Activity Of Tumor-infiltrating Lymphocytes Research Articles

Expansion of tumor-infiltrating lymphocytes in non-small cell lung cancer: Clinical potential and efficacy in EGFR mutation subsets

Our study aimed to expand tumor-infiltrating lymphocytes (TILs) from primary non-small cell lung cancers (NSCLCs) and evaluate their reactivity against tumor cells. We expanded TILs from 103 primary NSCLCs using histopathological analysis, flow cytometry, IFN-γ release assays, cell-mediated cytotoxicity assays, and in vivo efficacy tests. TIL expansion was observed in all cases, regardless of EGFR mutation status. There was also an increase in the median CD4+/CD8+ ratio during expansion. In post-rapid expansion protocol (REP) TILs, 13 out of 16 cases, including all three cases with EGFR mutations, exhibited a two-fold or greater increase in IFN-γ secretion. The cytotoxicity assay revealed enhanced tumor cell death in three of the seven cases, two of which had EGFR mutations. In vivo functional testing in a patient-derived xenograft model showed a reduction in tumor volume. The anti-tumor activity of post-REP TILs underscores their potential as a therapeutic option for advanced NSCLC, irrespective of mutation status.

3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

Abstract 4058: Nanoparticle delivery of interleukin-2 induces anti-tumor immune response and therapeutic efficacy in mouse tumor models

Abstract Immune checkpoint inhibitors (ICIs) have demonstrated robust responses in some solid tumor types. However, the low response rate, which is associated with lack of pre-existing tumor-infiltrating lymphocytes (TILs), remains a significant challenge. Interleukin-2 (IL-2) can effectively expand TILs and has been FDA-approved for metastatic renal cancer and melanoma. However, IL-2 has several drawbacks—including short circulation half-life, dose-limiting toxicities, and counterproductive activation of regulatory T cells (Tregs)—resulting in less than 10% complete response rate. Numerous efforts have been made to enhance IL-2 formulations, but none have yet received FDA approval. In this study, we utilized our proprietary nanoparticle platform (Pdx-NP™) to improve IL-2 formulation. Pdx-NP™ can load IL-2 with near-complete binding, resulting in a 160-nm nanoconstruct (IL2-NP), suitable for both local and intravenous administrations. To evaluate the efficacy, we utilized bilateral melanoma (B16F10) and colon (MC38) tumor models, in which the local tumor was treated intratumorally for 3 doses and the distal tumor was left untreated to assess the systemic anti-tumor immune response. In the B16F10 model, IL2-NP treatment demonstrated favorable immune profiles in local and distal tumors and the draining lymph nodes, such as increased CD8+ T cell proliferation, CD8+ T cells/Treg ratio, and recruitment of dendritic cells and macrophages, compared to saline or free IL-2 counterparts. These positive immune responses translated into significant growth reduction of IL2-NP treated tumors and improvement in survival of the mice compared to free IL-2. In the MC38 tumor model, the IL2-NP treatment resulted in complete regression of both local and distal tumors in 43% of the mice. When combined with ICIs (αPD-1 and αCTLA-4), the treatment achieved a cure rate of 100%, whereas ICIs alone yielded only a 29% cure rate. In conclusion, IL2-NP induces robust proliferation and activation of TILs and is efficacious both as a monotherapy and in combination with ICIs. This is due to the ability of Pdx-NP™ to protect and retain therapeutics within tumors, with an 8-fold increase in tumor accumulation over the free drug counterpart. Our ongoing efforts are focused on the targeted delivery of IL-2 to tumors upon systemic administration, using αPD-L1 antibody-conjugated Pdx-NP™. This antibody-conjugated nanoparticle has an elimination half-life (t1/2) of about 25 hrs in non-human primates. Paired with αPD-L1 for tumor homing, this nanoconstruct is hypothesized to enhance IL-2 accumulation in tumors compared to free IL-2, which has t1/2 of only 1.5 hrs in human. The current clinical landscape of cancer immunotherapy focuses on combinational approaches, and our versatile Pdx-NP™ platform, which is capable of co-delivering different classes of therapeutics, is well suited for this endeavor. Citation Format: Ruijie Wang, Alyssa Wallstrum, Natalie White, Pramod Kumar, Tongchatra Watcharawittayakul, Noah A. Crumrine, Cole Baker, Jeremy Saito, Moataz Reda, Worapol Ngamcherdtrakul, Wassana Yantasee. Nanoparticle delivery of interleukin-2 induces anti-tumor immune response and therapeutic efficacy in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4058.

Signaling via a CD28/CD40 chimeric costimulatory antigen receptor (CoStAR™), targeting folate receptor alpha, enhances T cell activity and augments tumor reactivity of tumor infiltrating lymphocytes.

Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.

Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities.

Tumor infiltrating lymphocytes (TILs) usually become exhausted and dysfunctional owing to chronic contact with tumor cells and overexpression of multiple inhibitor receptors. Activation of TILs by targeting the inhibitory and stimulatory checkpoints has emerged as one of the most promising immunotherapy prospectively. We investigated whether triggering of CD28, 4-1BB, and PD-1 checkpoints simultaneously or alone could enhance the immune response capacity of lymphocytes. In this regard, anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins were designed and produced in CHO-K1 cells as an expression host. Following confirmation of the Fc fusion proteins' ability to bind to native targets expressed on engineered CHO-K1 cells (CHO-K1/hPD-1, CHO-K1/hCD28, CHO-K1/hCTLA4, and CHO-K1/h4-1BB), the effects of each protein, on its own and in various combinations, were assessed in vitro on T cell proliferation, cytotoxicity, and cytokines secretion using the Mixed lymphocyte reaction (MLR) assay, 7-AAD/CFSE cell-mediated cytotoxicity assay, and a LEGENDplex™ Human Th Cytokine Panel, respectively. MLR results demonstrated that T cell proliferation in the presence of the combinations of anti-PD-1/CD80-Fc, CD80-Fc/4-1BBL-Fc, and anti-PD-1/CD80-Fc/4-1BBL-Fc proteins was significantly higher than in the untreated condition (1.83-, 1.91-, and 2.02-fold respectively). Furthermore, anti-PD-1 (17%), 4-1BBL-Fc (19.2%), anti-PD-1/CD80-Fc (18.6%), anti-PD-1/4-1BBL-Fc (21%), CD80-Fc/4-1BBL-Fc (18.5%), and anti-PD-1/CD80-Fc/4-1BBL-Fc (17.3%) significantly enhanced cytotoxicity activity compared to untreated condition (7.8%). However, concerning the cytokine production, CD80-Fc and 4-1BBL-Fc alone or in combination significantly increased the secretion of IFN-γ, TNF-α, and IL-2 compared with the untreated conditions. In conclusion, this research establishes that the various combinations of produced anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins can noticeably induce the immune response in vitro. Each of these combinations may be effective in killing or destroying cancer cells depending on the type and stage of cancer.

Tumor-infiltrating lymphocytes for treatment of solid tumors: It takes two to tango?

Tumor-infiltrating lymphocytes (TILs), frontline soldiers of the adaptive immune system, are recruited into the tumor site to fight against tumors. However, their small number and reduced activity limit their ability to overcome the tumor. Enhancement of TILs number and activity against tumors has been of interest for a long time. A lack of knowledge about the tumor microenvironment (TME) has limited success in primary TIL therapies. Although the advent of engineered T cells has revolutionized the immunotherapy methods of hematologic cancers, the heterogeneity of solid tumors warrants the application of TILs with a wide range of specificity. Recent advances in understanding TME, immune exhaustion, and immune checkpoints have paved the way for TIL therapy regimens. Nowadays, TIL therapy has regained attention as a safe personalized immunotherapy, and currently, several clinical trials are evaluating the efficacy of TIL therapy in patients who have failed conventional immunotherapies. Gaining favorable outcomes following TIL therapy of patients with metastatic melanoma, cervical cancer, ovarian cancer, and breast cancer has raised hope in patients with refractory solid tumors, too. Nevertheless, TIL therapy procedures face several challenges, such as high cost, timely expansion, and technical challenges in selecting and activating the cells. Herein, we reviewed the recent advances in the TIL therapy of solid tumors and discussed the challenges and perspectives.

Autophagic flux restoration enhances the antitumor efficacy of tumor infiltrating lymphocytes

BackgroundAlthough adoptive cell therapy with tumor infiltrating lymphocytes (TILs) has mediated effective antitumor responses in several cancers, dysfunction and exhaustion of TILs significantly impair the therapeutic effect of TILs. Thus,...

Membrane anchored IL-18 linked to constitutively active TLR4 and CD40 improves human T cell antitumor capacities for adoptive cell therapy

BackgroundAdoptive transfer of tumor-infiltrating lymphocytes (TILs) or blood T cells genetically redirected by an antitumor TCR or CAR induces a strong antitumor response in a proportion of patients with cancer;...

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit Tcell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of Tcell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human Tcell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood Tcells and TIL. Tumor-specific Tcells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. CISH expression was associated with Tcell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing Tcell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade invivo. CISH negatively regulates human Tcell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

The Novel Immune Checkpoint GPR56 Is Expressed on Tumor-Infiltrating Lymphocytes and Selectively Upregulated upon TCR Signaling.

Simple SummaryDespite the clinical efficacy of so-called immune checkpoint inhibitors (ICIs) in various cancers, some cancer types, including epithelial ovarian cancer (EOC), do not effectively respond to current therapeutics. Thus, the identification of new immune checkpoints that regulate T cell immunity remains of great interest. One as yet largely uninvestigated checkpoint of potential interest is the G protein-coupled receptor 56 (GPR56), which belongs to the adhesion GPCR family. We identified that GPR56 is expressed on tumor infiltrating lymphocytes (TILs) and investigated its role as a potential immune checkpoint within the context of cancer. Based on our data, GPR56 indeed appears to function as an immune checkpoint in TILs and may thus provide a novel immunotherapeutic target for the reactivation of tumor-infiltrating and tumor-reactive lymphocytes.High levels of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are associated with a survival benefit in various cancer types and the targeted (re)activation of TILs is an attractive therapeutic anti-cancer approach that yields curative responses. However, current T cell targeting strategies directed at known immune checkpoints have not increased objective response rates for all cancer types, including for epithelial ovarian cancer (EOC). For this reason, the identification of new immune checkpoints that regulate T cell immunity remains of great interest. One yet largely uninvestigated checkpoint of potential interest is the G protein-coupled receptor 56 (GPR56), which belongs to the adhesion GPCR family. GPR56 was originally reported to function in cerebral cortical development and in anti-depressant response, but also in cancer. Recently, GPR56 was identified as an inhibitory receptor expressed on human NK cells that by cis-interaction with the tetraspanin CD81 attenuated the cytotoxic activity of NK cells. This NK cell checkpoint could be blocked by an GPR56 antibody, leading to increased cytotoxicity. Interestingly, GPR56 expression has also been reported on cytokine producing memory CD8 T lymphocytes and may thus represent a T cell checkpoint as well. Here, GPR56 mRNA expression was characterized in the context of TILs, with GPR56 expression being detected predominantly in tumor infiltrating CD8 T cells with a cytotoxic and (pre-)exhausted phenotype. In accordance with this mRNA profile, TILs from ovarian cancer patients expressed GPR56 primarily within the effector memory and central memory T cell subsets. On T cells from healthy donors the expression was limited to effector memory and terminally differentiated T cells. Notably, GPR56 expression further increased on TILs upon T cell receptor (TCR)-mediated stimulation in co-cultures with cancer cells, whereas GPR56 expression on healthy primary human T cells did not. Further, the ectopic expression of GPR56 significantly reduced the migration of GPR56-positive T cells. Taken together, GPR56 is a potential immune-checkpoint in EOC found on (pre-)exhausted CD8 TILs that may regulate migratory behavior.

Abstract LB212: Allogeneic, IL-2-independent tumor-infiltrating lymphocytes expressing membrane-bound IL-15 (cytoTIL15࣪) eradicate tumors in a melanoma PDX model through recognition of shared tumor antigens

Abstract Standard tumor-infiltrating lymphocyte (TIL) therapy requires IL-2 administration to support TIL expansion and survival, but this cytokine is associated with T cell exhaustion and can result in severe toxicities that limit patient eligibility (1). To this end, we genetically engineered TIL to express membrane-bound IL-15 (mbIL15) under the control of Obsidian’s cytoDRIVE® technology (cytoTIL15࣪), which allows regulation of protein expression via a drug-responsive domain upon acetazolamide (ACZ) administration. IL-15 is a preferred cytokine over IL-2 to mediate TIL activation and expansion, because it does not result in CD8 T cell exhaustion or stimulate regulatory CD4 T cells, and enhances development of a memory T-cell phenotype. We have previously demonstrated IL-2-independent, 3-6-fold increased cytoTIL15 persistence in an antigen-independent setting relative to unengineered TIL therapy with IL-2 (uTIL) (2). Due to the challenge of generating autologous tumor/TIL-matched pairs and most importantly, to assess cytoTIL15 cell’s functional impact on anti-tumor growth across multiple donors, we developed an allogeneic patient-derived xenograft (PDX) model. To establish the model, different melanoma tumor digests were co-incubated in vitro with select HLA-A*02-matched, allogeneic melanoma TIL donors to assess their reactivity. Tumors were screened for expression of shared antigens, such as gp100 and MART1, and TIL donor TCRs were screened with tetramers. Once established, serially passaged tumor fragments were grown, measured, and randomized into groups to receive intravenous transfer of TIL (n=8/cohort). Mice receiving uTIL were treated with four saturating doses of recombinant IL-2, and mice receiving cytoTIL15 cells received either vehicle or oral 200 mg/kg ACZ daily for the entire study, without any IL-2. Three of four cytoTIL15 cell preparations from different donors dosed with ACZ achieved significant tumor growth inhibition compared to uTIL. Four mice developed complete responses as early as 17 days post cytoTIL15 cell transfer. The level of anti-tumor response was associated with increased frequency of MART1-reactive cytoTIL15 cells. On day 20 after TIL transfer, tumors and secondary lymphoid organs were collected (n=4/cohort). Tumors treated with cytoTIL15 cells + ACZ showed an 8-10-fold increased TIL infiltration compared to uTIL or cytoTIL15 cells + vehicle. Moreover, enhanced cytoTIL15 cell infiltration and anti-tumor activity was associated with increases in pro-inflammatory cytokines (e.g., IFNγ). Taken together, these data clearly demonstrate the superiority of cytoTIL15 cells over uTIL for controlling or eradicating melanoma tumor outgrowth and the utility of an allogeneic PDX model for comparative evaluation of tumor-antigen specific TIL reactivity.

A Platform of Patient-Derived Microtumors Identifies Individual Treatment Responses and Therapeutic Vulnerabilities in Ovarian Cancer.

Simple SummaryFor personalized oncology, it is crucial to develop appropriate patient-derived tumor models that allow individualized validation of the most effective cancer therapy. The objective of this study was to develop and characterize a new patient-derived ovarian cancer tumor model composed of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TIL). In contrast to other preclinical tumor models, such as patient-derived organoids, PDM are generated within 24 h from fresh ovarian tumor samples. From immunohistochemical comparison with the original primary tumor, we conclude that the histopathological features of the original tumor are essentially preserved. Importantly, we successfully identified treatment-sensitive and treatment-resistant tumor models for standard platinum-based therapy by reverse-phase protein array (RPPA) analysis of PDM. Furthermore, we were able to evaluate the efficacy of cancer immunotherapy by co-culturing PDM and autologous TILs. PDM and TILs may therefore serve as a preclinical platform to identify individualized, tailored cancer treatments in the future.In light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease, we developed a 3D preclinical tumor model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) to identify individual treatment vulnerabilities and validate chemo-, immuno- and targeted therapy efficacies. Enzymatic digestion of primary ovarian cancer tissue and cultivation in defined serum-free media allowed rapid and efficient recovery of PDM, while preserving histopathological features of corresponding patient tumor tissue. Reverse-phase protein array (RPPA)-analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Co-cultures of PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Combining protein pathway analysis and drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision making.

A Fraction of CD8+ T Cells from Colorectal Liver Metastases Preferentially Repopulate Autologous Patient-Derived Xenograft Tumors as Tissue-Resident Memory T Cells.

Simple SummaryTreatment options for colorectal cancer (CRC) patients with liver metastases are often limited to liver surgery with or without chemotherapy. However, not all patients present operable colorectal liver metastases (CRLMs). Thus, alternative therapies that exploit the anti-tumor potential of tumor-infiltrating lymphocytes (TILs) are being evaluated. The establishment of markers connecting the phenotype to the function of tumor-reactive CD8+ TILs could aid diagnostic and therapeutic advances. In this regard, tissue-resident memory T cells (TRM cells) could be a potential candidate for therapies targeting TILs. Putative tumor-reactive TRM cells among CD8+ TILs likely co-express CD103 and CD39, since these markers indicate stable tumor residency and repeated response to antigens from the tumor environment, respectively. Our phenotypic and functional analyses of TILs in CRLM, with a specific focus on CD103+CD8+ TRM cells, may guide the improvement of TIL-mediated CRC treatments.The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ TRM cells predominantly in CRLM, which prompted further assessments. These TRM cells responded to cognate antigens in vitro. As functional activities of autologous TILs are central to the implementation of personalized cancer treatments, we applied a patient-derived xenograft (PDX) model to monitor TILs’ capacity to control CRLM-derived tumors in vivo. We established PDX mice with CRLMs from two patients, and in vitro expansion of their respective TILs resulted in opposing CD4+ vs. CD8+ TIL ratios. These CRLMs also displayed mutated KRAS, which enabled trametinib-mediated inhibition of MEK. Regardless of the TIL subset ratio, persistent or transient control of CRLM-derived tumors of limited size by the transferred TILs was observed only after trametinib treatment. Of note, a portion of transferred TILs was observed as CD103+CD8+ TRM cells that strictly accumulated within the autologous CRLM-derived tumor rather than in the spleen or blood. Thus, the predominance of CD103+CD39+CD8+ TRM cells in CRLM relative to the adjacent liver and the propensity of CD103+CD8+ TRM cells to repopulate the autologous tumor may identify these TILs as strategic targets for therapies against advanced CRC.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC

BackgroundChimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity.MethodsTo bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model.ResultsF i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation.ConclusionsOur next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

Tumor-infiltrating lymphocyte: features and prognosis of lymphocytes infiltration on colorectal cancer

ABSTRACT Tumor-infiltrating lymphocytes (TILs) are vital elements of the tumor microenvironment (TME), and the anti-tumor activity of TILs on colorectal cancer (CRC) has been a topic of concern. However, the characteristics and prognosis of the various types of lymphocyte infiltration in CRC have not been fully explained. Our study aimed to identify distinct features and prognosis of TILs. We integrated multiple-cohort databases to illustrate the features, proportions, and prognosis of TILs on CRC. We found that macrophages were significantly enriched in CRC. When we used the scRNA-seq database to further evaluate the proportion of TILs, we noticed markedly higher numbers of CD4 + T cell, B cell, and CD8 + T cell in four Gene Expression Omnibus Series (GSE) CRC cohorts. Interestingly, we found that the infiltrating level of TIL subgroups from highest to lowest is always dendritic cells, CD8 + T cells, CD4 + T cells, neutrophils, B cells, and macrophages; the proportion of infiltration is largely constant regardless of mutations in specific genes or somatic copy number variation (sCNV). In addition, the data corroborated that CD4+ TILs and CD8+ TILs have certain application values in the prognosis of CRCs, and age negatively related to CD8+ TILs and B plasma infiltration. Finally, patients with CRC who are older than 70 years have a better response to immune-checkpoint blockade.

Enriching for tumor-reactive CD8 TIL (AGX148) leads to effective tumor clearance in a patient-derived xenograph model

Abstract Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a subset of patients with melanoma; however, the efficacy of standard bulk TIL therapy can be limited. We hypothesize that the therapeutic efficacy of TIL ACT is dependent on the frequency and functionality of tumor-reactive T cells when infused back into patients. Our approach enhances the activity of the ACT product by enriching for the tumor-reactive CD8 TIL prior to ex vivo expansion by sorting on cells that co-express CD103 and CD39. The CD103/CD39 double positive (DP) CD8 TIL can be found in most human solid tumors but at varying frequencies in any given patient. Surgically resected human tumor samples were provided through a collaboration with the EACRI. Tumor samples were flow sorted and expanded ex vivo and patient-matched autologous tumor lines were generated. Tumors were then cocultured with the expanded autologous ACT product (AGX148) in vitro or used in xenograph mouse experiments. The AGX148 TIL products are effective at recognizing and killing autologous tumor cells, in vitro. Upon adoptive transfer into patient-derived tumor-bearing huIL-2 NOG mice, the transferred human T cells increased in frequency, migrated to the tumor, became activated in the tumor and recognized/destroyed established tumors. Interestingly there was little to no in vivo activity of CD8 TIL that did not express these two markers. We have generated a potent therapeutic ACT TIL product (AGX148) through the selective isolation and expansion of tumor-reactive T cells. This TIL product effectively induces durable curative responses in hosts harboring autologous tumors providing rationale for clinical investigation.

Granzyme-B Expression as a Predictor of Immune Surveillance (Correlation Study in Patient with Squamous Cell Carcinoma and Uterine Cervical Dysplasia)

Background: The most common histologic type of cervical cancer is squamous cell carcinoma (SCC), with the risk of recurrence depending on immune surveillance with granzyme-B markers, and tumor infiltrating lymphocytes (TIL) is an immunological determinant associated with a better prognosis. Granzyme-B is produced by TIL. The study was aimed to prove the difference in expression of granzyme-B in SCC epithelial cells with cervical dysplasia precancerous lesions as a predictor of immune surveillance. Methods: Correlation analytic study with cross-sectional design on paraffin block patients with SCC and cervical epithelial dysplasia at the Anatomical Pathology Laboratory, RSUP Dr. Kariadi, Semarang, during 2018 and 2019, using granzyme-B immunohistochemistry assays, assessed TIL activation. Results: Most cases of cervical SCC were 40 years old, 50% were stage IIIB, 20% with the final condition died on observation 1 year after the initial diagnosis was made. The uterine cervical SCC group expressed granzyme-B with a mean score of 5.81±1.17 (p=0.001) and the dysplastic epithelial lesion group with a mean score of 6.83±0.95 (p=022), the Mann-Whitney test showed p value = 0.009, so that the test results are statistically significant. Conclusion: Expression of granzyme-B in dysplasia is higher than in uterine cervical SCC.

CD247, a Potential T Cell-Derived Disease Severity and Prognostic Biomarker in Patients With Idiopathic Pulmonary Fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) has high mortality worldwide. The CD247 molecule (CD247, as known as T-cell surface glycoprotein CD3 zeta chain) has been reported as a susceptibility locus in systemic sclerosis, but its correlation with IPF remains unclear.MethodsDatasets were acquired by researching the Gene Expression Omnibus (GEO). CD247 was identified as the hub gene associated with percent predicted diffusion capacity of the lung for carbon monoxide (Dlco% predicted) and prognosis according to Pearson correlation, logistic regression, and survival analysis.ResultsCD247 is significantly downregulated in patients with IPF compared with controls in both blood and lung tissue samples. Moreover, CD247 is significantly positively associated with Dlco% predicted in blood and lung tissue samples. Patients with low-expression CD247 had shorter transplant-free survival (TFS) time and more composite end-point events (CEP, death, or decline in FVC >10% over a 6-month period) compared with patients with high-expression CD247 (blood). Moreover, in the follow-up 1st, 3rd, 6th, and 12th months, low expression of CD247 was still the risk factor of CEP in the GSE93606 dataset (blood). Thirteen genes were found to interact with CD247 according to the protein–protein interaction network, and the 14 genes including CD247 were associated with the functions of T cells and natural killer (NK) cells such as PD-L1 expression and PD-1 checkpoint pathway and NK cell-mediated cytotoxicity. Furthermore, we also found that a low expression of CD247 might be associated with a lower activity of TIL (tumor-infiltrating lymphocytes), checkpoint, and cytolytic activity and a higher activity of macrophages and neutrophils.ConclusionThese results imply that CD247 may be a potential T cell-derived disease severity and prognostic biomarker for IPF.

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

Coadministration of a Clinically Relevant Dexamethasone Dosage With Ablative Radiation Therapy Reduces Peripheral Lymphocytes But Does Not Alter In Vivo Intratumoral Lymphocyte Phenotype or Inhibit Efficacy of Radiation Therapy in a Murine Colorectal Tumor Model.

Dexamethasone is commonly given during radiation therapy (RT) to manage toxicities. Our study examines if dexamethasone coadministration with RT inhibits the RT-induced antitumor T cell response in mouse. Intramuscularly implanted MC38 tumors were irradiated with 15 Gy after establishing for 7 days. Tumor bearing mice were administered dexamethasone using multiple schedules and doses. Peripheral lymphocyte reduction was monitored by complete blood count and intratumoral and tumor draining lymph node (tdLN) populations by flow cytometry. Effector phenotype and function of ex vivo stimulated tumor-infiltrating lymphocytes (TILs) and naïve splenocytes as well as in vivo TILs with or without dexamethasone were monitored by flow cytometry and ELISA. Long course high dose, short course high dose, and short course human equivalent dose dexamethasone reduced peripheral lymphocytes yet did not inhibit survival after irradiation. Short course high dose administration decreased TIL and tdLN lymphocyte activation as well as tdLN mass but did not affect TIL frequencies or change tdLN cell population composition. Dexamethasone inhibited effector function of ex vivo stimulated naïve splenocytes and TILs, but magnitude of IFN-γ secretion was consistently higher in TILs regardless of dexamethasone dose. In vivo analysis of TILs after irradiation and HE dexamethasone treatment showed that TILs had a similar effector phenotype compared with vehicle controls. Dexamethasone reduces blood and tdLN lymphocytes. Dexamethasone also suppresses TIL activation/effector function yet does not affect survival in irradiated MC38 tumor bearing mice, which depend on RT-induced immune responses for therapy efficacy. Additional study in human subjects is warranted.