma patients have met with only limited success. Although melanoma-specific CD8+ T-cell responses can often be generated in patients naturally or through vaccination regimens, tumors frequently continue to grow unabated, suggesting that tumor-specific immune responses may be actively dampened in vivo. Research over the past decade has brought to light several mechanisms used by melanomas and other tumors to suppress tumor-specific immune responses. These include the presence of regulatory immune cells within the tumor microenvironment and draining lymph nodes that serve to shut down effector T-cell function. In addition, melanoma tumors themselves express a number of soluble and membrane-bound molecules that are responsible for inhibiting activated immune cells. The identification of these suppressive mechanisms has provided significant opportunities for designing novel therapeutic interventions that could augment current vaccination and adoptive transfer approaches for treatment of melanoma.

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter.

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%-50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation.Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs.Results: CD8+BTLA- TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA- T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened "serial killing" capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation.Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151-64. ©2017 AACR.

4-1BB-Enhanced Expansion of CD8+ TIL from Triple-Negative Breast Cancer Unveils Mutation-Specific CD8+ T Cells.

Triple-negative breast cancer (TNBC) highly infiltrated with CD8+ tumor-infiltrating lymphocytes (TIL) has been associated with improved prognosis. This observation led us to hypothesize that CD8+ TIL could be utilized in autologous adoptive cell therapy for TNBC, although this concept has proven to be challenging, given the difficulty in expanding CD8+ TILs in solid cancers other than in melanoma. To overcome this obstacle, we used an agonistic antibody (urelumab) to a TNFR family member, 4-1BB/CD137, which is expressed by recently activated CD8+ T cells. This approach was first utilized in melanoma and, in this study, led to advantageous growth of TILs for the majority of TNBC tumors tested. The agonistic antibody was only added in the initial setting of the culture and yet favored the propagation of CD8+ TILs from TNBC tumors. These expanded CD8+ TILs were capable of cytotoxic functions and were successfully utilized to demonstrate the presence of immunogenic mutations in autologous TNBC tumor tissue without recognition of the wild-type counterpart. Our findings open the way for a successful adoptive immunotherapy for TNBC. Cancer Immunol Res; 5(6); 439-45. ©2017 AACR.

Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function.

The interaction between tumor and the immune system is still poorly understood. Significant clinical responses have been achieved in cancer patients treated with antibodies against the CTLA4 and PD-1/PD-L1 checkpoints; however, only a small portion of patients responded to the therapies, indicating a need to explore additional co-inhibitory molecules for cancer treatment. B7-H3, a member of the B7 superfamily, was previously shown by us to inhibit T-cell activation and autoimmunity. In this study, we have analyzed the function of B7-H3 in tumor immunity. Expression of B7-H3 was found in multiple tumor lines, tumor-infiltrating dendritic cells, and macrophages. B7-H3-deficient mice or mice treated with an antagonistic antibody to B7-H3 showed reduced growth of multiple tumors, which depended on NK and CD8+ T cells. With a putative receptor expressed by cytotoxic lymphocytes, B7-H3 inhibited their activation, and its deficiency resulted in increased cytotoxic lymphocyte function in tumor-bearing mice. Combining blockades of B7-H3 and PD-1 resulted in further enhanced therapeutic control of late-stage tumors. Taken together, our results indicate that the B7-H3 checkpoint may serve as a novel target for immunotherapy against cancer.

Defective STAT1 activation associated with impaired IFN-γ production in NK and T lymphocytes from metastatic melanoma patients treated with IL-2.

High dose (HD) IL-2 therapy has been used for almost two decades as an immunotherapy for metastatic melanoma. IL-2 promotes the proliferation and effector function of T and NK cells through the tyrosine phosphorylation and activation of signal transducer and activator of transcription factors (STAT), especially STAT5. However, whether any defects in STAT activation exist in T and NK lymphocytes from melanoma patients are under debate. Here, we measured the extent of HD IL-2-induced phosphorylation of STAT5 and STAT1 in lymphocyte subsets from metastatic melanoma patients and healthy controls at a single cell level using flow cytometry. We found no defects in IL-2-induced STAT5 phosphorylation and induction of proliferation in T and NK cell subsets in vitro. This was confirmed by measuring ex vivo STAT5 activation in whole blood collected from patients during their first bolus HD IL-2 infusion. IL-2 also induced STAT1 phosphorylation via IFN-γ receptors in T and NK cell subsets through the release of IFN-γ by CD56hi and CD56lo NK cells. Further analysis revealed that melanoma patients had a sub-optimal STAT1 activation response linked to lower IL-2-induced IFN-γ secretion in both CD56hi and CD56low NK cell subsets. STAT1 activation in response to IL-2 also showed an age-related decline in melanoma patients not linked to tumor burden indicating a premature loss of NK cell function. Taken together, these findings indicate that, although STAT5 activation is normal in metastatic melanoma patients in response to IL-2, indirect STAT1 activation is defective owing to deficiencies in the NK cell response to IL-2.

