Tumor-infiltrating Lymphocytes Populations Research Articles

Tumor-Infiltrating T Cells in Skin Basal Cell Carcinomas and Squamous Cell Carcinomas: Global Th1 Preponderance with Th17 Enrichment-A Cross-Sectional Study.

Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) are high-incidence, non-melanoma skin cancers (NMSCs). The success of immune-targeted therapies in advanced NMSCs led us to anticipate that NMSCs harbored significant populations of tumor-infiltrating lymphocytes with potential anti-tumor activity. The main aim of this study was to characterize T cells infiltrating NMSCs. Flow cytometry and immunohistochemistry were used to assess, respectively, the proportions and densities of T cell subpopulations in BCCs (n = 118), SCCs (n = 33), and normal skin (NS, n = 30). CD8+ T cells, CD4+ T cell subsets, namely, Th1, Th2, Th17, Th9, and regulatory T cells (Tregs), CD8+ and CD4+ memory T cells, and γδ T cells were compared between NMSCs and NS samples. Remarkably, both BCCs and SCCs featured a significantly higher Th1/Th2 ratio (~four-fold) and an enrichment for Th17 cells. NMSCs also showed a significant enrichment for IFN-γ-producing CD8+T cells, and a depletion of γδ T cells. Using immunohistochemistry, NMSCs featured denser T cell infiltrates (CD4+, CD8+, and Tregs) than NS. Overall, these data favor a Th1-predominant response in BCCs and SCCs, providing support for immune-based treatments in NMSCs. Th17-mediated inflammation may play a role in the progression of NMSCs and thus become a potential therapeutic target in NMSCs.

Translational and oncologic significance of tertiary lymphoid structures in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is an aggressive tumor with poor survival and limited treatment options. PDAC resistance to immunotherapeutic strategies is multifactorial, but partially owed to an immunosuppressive tumor immune microenvironment (TiME). However, the PDAC TiME is heterogeneous and harbors favorable tumor-infiltrating lymphocyte (TIL) populations. Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop within non-lymphoid tissue under chronic inflammation in multiple contexts, including cancers. Our current understanding of their role within the PDAC TiME remains limited; TLS are complex structures with multiple anatomic features such as location, density, and maturity that may impact clinical outcomes such as survival and therapy response in PDAC. Similarly, our understanding of methods to manipulate TLS is an actively developing field of research. TLS may function as anti-tumoral immune niches that can be leveraged as a therapeutic strategy to potentiate both existing chemotherapeutic regimens and potentiate future immune-based therapeutic strategies to improve patient outcomes. This review seeks to cover anatomy, relevant features, immune effects, translational significance, and future directions of understanding TLS within the context of PDAC.

Galectin-9 as a regulator of immune interactions in the tumor microenvironment and novel therapeutic target in biliary tract cancer.

555 Background: While results of the TOPAZ-1 trial led to inclusion of anti-PD-1 therapy with standard of care gemcitabine/cisplatin chemotherapy for patients with unresectable biliary tract cancer (BTC), the improvement in overall survival is modest: 12.8 vs 11.5 mos. Identification of novel immunotherapeutic strategies remains a high priority. We discovered Galectin-9 (Gal-9), a carbohydrate-binding protein, is expressed in BTC; it has various immunosuppressive effects, including induction of apoptosis in T cells upon binding to its receptor Tim-3. Defining the role of Gal-9 in the BTC microenvironment will inform how to leverage it as a novel therapeutic target. Methods: Tissue microarrays (TMAs) were constructed from tumors of 66 patients who underwent curative-intent resection of BTC from 2000- 2015 at our institution. These TMAs underwent immunohistochemical staining for CD4, CD8, Gal-9 & Tim-3; the percentage of cells staining positive for each marker was quantified. For each marker, martingale residual plots and bias-adjusted log rank tests were used to identify a cut-off value of “low” versus “high” expression where survival difference was maximal. For in vivo mouse experiments, the syngeneic BTC cell line URCCA4.3 was injected subcutaneously into the flank of C57BL/6 mice, followed by 3 weeks of treatment with anti-Galectin-9 or anti-Tim-3 antibody alone or in combination with anti-PD-1 antibody. Tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Results: Median overall survival (mOS) was significantly improved in patients with high Gal-9 expression in their tumors, compared to those with low (32.5 mos vs 17.6 mos, p=0.018). However, when patients with high Gal-9 expression were further stratified according to Tim-3, patients with both high Gal-9 & high Tim-3 expression had significantly worse mOS than those with low Tim-3 (22.7 mos vs 113.9 mos, p=0.031), implying that interaction between Gal-9 and Tim-3 wields significant impact on TIL function. There was no difference in OS based on Tim-3 expression alone. When mice bearing BTC tumors were treated with Gal-9 or Tim-3 antibody monotherapy, there was no significant change in tumor growth versus control, but when either was used in combination with PD-1 inhibition, there was a significant reduction in tumor burden at study endpoint. Mice treated with either dual therapy had a significant decrease in CD4+ Treg and increase in CD4+ effector memory TIL populations compared to monotherapy (p<0.05). Conclusions: This study provides unique insight into the role for targeting the Gal-9/Tim-3 pathway in BTC. Our clinical data highlights that co-expression of these markers in BTC tumors is associated with significantly worse OS. Our in vivo pre-clinical studies demonstrate that blockade of either Gal-9 or Tim-3 synergizes with PD-1 targeted antibodies for superior anti-tumor efficacy.

Epigenetic modulation of antitumor immunity and immunotherapy response in breast cancer: biological mechanisms and clinical implications.

Breast cancer (BC) is the most common non-skin cancer and the second leading cause of cancer death in American women. The initiation and progression of BC can proceed through the accumulation of genetic and epigenetic changes that allow transformed cells to escape the normal cell cycle checkpoint control. Unlike nucleotide mutations, epigenetic changes such as DNA methylation, histone posttranslational modifications (PTMs), nucleosome remodeling and non-coding RNAs are generally reversible and therefore potentially responsive to pharmacological intervention. Epigenetic dysregulations are critical mechanisms for impaired antitumor immunity, evasion of immune surveillance, and resistance to immunotherapy. Compared to highly immunogenic tumor types, such as melanoma or lung cancer, breast cancer has been viewed as an immunologically quiescent tumor which displays a relatively low population of tumor-infiltrating lymphocytes (TIL), low tumor mutational burden (TMB) and modest response rates to immune checkpoint inhibitors (ICI). Emerging evidence suggests that agents targeting aberrant epigenetic modifiers may augment host antitumor immunity in BC via several interrelated mechanisms such as enhancing tumor antigen presentation, activation of cytotoxic T cells, inhibition of immunosuppressive cells, boosting response to ICI, and induction of immunogenic cell death (ICD). These discoveries have established a highly promising basis for using combinatorial approaches of epigenetic drugs with immunotherapy as an innovative paradigm to improve outcomes of BC patients. In this review, we summarize the current understanding of how epigenetic processes regulate immune cell function and antitumor immunogenicity in the context of the breast tumor microenvironment. Moreover, we discuss the therapeutic potential and latest clinical trials of the combination of immune checkpoint blockers with epigenetic agents in breast cancer.

Membrane (CD8⁺PD-1⁺ and CD4+PD-1⁺) and soluble (sPD-1 and sPD-L1) forms of immune checkpoints in melanoma, breast cancer, and oral mucosal cancer patients: A observational study

Background. The PD-1/PD-L1 pathway plays an important role in tumor evasion from immunological surveillance. In addition to the membrane forms of PD-1 and PD-L1, there are soluble variants: sPD-1 and sPD-L1. Both membrane and soluble forms have immunoregulatory properties and can affect the function and number of different immune cell populations.
 Aim. To study the relationship between the initial level of CD8⁺PD-1⁺ and CD4+PD-1⁺ lymphocytes and soluble forms of sPD-1 and sPD-L1 with the percentage of the main effector and regulatory populations of peripheral blood (PB) lymphocytes and tumor-infiltrating lymphocytes.
 Materials and methods. The study included melanoma, breast cancer and the oral mucosa cancer patients. The percentage of cell populations of PB lymphocytes and tumor-infiltrating lymphocytes was determined by flow cytometry before treatment. The concentrations of sPD-1 and sPD-L1 proteins were studied in blood serum using enzyme immunoassay.
 Results. The relationship of the level of CD8⁺PD-1⁺ cells with certain populations of CD8-lymphocytes in PB and tumor tissue was found. In the PB of melanoma patients with CD8⁺CD11b⁺CD28⁺ and CD8⁺CD11b⁻CD28⁻ T cells, in breast cancer patients with a population of CD8⁺CD11b⁺CD28⁺ lymphocytes. In the tumor tissue of all patients there was a positive correlation with a population of regulatory CD8⁺CD11b⁻CD28⁻ T cells. The immunoregulatory properties of sPD-1 and sPD-L1 were confirmed. Both sPD-1 and sPD-L1 levels were positively correlated with the number of suppressor CD8⁺CD11b⁻CD28⁻ T cells and negatively with the level of CD8 lymphocytes, CD8⁺CD11b⁺CD28⁺ cytotoxic/memory T cells, B cells and activated CD25 lymphocytes.
 Conclusion. The results of the study can make a certain contribution to the study of the prognostic significance of membrane and soluble forms of PD-1 and PD-L1, taking into account the peculiarities of their relationship with suppressor and effector populations of lymphocytes of systemic and local immunity.

Abstract B004: Neoantigen adenoviral cancer vaccine generates improved CD8+ T-cell responses compared to adjuvanted peptide vaccine

Abstract Development of large, functional CD8+ T cell responses is crucial for successful cancer vaccine therapeutics. Neoantigens have demonstrated their potential as cancer vaccine immunogens, but improvements in delivery method are still necessary. In this work, we characterized five candidate Adenovirus Serotype 26 (Ad26) cancer vaccines for prophylactic efficacy in the MC-38 cancer model. All five single shot vaccines generated cellular immune responses at a higher level than prime boost peptide vaccination. The vaccines generated varying degrees of protection against tumor growth. The best Ad26 vaccine candidates showed improved protection compared to long synthetic peptide vaccines containing the same immunogens, the current standard in the field. Ad26 expressing seven neoantigens conjugated to the Herpes Viral Protein 22 (VP22) (Ad26.VP22.7Epi) was found to reduce tumor growth the most. Ad26.VP227Epi was compared to peptide vaccination in immune recall post challenge studies, in both the spleen and the tumor. Both Ad26.VP22.7Epi and Peptide showed a clear recall response after challenge in the spleen. This response was primarily focused on the Adpgk neoantigen but Ad26.VP22.7Epi additionally induced responses to the Irgq neoantigen while the peptide vaccine had stronger responses against the Reps1 neoantigen. The Ad26.VP227Epi vaccine generated increased numbers of infiltrating CD8+ T-cells, IFN+ CD8+ T-cells, and CD107a+ CD8+ T-cells, which all correlated with reduced tumor growth. We used single cell RNA-seq (scRNA-seq) to characterize tumor infiltrating lymphocyte populations (defined as CD45+) generated by the two platforms. We found that both Ad26.VP22.7Epi and Peptide induced higher numbers of infiltrating CD8+ T-cells within the tumor as compared to Sham. Single-cell analysis also showed higher numbers of tumor infiltrating Tregs in peptide vaccinated mice. Higher numbers of CD8+ T-cells correlated with protection as expected but interestingly increased numbers of Tregs positively correlated with tumor volume only among vaccinated mice, which could further explain differences between Ad vector and peptide vaccination. Differential gene expression analysis of CD8+ T-cells showed an upregulation of Th1 pathways and general T cell activation when comparing Ad vector vaccinated mice to peptide vaccinated. Peptide vaccinated CD8+ T-cells showed upregulation in the IL-10 pathway, potentially caused by an increased Tregs. Further investigation of these differences among CD8+ T cell and Treg populations could further elucidate potential mechanisms explaining the differences in platform efficacy. Citation Format: Gabriel Dagotto, Alessandro Colarusso, Robert Patio, David Li, Tochi Anioke, Victoria Giffin, Malika Aid, Dan Barouch. Neoantigen adenoviral cancer vaccine generates improved CD8+ T-cell responses compared to adjuvanted peptide vaccine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B004.

Concomitant Expression of CD39, CD69, and CD103 Identifies Antitumor CD8+ T cells in Breast Cancer Implications for Adoptive Cell Therapy.

In cancer, an effective immune response involves the action of several different cell types, among which CD8 T cells play a major role as they can specifically recognize and kill cancer cells via the release of cytotoxic molecules and cytokines, being of major importance for adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). The inflammation resulting from the tumor growth attracts both activated and bystander T cells. For an effective antitumor response, the T cell must express a specific group of chemokine receptors and integrins which include CD103, CD39, CD69, and CD25. These markers had already been analyzed in various cancers, not including breast cancer and their subsequent subtypes, until now. To analyze, the key receptors on ex vivo expanded tumor-infiltrating lymphocytes in luminal A and luminal B breast cancer (BC) subtypes. We were successful in expanding TILs ex vivo using a standard TIL culture condition from a cohort study of 15 primary luminal A and luminal B breast cancer patients. Furthermore, we examined the expression of CD103, CD39, CD69, and CD25 biomarkers after the expansion by flow cytometry. We found that the information about the percentage of TILs obtainable after the ex vivo expansion is not associated to nor it is dependent on the heterogeneity of the TIL population before the expansion and does not differ by the molecular subtype (p>0.05). We also found that there is a major population of memory-resident antitumor CD8+CD103+CD39+ and CD8+CD103+CD69+ TILs present in the stroma after the expansion when compared to CD4 immunosubtypes (p<0.0001). Only the CD8+CD103+CD39+ subpopulation was related to BC subtype (0.0009). Evidence from our study suggests that CD8 TILs present in the stroma of luminal A and luminal B breast cancer patients can be quantified and phenotyped by flow cytometry and be further expanded ex vivo. The immuno-phenotyping of these markers may be targeted to improve the success of immunotherapeutic approaches, such as adoptive cellular therapy (ACT) in patients with BC.

Abstract B023: Using hypoxia to improve tumor infiltrating lymphocytes therapy in different subtypes of renal cell carcinoma

Abstract BACKGROUND: Improving the long-term efficacy for advanced renal cell carcinoma (RCC) remains challenging after progression on first line therapy. Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) is a promising personalized immunotherapeutic approach to treating solid tumors. Previously, TIL therapy for clear cell RCC patients showed moderate success but did not account for the distinct immune microenvironments in different subtypes of RCC such as clear cell (ccRCC), papillary (pRCC) or unclassified (uRCC) known to have different response to immunotherapy. In addition, hypoxia plays a critical role in tumor progression and therapy resistance in RCC. Therefore, the aim of this study was to evaluate TIL expansion from different subtypes of RCC under varying oxygen concentrations. METHODS: Tumor types collected from 48 patients included ccRCC (81.25%), pRCC (16.7%) and uRCC (8.3%). Tumors were minced into fragments and cultured for 4 weeks in media supplemented with high dose IL-2 (6000 IU/mL). At the end of the 4 weeks, TIL were further expanded using a rapid expansion protocol (REP) in either 20% or 5% oxygen. TIL expansion, reactivity to autologous tumor, and phenotype were evaluated in pre-REP and post-REP samples. RESULTS: Among the ccRCC, 91.7% of the samples expanded, while 100% of the pRCC and uRCC expanded. pRCC expanded more TIL than ccRCC (1.05e8 vs 6.7e7 total TIL expanded, respectively) while uRCC expanded less than the other subtypes (2.03e7). Pre-REP reactivity against autologous tumors was high for all the subtypes (88.3%, 75% and 100% for ccRCC, pRCC and uRCC respectively). The T-cell phenotype across subtypes showed a tendency of more CD4+ T-cells and less CD8+ T-cells for pRCC and uRCC, compared to ccRCC. All subtypes that underwent REP were able to expand in both 20% and 5% O2 (hypoxia) conditions. TIL expanded in hypoxia had a significantly higher percentage of tissue resident memory T-cells (CD69+CD103+; 15.1%, p&amp;lt;0.0001) when compared to the pre-REP TIL population (2%) or TIL expanded at 20% O2 (3.1%). CONCLUSION: These results demonstrate the feasibility of expanding tumor-reactive TIL from different subtypes of RCC for the first time, and highlight the advantage of exposing TIL to hypoxic conditions to enhance the differentiation of resident memory T-cells in TIL products. Citation Format: Marine Potez, Mohammed Alkhouli, Johannes Ali, Michael Carter, Matthew Beatty, Shari Pilon-Thomas, Jad Chahoud. Using hypoxia to improve tumor infiltrating lymphocytes therapy in different subtypes of renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B023.

Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors

BackgroundImmunologically cold tumors with an ‘immune desert’ phenotype lack tumor-infiltrating lymphocytes (TILs) and are typically impervious to systemic immune checkpoint blockade (ICB). Intratumoral treatment of tumors with immunomodulatory agents can...

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.

Abstract 4052: Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells

Abstract Background: The autologous cell therapy field has investigated numerous novel strategies and processes to improve response rates and expand the use of adoptive cell therapies (ACT) to patient suffering from a broad range of cancers. Tumor infiltrating lymphocytes (TIL) can be harvested from tumors, expanded in vitro, and infused to patients leading to substantial clinical benefits in some patient populations. Tumor reactivity of TIL can vary greatly, representing a key limiting factor of bulk TIL therapies. The ability to generate an autologous cell product enriched in tumor reactive cells while limiting the presence of potentially detrimental and/or competitive non-reactive cells is highly desirable for next generation TIL products. Here we demonstrate that direct selection of antigen experienced TIL from tumor dissociates is feasible and allows to obtain functional cells enriched for tumor reactivity from various tumor indications. Methods: Patient-derived tumor material from various indications, including ovarian, kidney, colorectal cancer and lung tumors were first processed into single cell suspensions, maximizing TIL recovery and viability. Antigen experienced TIL were immediately selected based on the co-expression of PD-1 and CD39, using fluorescence-activated single cell sorting (FACS). The sorted cells or the unselected counterparts were expanded in a rapid expansion protocol (REP), then analyzed for phenotypic and functional characteristics and reactivity against autologous tumor cells. Results: The PD-1+ CD39+ selection strategy consistently allowed sorting of a population of viable TIL that was amenable to in vitro culture from tumors of various cancer indications. The selected TIL successfully expanded in a rapid expansion protocol. TCR sequencing analysis revealed that the PD-1+ CD39+ sorted populations were enriched in unique clones compared to the unselected populations. Expanded PD-1+ CD39+ selected cells demonstrated the ability to produce key effector cytokines upon restimulation in polyclonal assays. Importantly, the PD-1+ CD39+ expanded TIL were enriched for tumor reactive T cells and showed improved cytotoxic activity against autologous tumors. Conclusions: PD-1+ CD39+ selected TIL can be successfully isolated and expanded in vitro, generating a TIL product of superior reactivity in multiple cancer indications. PD-1+ CD39+ selected TIL showed increased cytokine secretion and cytotoxic activity against autologous material, indicating that this selection strategy enriches for functional tumor-reactive lymphocytes, which is likely to be a key feature of successful ACT. Citation Format: Christophe Pedros, Larissa Pikor, Anna Fritzsche, Zachary K. Jilesen, Bethany Macleod, Niloufar Khojandi, Bryant Thompson, Matthew Thayer, Nikolas Bryan, Emily Carron, Sowbarnika S Ratliff, April Fraley, Jake Nikota, Robert Fisher, Sebastien Delpeut, Anna Kluew, Madysson Scott, Christian Laing, Leo He, Antoine Bernard, Nathalie Brassard, Simon Turcotte, Timothy J Langer, David Stojdl, Stewart Abbot, Barbara Sennino. Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4052.

Abstract 2828: Impact of sequencing of immune checkpoint blockade and targeted radionuclide therapy on murine tumor response

Abstract Background: Sequencing of immune checkpoint inhibitors (ICI) and external beam radiation therapy (EBRT) for cancer treatment has been studied, but the optimal sequencing has yet to be determined. While some studies have noted therapeutic advantages of priming the tumor immune microenvironment with EBRT prior to ICI, others have described the benefit of modulating the tumor infiltrating lymphocyte (TIL) population with ICI before EBRT. Targeted radionuclide therapy (TRT) approaches allow investigation of how irradiation by a tumor-targeted radionuclide and differences in emission type, linear energy transfer, and dose rate affect optimal timing of ICI administration. NM600 is an alkylphosphocholine analog selectively taken up by tumors capable of chelating numerous radionuclides for comparative studies. Objective: We use two immunologically cold tumor models, MOC2 head and neck squamous cell carcinoma and B78 melanoma, to describe the influence of dose rate on type I interferon (IFN1) signaling and the effect of ICI and 90Y-, 177Lu-, and 225Ac-NM600 TRT sequences on tumor response. Methods: 90Y, 177Lu, or 225Ac were added to culture media in activities estimated using GEANT4 Monte Carlo to deliver 12 Gy to the cell monolayer. qPCR was performed on cDNA from cells irradiated with EBRT, 90Y, 177Lu, or 225Ac, and harvested on days 1, 3, or 7. In vivo dosimetry was performed using the Monte Carlo-based RAPID platform utilizing serial PET/CT or SPECT/CT imaging and/or longitudinal biodistribution. Differences over time (days 4, 7, 14, 21, 28 after RT) in TIL and systemic immune cell populations were measured by flow cytometry following no treatment, 12 Gy EBRT, or 90Y-, 177Lu-, or 225Ac-NM600 in MOC2 tumors. Mice bearing B78 tumors received 1.5 Gy 90Y-, 177Lu-, or 225Ac-NM600, or no radiation on day 1 +/- ICI (anti-CTLA4 + anti-PDL1) on days -3/0/3 (early), 4/7/10 (middle), or 11/14/17 (late). Mice were monitored for tumor growth and survival. Results: TRT and EBRT induced IFN1 responses in MOC2 cells. MOC2 cells treated every 24h with EBRT-matched 90Y/225Ac dose rates led to upregulation of IFN1-associated Ifnb1 and Mx1, mimicking radionuclide-induced responses. Increased tumor CD8/Treg ratios and decreased Tregs were observed at day 7 following all RT forms in MOC2 tumors. Long half-life 225Ac-NM600 (90Y: 65h; 177Lu: 161h; 225Ac: 240h) induced similar TIL changes at day 21. For 1.5 Gy 90Y-, 177Lu-, and 225Ac-NM600, B78 tumor growth delay and statistically significant overall survival benefit over respective TRT monotherapy and control groups was observed with early (day -3/0/3) dual ICI administration. Conclusions: These studies demonstrate novel immunomodulatory effects of α- and β- emitting TRT and the capacity to achieve substantial antitumor responses with appropriate TRT + ICI sequencing. These results may inform clinical trial design of TRT + ICI regimens for patients with metastatic cancers. Citation Format: Caroline P. Kerr, Joseph J. Grudzinski, Carolina A. Ferreira, David Adam, Julia Sheehan-Klenk, Amber M. Bates, Won Jong Jin, Ohyun Kwon, Justin C. Jagodinsky, Maria Powers, Raghava N. Sriramaneni, Paul A. Clark, Luke Zangl, Thanh Phuong T. Nguyen, Anatoly N. Pinchuk, Cynthia Choi, Christopher F. Massey, Reinier Hernandez, Bryan Bednarz, Jamey P. Weichert, Zachary S. Morris. Impact of sequencing of immune checkpoint blockade and targeted radionuclide therapy on murine tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2828.

Abstract 4060: Selective Neoantigens Peptide TILs (snapTILs): A next generation tumor infiltrating lymphocytes (TIL) therapy platform

Abstract Background: Immunotherapies using autologous tumor infiltrating lymphocytes (TILs) have become effective treatment modalities for several patients with solid tumors, yielding a response rate of around 49% in patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TILs from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of these administered TILs recognize tumor antigens and most are non-mutated public antigens. Moreover, expression of these antigens on normal cells can trigger central and peripheral tolerance mechanisms. Against this background, cancer neoantigens derived from private mutations represent an ideal class of cancer antigens to target because they are highly tumor specific by nature, therefore reducing the potential induction of central and peripheral tolerance. We recently developed a new technology to enrich TILs with predefined neoantigen specificities, which we termed Selective Neo Antigens Peptides TILs (snapTILs). Here we describe the efficacy studies of a melanoma and pancreatic cancer patient’s snapTILs done in autologous settings both ex vivo and in vivo. Methods: Our workflow consisted of a multistep process performed on a patient-specific basis. Briefly, mutations were called by whole-exome sequencing of tumor vs. normal DNA to generate a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay. Selected peptides were then incubated with TILs isolated from the patient’s tumor biopsy, resulting in TIL population enriched of T cells that recognize the neopeptides (snapTILs). After rapid expansion, snapTIL efficacy was tested by checking their ability to infiltrate in patient-derived tumor organoids, their activation status (IFNγ), cytotoxicity (Granzyme B), and T-cell exhaustion markers. Lastly, snapTILs in vivo efficacy was tested in human melanoma and pancreatic cancer xenograft models using hIL-2 transgenic mice. Results: Our initial data showed that the levels of infiltration of snapTILs into tumor organoids was significantly higher compared to IL2-stimulated TILs in both cancer models. Also, snapTILs recognized tumors with great specificity as they showed significantly less infiltration in organoids generated from generally uninvolved tissues from the same patients. Moreover, snapTILs were found to be more cytotoxic in nature than the IL2-stimulated TILs in terms of IFNγ and Granzyme B secretion after coculture with autologous tumor cells. Our in vivo data confirmed these observations by showing 70% tumor growth inhibition in highly immunogenic melanoma and 50% tumor growth restriction in a poorly immunogenic pancreatic tumor model. In conclusion, our approach proves the potential for personally tailored immunotherapies to yield more effective and safer cancer treatments using snapTILs. Citation Format: Tithi Ghosh Halder, Shelby Rheinschmidt, Sydney Adamson, Anna Larson, Taylor Bargenquast, Ryan Rodriguez del Villar, Serina Ng, Kate Gutowsky, Alexis Weston, Trason Thode, Mohan Kaadige, Erin Kelley, Jorge Soria-Bustos, Jorge Giron, John Altin, Raffaella Soldi, Sunil Sharma. Selective Neoantigens Peptide TILs (snapTILs): A next generation tumor infiltrating lymphocytes (TIL) therapy platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4060.

Terminally exhausted CD8+ T cells resistant to PD-1 blockade promote generation and maintenance of aggressive cancer stem cells.

Heterogeneity within the tumor infiltrating lymphocyte (TIL) population limits immunotherapeutic efficacy against cancer. Between two subpopulations of exhausted CD8+ TILs (progenitor-exhausted, TPEX; terminally-exhausted, TTEX), TTEX cells remain unresponsive to anti-PD1 therapy. Deciphering whether and how PD1-resistant TTEX cells engage in tumor promotion could improve the response to immunotherapy. Here, we report that TTEX cells actively participate in tumor progression by modulating cancer stem cells (CSC). TTEX cells strongly correlated with elevated CSC frequency in poorly immune-infiltrated (CD8+ TIL low) advanced human breast and ovarian carcinomas. TTEX directly upregulated CSC frequency in vitro, which was not affected by anti-PD1 treatment. The TTEX-influenced CSCs were highly clonogenic and exhibited a multi-drug resistant phenotype, overexpressing drug efflux pumps like ABCC1 and ABCB1. These CSCs were highly invasive, displaying increased invadopodia development and elevated cofilin, CXCR4, and MMP7 expression. The invasive properties along with epithelial-mesenchymal plasticity of TTEX-educated CSCs increased metastasis in vivo. TTEX increased cell surface levels and activation of VEGFR2 in CSCs, and silencing or inhibition of VEGFR2 reversed the CSC-stimulatory effects of TTEX. LAMP3 and NRP1 on the surface of TTEX stimulated VEGFR2 in CSCs to promote aggressiveness. Cumulatively, these findings suggest that screening carcinoma patients for both CD8+ TIL and TTEX frequency prior to anti-PD1-therapy could improve patient outcomes. Additionally, targeting the LAMP3/NRP1-VEGFR2 axis could be a therapeutic strategy in advanced carcinoma patients with limited CD8+ T cell infiltration and high TTEX frequency.

CD103 and CD39 coexpression identifies neoantigen-specific cytotoxic T cells in colorectal cancers with low mutation burden

BackgroundExpression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific...

Tissue-Resident Memory T Cells in Pancreatic Ductal Adenocarcinoma Coexpress PD-1 and TIGIT and Functional Inhibition Is Reversible by Dual Antibody Blockade.

Pancreatic ductal adenocarcinoma (PDAC) has a poor clinical outlook. Responses to immune checkpoint blockade are suboptimal and a much more detailed understanding of the tumor immune microenvironment is needed if this situation is to be improved. Here, we characterized tumor-infiltrating T-cell populations in patients with PDAC using cytometry by time of flight (CyTOF) and single-cell RNA sequencing. T cells were the predominant immune cell subset observed within tumors. Over 30% of CD4+ T cells expressed a CCR6+CD161+ Th17 phenotype and 17% displayed an activated regulatory T-cell profile. Large populations of CD8+ tissue-resident memory (TRM) T cells were also present and expressed high levels of programmed cell death protein 1 (PD-1) and TIGIT. A population of putative tumor-reactive CD103+CD39+ T cells was also observed within the CD8+ tumor-infiltrating lymphocytes population. The expression of PD-1 ligands was limited largely to hemopoietic cells whilst TIGIT ligands were expressed widely within the tumor microenvironment. Programmed death-ligand 1 and CD155 were expressed within the T-cell area of ectopic lymphoid structures and colocalized with PD-1+TIGIT+ CD8+ T cells. Combinatorial anti-PD-1 and TIGIT blockade enhanced IFNγ secretion and proliferation of T cells in the presence of PD-1 and TIGIT ligands. As such, we showed that the PDAC microenvironment is characterized by the presence of substantial populations of TRM cells with an exhausted PD-1+TIGIT+ phenotype where dual checkpoint receptor blockade represents a promising avenue for future immunotherapy.

Association of Genomic Instability Score, Tumor Mutational Burden, and Tumor-Infiltrating Lymphocytes as Biomarkers in Uterine Serous Carcinoma.

Background: Uterine serous carcinomas represent 10% of uterine carcinomas but account for nearly 40% of deaths from the disease. Improved molecular characterization of these tumors is instrumental in guiding targeted treatment and improving outcomes. This study assessed the genomic instability score (GIS), tumor mutational burden (TMB), and tumor-infiltrating lymphocytes (TILs) in patients with USC. Methods: A retrospective cohort study evaluated patients with USC following staging surgery. The GIS and TMB were determined from archived specimens. We evaluated the tumoral expression of CD3, CD4, CD8, FOXP3, and CD68 using immunohistochemistry. T-tests were used to assess associations of TILs with the GIS. Results: We evaluated 53 patients with USC. The median GIS was 31 (range: 0−52) and a higher GIS was not associated with progression-free (PFS) or overall survival (OS). The median TMB was 1.35 mt/Mb; patients with TMB > 1.35 mt/Mb had improved PFS and OS (p = 0.005; p = 0.002, respectively). Tumors with increased CD3+ and CD4+ immune cells had a higher mean GIS (p = 0.013, p = 0.002). Conclusions: TMB > 1.35 mt/Mb was associated with improved survival in USC patients, whereas the GIS was not. Lower TMB thresholds may provide prognostic value for less immunogenic tumors such as USC. In this limited cohort, we observed that increased TIL populations were correlated with a higher GIS.

Canine melanoma: A review of diagnostics and comparative mechanisms of disease and immunotolerance in the era of the immunotherapies.

Melanomas in humans and dogs are highly malignant and resistant to therapy. Since the first development of immunotherapies, interest in how the immune system interacts within the tumor microenvironment and plays a role in tumor development, progression, or remission has increased. Of major importance are tumor-infiltrating lymphocytes (TILs) where distribution and cell frequencies correlate with survival and therapeutic outcomes. Additionally, efforts have been made to identify subsets of TILs populations that can contribute to a tumor-promoting or tumor-inhibiting environment, such as the case with T regulatory cells versus CD8 T cells. Furthermore, cancerous cells have the capacity to express certain inhibitory checkpoint molecules, including CTLA-4, PD-L1, PD-L2, that can suppress the immune system, a property associated with poor prognosis, a high rate of recurrence, and metastasis. Comparative oncology brings insights to comprehend the mechanisms of tumorigenesis and immunotolerance in humans and dogs, contributing to the development of new therapeutic agents that can modulate the immune response against the tumor. Therapies that target signaling pathways such as mTOR and MEK/ERK that are upregulated in cancer, or immunotherapies with different approaches such as CAR-T cells engineered for specific tumor-associated antigens, DNA vaccines using human tyrosinase or CGSP-4 antigen, anti-PD-1 or -PD-L1 monoclonal antibodies that intercept their binding inhibiting the suppression of the T cells, and lymphokine-activated killer cells are already in development for treating canine tumors. This review provides concise and recent information about diagnosis, comparative mechanisms of tumor development and progression, and the current status of immunotherapies directed toward canine melanoma.

NIMG-58. IMMUNOPET OF 89ZR-DFO-CD69 AB VISUALIZES T-CELL ACTIVATION AND PREDICTS SURVIVAL FOLLOWING IMMUNOTHERAPY IN MURINE GBM MODEL

Abstract INTRODUCTION Glioblastoma (GBM) is the most malignant brain tumor in adults, with a dismal prognosis despite aggressive therapy. Immunotherapy is currently being evaluated as a treatment modality for recurrent GBM. MRI is not adequate for response assessment to immunotherapy even after using refined response assessment criteria. Thus, there is a need for the development of neuroimaging techniques for response assessment. T-cells are key mediators of cancer immunotherapy responses and upregulation of CD69 is a marker of T-cell activation. Our aim is to use PET/CT imaging to non-invasively quantify CD69 in vivo, following immunotherapy, and correlate the expression to survival. METHODS CD69 was evaluated by flow cytometry and immunofluorescence staining on human and mouse in vitro activated T-cells and on dissociated tumors from GL261 glioma-bearing mice treated with anti-PD1/anti-CTLA4 immunotherapy (ICI). Single-cell RNA sequencing (ScRNAseq) datasets from recurrent GBM patients receiving (n=20) or not receiving (n=22) ICI were examined for CD69 expression on tumor infiltrating lymphocyte (TIL) populations. PET/CT was performed on mice (n=30) receiving radiolabeled anti-CD69 antibody (89Zr-DFO-anti-CD69) to evaluate response to ICI therapy. Standard uptake values (SUV) were compared between ICI and controls and in relationship to survival. RESULTS We confirmed CD69 upregulation upon T-cell activation in vitro. Ex vivo, CD69 expression significantly increased on TILs early after ICI treatment compared to control (63.46% vs 24.37% CD69+/TILs, respectively; p=0.017). ScRNAseq demonstrated significant elevated CD69 expression in almost all TIL populations tested in recurrent GBM patients treated with ICI compared with a control group. ImmunoPET demonstrated significantly higher anti-CD69 tracer uptake in ICI-treated mice compared with controls. Most importantly, we observed a strong positive correlation between survival and immunoPET SUV (r=0.9425, p=0.016) in the ICI-treatment group, but not within the control group. CONCLUSIONS Our study demonstrates the potential incorporation of CD69 ImmunoPET as response assessment to ICI for GBM patients.

Glioma cancer stem cells modulating the local tumor immune environment.

Glioma stem cells (GSCs) drive the resistance mechanism in glioma tumors and mediate the suppression of innate and adaptive immune responses. Here we investigate the expression of mesenchymal-epithelial transition factor (c-Met) and Fas receptor in GSCs and their role in potentiating the tumor-mediated immune suppression through modulation of tumor infiltrating lymphocyte (TIL) population. Tumor tissues were collected from 4 patients who underwent surgery for glioblastoma. GSCs were cultured as neurospheres and evaluated for the co-expression of CD133, c-Met and FasL through flow cytometry. TILs were isolated and evaluated for the lymphocyte subset frequencies including CD3 +, CD4 +, CD8 +, regulatory T cells (FOXP3 + CD25) and microglia (CD11b + CD45) using flow cytometry. Our findings revealed that a significant population of GSCs in all four samples expressed c-Met (89–99%) and FasL (73–97%). A significantly low microglia population was found in local immune cells ranging from 3 to 5%. We did not find a statistically significant correlation between expressions of c-Met + GSC and FasL + GSC with local and systemic immune cells. This may be regarded to the small sample size. The percent c-Met + and FasL + GSC population appeared to be related to percent cytotoxic T cells, regulatory T cells and microglia populations in glioblastoma patients. Further investigation is warranted in a larger sample size.