The beneficial effects of a gas-permeable flask for expansion of Tumor-Infiltrating lymphocytes as reflected in their mitochondrial function and respiration capacity.

Adoptive transfer of autologous ex vivo expanded tumor-infiltrating lymphocytes (TIL) is a highly successful cell therapy approach in the treatment of late-stage melanoma. Notwithstanding the success of this therapy, only very few centers worldwide can provide it. To make this therapy broadly available, one of the major obstacles to overcome is the complexity of culturing the TIL. Recently, major efforts have been deployed to resolve this issue. The use of the Gas-permeable flask (G-Rex) during the REP has been one application that has facilitated this process. Here we show that the use of this new device is able to rescue poor TIL growth and maintain clonal diversity while supporting an improved mitochondrial function.

Tumor-Infiltrating Lymphocyte Therapy: Addressing Prevailing Questions.

Autologous adoptive T-cell therapies have made tremendous strides over the last few years with excitement currently being generated by technologies that can reprogram T-cell specificities toward any desired antigen including chimeric antigen receptors and recombinant T-cell receptors. Time will tell whether these new genetically engineered T-cell technologies will be effective as advertised, especially in solid tumors, considering the limited availability of specific antigens and the difficulty in managing the unpredictable on-target, off-tissue toxicities. However, a form of T-cell therapy that has been utilized in patients more than any other and has left a lasting mark in the field is tumor-infiltrating lymphocytes (TILs). Tumor-infiltrating lymphocyte therapy has consistently yielded durable clinical responses in selected patients with metastatic melanoma and is now being increasingly applied to treat other solid tumors, including head and neck squamous cell carcinoma, cervical cancer, breast cancer, and lung cancer. Despite its long history in the clinic and key developments over the last few decades that have augmented response rates and have made TIL manufacturing more streamlined, a number of key outstanding conceptual questions remain to be answered in the TIL therapy field. In this review, we address critical questions, including the mechanism of action of TILs and active T-cell subsets, the current need for lymphoablative preconditioning, predictive biomarkers, the role of combination therapy such as checkpoint blockade, new excitement over the recognition of mutated antigens (the "mutanome") by TILs, and issues in developing TILs for nonmelanoma indications. In each case, we will critically discuss the main issues and concerns and how they can affect the eventual positioning of TIL therapy in the mainstream of cancer care.

Peptide-based vaccination and induction of CD8+ T-cell responses against tumor antigens in breast cancer.

Tumor-associated antigens (TAAs) have been identified in many malignant tumors. Within these TAAs are peptide sequences that bind major histocompatibility complex (MHC) class I and class II molecules recognized by T cells triggering antigen-specific CD8+ cytotoxic T-cell and CD4+ T-helper cell responses. Efforts to develop vaccines for breast cancer have been underway for more than 20 years, including peptide and whole inactivated tumor cell vaccines as well as antigen-loaded dendritic cell vaccines. The majority of vaccine trials have used peptides, including single-peptide and multiple-peptide formulations using either MHC class I and class II epitopes in oil-based emulsions alone or in combination with an adjuvant, such as granulocyte-macrophage colony-stimulating factor, and Toll-like receptor agonists. Preclinical research in vitro and in animal models has been aimed at improving vaccine efficacy by identifying more immunogenic peptides and combinations of peptides and adjuvants and cytokine adjuvants that induce stronger immune responses and prolong T-cell memory. Clinical studies investigating the therapeutic potential of active immunization using peptide vaccines has found no serious side effects. In this review, we examine TAA peptide-based vaccination regimens showing promise in breast cancer patients that are also being investigated in clinical trials of safety and efficacy. We also discuss the current limitations in the peptide vaccination field and areas for future development.

BTLA marks a less-differentiated tumor-infiltrating lymphocyte subset in melanoma with enhanced survival properties.

In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+ T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+ TIL subset and their CD8+BTLA- counterparts. We found that the CD8+ BTLA+ TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longer in vivo after infusion. In contrast, CD8+BTLA- TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+ TILs from apoptosis. Our results point to a new role of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+ T cells. These attributes may explain our previous observation that BTLA expression on CD8+ TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma.