Resident Memory T-cells Research Articles

Evaluation of anti-vector immune responses to adenovirus-mediated lung gene therapy and modulation by αCD20

Although the last decade has seen tremendous progress in drugs that treat cystic fibrosis (CF) due to mutations that lead to protein misfolding, there are approximately 8%-10% of subjects with mutations that result in no significant CFTR protein expression demonstrating the need for gene editing or gene replacement with inhaled mRNA or vector-based approaches. A limitation for vector-based approaches is the formation of neutralizing humoral responses. Given that αCD20 has been used to manage post-transplant lymphoproliferative disease in CF subjects with lung transplants, we studied the ability of αCD20 to module both T and B cell responses in the lung to one of the most immunogenic vectors, E1-deleted adenovirus serotype 5. We found that αCD20 significantly blocked luminal antibody responses and efficiently permitted re-dosing. αCD20 had more limited impact on the Tcell compartment, but reduced tissue resident memory Tcell responses in bronchoalveolar lavage fluid. Taken together, these pre-clinical studies suggest that αCD20 could be re-purposed for lung gene therapy protocols to permit re-dosing.

Abstract 5563: Novel approaches to measuring tumor-mutational burden provide insights into tumor-immune interaction in primary melanoma

Abstract Background: Tumor mutation burden (TMB) has been widely explored in metastatic melanoma as a proxy of neoantigenic load. In contrast, the role of TMB on the biology of primary melanoma disease progression is less understood. In this study, we explored the associations between the immune context of melanoma subtypes and TMB as measured by several standard and novel criteria. Methods: Whole-genome-sequencing and multiplex immunohistochemistry (mIHC) were used to characterize the genomic and immune landscape of a cohort of 116 primary melanomas, including cutaneous non-acral (n=59), acral (n=39) and mucosal (n=18) subtypes, as well as 5 mucosal metastases. mIHC was used to identify intratumoral CD8+ T-cells, memory T-cells, macrophages, dendritic cells, B-cells and PD-L1 expression. Standard measures of TMB -total mutational burden (tTMB) and non-synonymous (ns) mutational burden (nsTMB)- as well as novel measures that incorporate multiplicity and clonality -ns persistent mutation burden (ns-pTMB), and ns mutation dosage (ns-dosTMB) were calculated, and their association with immune profiles and clinical-pathological factors established. Results: In cutaneous non-acral melanomas, ns-pTMB and ns-dosTMB were positively associated with CD8+ T cell proportions (p=0.02 for both comparisons). Clonal, but not subclonal ns-pTMB from mutations present in single-copy regions of the genome were positively associated with higher tumor-resident CD103+CD8 T-cell (p=0.01) and B-cell proportions (p=0.02). Higher single-copy ns-pTMB was also associated with tumors presenting early/intermediate regression compared to late regression. In mucosal melanomas, clonal ns-dosTMB had the strongest positive association with the presence of macrophages (CD68, p=0.005). Additionally, macrophages (p=0.01), tissue resident memory T-cells (p=0.02), PDL1+ cells (p=0.03) and B-cells (p=0.048) were found to be positively associated with clonal ns-pTMB from mutations present in multiple copies, which was also positively associated with the presence of lymphatic invasion (p=0.03). Mucosal patients who recurred had lower levels of single-copy ns-pTMB (p=0.03). In acral melanomas, all measures of TMB were positively associated with the presence of dendritic cells (CD11c), with no particular role for clonality observed. In both cutaneous non-acral (p=0.046) and acral melanomas (p=0.03), but not mucosal (p=0.4), clonal ns-dosTMB was found positively associated with tumor mitotic rate. Conclusion: Novel TMB measures reveal associations between tumor and immune context that are not captured by standard TMB measures alone in primary melanomas. Different TMB measures are linked to distinct immune populations across melanoma subtypes that harbor unique genomic and immune landscapes. Clonal mutations play a dominant role in the associations of TMB with immune cell populations. Citation Format: Eva R. Shteinman, Nigel Maher, Jordan W. Conway, Grace H. Attrill, Felicity Newell, Nic Waddell, Nicholas Hayward, John V. Pearson, Georgina V. Long, Richard A. Scolyer, James S. Wilmott, Ismael A. Vergara. Novel approaches to measuring tumor-mutational burden provide insights into tumor-immune interaction in primary melanoma [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 5563.

IPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer

Functionally rejuvenated human papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, leading to difficulty in treating patients with advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the major obstacle is rejection by the patient immune system. To overcome this, we develop HLA-A24&-E dual integrated HPV-rejTs after erasing HLA class I antigens. These rejTs effectively suppress recipient immune rejection while maintaining more robust cytotoxicity than original cytotoxic T lymphocytes. Single-cell RNA sequencing performed to gain deeper insights reveal that HPV-rejTs are highly enriched with tissue resident memory Tcells, which enhance cytotoxicity against cervical cancer through TGFβR signaling, with increased CD103 expression. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of Tcell receptor (TCR) and increased TCR-mediated target cell death. We believe that our work will contribute to feasible "off-the-shelf" Tcell therapy with robust anti-cervical cancer effects.

Why drug exposure is frequently associated with T-cell mediated cutaneous hypersensitivity reactions.

Cutaneous hypersensitivity reactions represent the most common manifestation of drug allergy seen in the clinic, with 25% of all adverse drug reactions appearing in the skin. The severity of cutaneous eruptions can vastly differ depending on the cellular mechanisms involved from a minor, self-resolving maculopapular rash to major, life-threatening pathologies such as the T-cell mediated bullous eruptions, i.e., Stevens Johnson syndrome/toxic epidermal necrolysis. It remains a significant question as to why these reactions are so frequently associated with the skin and what factors polarise these reactions towards more serious disease states. The barrier function which the skin performs means it is constantly subject to a barrage of danger signals, creating an environment that favors elicitation. Therefore, a critical question is what drives the expansion of cutaneous lymphocyte antigen positive, skin homing, T-cell sub-populations in draining lymph nodes. One answer could be the heterologous immunity hypothesis whereby tissue resident memory T-cells that express T-cell receptors (TCRs) for pathogen derived antigens cross-react with drug antigen. A significant amount of research has been conducted on skin immunity in the context of contact allergy and the role of tissue specific antigen presenting cells in presenting drug antigen to T-cells, but it is unclear how this relates to epitopes derived from circulation. Studies have shown that the skin is a metabolically active organ, capable of generating reactive drug metabolites. However, we know that drug antigens are displayed systemically so what factors permit tolerance in one part of the body, but reactivity in the skin. Most adverse drug reactions are mild, and skin eruptions tend to be visible to the patient, whereas minor organ injury such as transient transaminase elevation is often not apparent. Systemic hypersensitivity reactions tend to have early cutaneous manifestations, the progression of which is halted by early diagnosis and treatment. It is apparent that the preference for cutaneous involvement of drug hypersensitivity reactions is multi-faceted, therefore this review aims to abridge the findings from literature on the current state of the field and provide insight into the cellular and metabolic mechanisms which may contribute to severe cutaneous adverse reactions.

42788 Inhibition of the IL-15 receptor reduces tissue resident memory T-cells and prevents contact hypersensitivity

42788 Inhibition of the IL-15 receptor reduces tissue resident memory T-cells and prevents contact hypersensitivity

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<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.

Single-cell profiling of muscle-infiltrating T cells in idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIM) are rare autoimmune systemic diseases characterized by muscle weakness and the presence of muscle-infiltrating T cells. IIM represent a clinical challenge due to heterogeneity of symptoms and variability of response to immunosuppressive treatment. Here, we performed in-depth single-cell sequencing on muscle-infiltrating T cells and peripheral blood memory T cells in six patients with recently diagnosed IIM. We identified tissue resident memory T-cell (TRM ) signatures including the expression of HOBIT, XCL1 and CXCR6 in the muscle biopsies of all patients with IIM. Clonally expanded T-cell clones were mainly found among cytotoxic and TRM implying their role in the disease pathogenesis. Finally, identical expanded T-cell clones persisting at follow-up in the muscle tissue of two patients suggest their involvement in disease chronicity. Our study reveals a muscle tissue resident memory T-cell signature in patients with IIM and a transcriptomic map to identify novel therapeutic targets in IIM.

Effector memory CD8 T-cells as a novel peripheral blood biomarker for activated T-cell pediatric acute liver failure.

A distinct phenotype of pediatric acute liver failure (PALF) has been identified, labeled activated T-cell hepatitis. These patients, previously included within the indeterminate group, have evidence of systemic immune activation and liver biopsy specimens with dense infiltration of CD8+ T-cells. We aimed to evaluate the peripheral blood T-cell phenotype in PALF patients with activated T-cell hepatitis compared to indeterminate cause. PALF patients with unknown etiology age 1-17 years were prospectively enrolled between 2017-2020. Within the unknown group, patients were classified as either activated T-cell hepatitis if they had a liver biopsy with dense or moderate CD8 staining and an elevated soluble interleukin-2 receptor level, or they were classified as indeterminate if they did not meet these criteria. Whole blood was collected for flow cytometry and T-cell phenotyping. Four patients with activated T-cell hepatitis and 4 patients with indeterminate PALF were enrolled. Activated T-cell hepatitis patients had significantly greater percentage of CD8 T-cells that were effector memory (TEM) phenotype compared to indeterminate PALF patients (median 66.8% (IQR 57.4-68.7) vs 19.1% (IQR 13.4-25.2), P = 0.03). In addition, CD8+ TEM cells in activated T-cell hepatitis patients were significantly more likely to be CD103 positive, a marker of tissue resident memory T-cells, compared to indeterminate PALF patients (median 12.4% (IQR 9.5-14.7) vs 4.7% (IQR 4.5-5.3), P = 0.03). We found patients with activated T-cell hepatitis can be identified by the unique pattern of increased percentage of peripheral blood effector memory CD8+ CD103+ T-cells. These findings will guide future studies exploring the T-cell phenotype for these patients and whether they may respond to directed immunosuppressive therapies.

Activated tissue resident memory T-cells (CD8+CD103+CD39+) uniquely predict survival in left sided "immune-hot" colorectal cancers.

Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification. A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM. Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis. The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival.

006 Inhibition of tissue resident memory T-cells as a therapy for contact hypersensitivity

006 Inhibition of tissue resident memory T-cells as a therapy for contact hypersensitivity

A173 ELUCIDATING THE EFFECTS OF NOD2-MEDIATED SIGNALLING ON INTESTINAL RESIDENT-MEMORY T-CELL FORMATION AND FUNCTION

Abstract Background Aberrant resident memory T-cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T-cell responses. However, the role of NOD2 in establishing memory T-cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation. Purpose It is the main objective of this project to determine whether NOD2-mediated signalling affects: 1. Antigen-specific T-cell priming in vivo 2. Bona fide intestinal TRM generation 3. Bona fide intestinal TRM function Method To address the effects of NOD2-signalling on intestinal T-cell priming in vivo, wildtype (WT) mice were adoptively transferred 50,000 naïve LCMV-specific (SMARTA) CD4+ T-cells. Mice were subsequently infected with LCMV-Armstrong in the presence or absence of the NOD2 agonist; MDP. 5-days following infection, the numbers and percentage of LCMV-specific T-cells in the mesenteric lymph nodes and spleen were examined. To examine the effects of NOD2 on intestinal TRM generation, littermate WT and NOD2 KO mice were infected with LCMV-Armstrong. Thirty-six-days following infection, the percentage and number of LCMV-specific CD4+ T-cells were profiled in the small and large intestinal lamina-propria by means of gp66-77 class-II MHC-tetramer staining. In another set of experiments, littermate WT and NOD2 KO mice were re-infected with LCMV-C13 30-days following LCMV-Armstrong immunization, and the interferon-response in the small intestine was profiled by quantitative PCR to assess the effect of NOD2-deficiency on antigen recall responses. Result(s) NOD2-stimulation by means of MDP injection increased the percentage and number of adoptively transferred SMARTA CD4+ T-cells in the mesenteric lymph nodes upon LCMV infection. Furthermore, NOD2-deficiency did not alter intestinal LCMV-specific CD4+ TRM seeding in the small and large intestinal lamina propria 36 days after infection. However, in vivo antigen recall experiments showed a decreased intestinal IFN response in NOD2 KO mice. Conclusion(s) Our findings reveal a potential role of NOD2 in the intestinal CD4+ T-cell priming and subsequent Ag-specific memory response. Disclosure of Interest None Declared

Ablation of Adar1 in myeloid cells imprints a global antiviral state in the lung and heightens early immunity against SARS-CoV-2

Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory Tcells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

Markers of immune dysregulation in response to the ageing gut: insights from aged murine gut microbiota transplants

BackgroundPerturbations in the composition and diversity of the gut microbiota are accompanied by a decline in immune homeostasis during ageing, characterized by chronic low-grade inflammation and enhanced innate immunity. Genetic insights into the interaction between age-related alterations in the gut microbiota and immune function remain largely unexplored.MethodsWe investigated publicly available transcriptomic gut profiles of young germ-free mouse hosts transplanted with old donor gut microbiota to identify immune-associated differentially expressed genes (DEGs). Literature screening of the Gene Expression Omnibus and PubMed identified one murine (Mus musculus) gene expression dataset (GSE130026) that included small intestine tissues from young (5–6 weeks old) germ-free mice hosts that were compared following 8 weeks after transplantation with either old (~ 24-month old; n = 5) or young (5–6 weeks old; n = 4) mouse donor gut microbiota.ResultsA total of 112 differentially expressed genes (DEGs) were identified and used to construct a gut network of encoded proteins, in which DEGs were functionally annotated as being involved in an immune process based on gene ontology. The association between the expression of immune-process DEGs and abundance of immune infiltrates from gene signatures in normal colorectal tissues was estimated from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) project. The analysis revealed a 25-gene signature of immune-associated DEGs and their expression profile was positively correlated with naïve T-cell, effector memory T-cell, central memory T-cell, resident memory T-cell, exhausted T-cell, resting Treg T-cell, effector Treg T-cell and Th1-like colorectal gene signatures.ConclusionsThese genes may have a potential role as candidate markers of immune dysregulation during gut microbiota ageing. Moreover, these DEGs may provide insights into the altered immune response to microbiota in the ageing gut, including reduced antigen presentation and alterations in cytokine and chemokine production.

Concomitant immunity to M. tuberculosis infection

Some persistent infections provide a level of immunity that protects against reinfection with the same pathogen, a process referred to as concomitant immunity. To explore the phenomenon of concomitant immunity during Mycobacterium tuberculosis infection, we utilized HostSim, a previously published virtual host model of the immune response following Mtb infection. By simulating reinfection scenarios and comparing with data from non-human primate studies, we propose a hypothesis that the durability of a concomitant immune response against Mtb is intrinsically tied to levels of tissue resident memory T cells (Trms) during primary infection, with a secondary but important role for circulating Mtb-specific T cells. Further, we compare HostSim reinfection experiments to observational TB studies from the pre-antibiotic era to predict that the upper bound of the lifespan of resident memory T cells in human lung tissue is likely 2–3 years. To the authors’ knowledge, this is the first estimate of resident memory T-cell lifespan in humans. Our findings are a first step towards demonstrating the important role of Trms in preventing disease and suggest that the induction of lung Trms is likely critical for vaccine success.

CGAMP-adjuvanted multivalent influenza mRNA vaccines induce broadly protective immunity through cutaneous vaccination in mice

Increasing preclinical and clinical results have demonstrated that mRNA vaccines efficiently prevent infectious diseases and are safe in animal models and humans. In this study, we fabricated a multivalent influenza mRNA lipid nanoparticle (LNP) vaccine with mRNAs of hemagglutinins from influenza H1N1 and H3N2 viruses, matrix protein 1, and nucleoprotein. We found that cutaneous immunization with mRNA LNPs induced strong Th1 and Th2 cellular immunity with robust antigen-specific antibody titers and increased cytokine-secreting splenocytes and antibody-secreting cells. The supplement of cGAMP improved the immunogenicity of mRNA LNPs. Compared with αGC or cGAMP/αGC adjuvanted mRNA LNP formulations in our study, cGAMP mRNA LNPs induced more robust antibody responses. Enhanced cellular immunity with more IL-4 and IFN-γ secreting cells and effector memory Tcell populations in spleens, as well as increased CD4+ resident memory (TRM) Tcells in lungs were observed in cGAMP mRNA LNPs immunized group. These results demonstrated that cGAMP is an effective adjuvant for cutaneous vaccination of multivalent mRNA LNP vaccines in mice to induce stronger immune responses in the spleen and lung, and the cGAMP-adjuvanted mRNA LNPs protected against homologous and heterologous viral infection.

Abstract A007: Resident memory T cells express PD-1 in high grade liposarcoma

Abstract Objective: Memory T cells play crucial roles in anti-tumor immunity. Tumor-associated resident memory T cells (TRM) have been associated with durable immune response and improved patient survival across multiple tumor types. SARC028, a phase II, multicenter trial of pembrolizumab, a programmed death 1 (PD-1) checkpoint inhibitor immunotherapy, showed promising activity in select histologic subtypes of advanced soft tissue sarcoma (STS), including dedifferentiated liposarcoma (DDLPS). However, there was only a 20% overall response rate. Notably, the patients who did respond to immunotherapy had durable responses, supporting our enthusiasm to understand the potential role for TRM in liposarcoma anti-tumor immunity. The characterization of memory T cells in liposarcoma, and whether TRM are present has not been explored. Design: Fresh retroperitoneal liposarcoma specimens from 8 patients: 3 well-differentiated liposarcoma (WDLPS) and 5 DDLPS were collected. Lymphocytes were analyzed by flow cytometry using T-cell (CD45, CD3, CD4, CD8) and phenotypic markers (CD69, CD62L, CD103, CD49a, PD-1, TIM-3). Results: DDLPS had a 4-fold higher overall T-cell infiltration and a 2.5-fold higher CD8+/CD4+ T-cell ratio than WDLPS. DDLPS contained CD8+ T cells with a CD45RA− CCR7− CD69hi CD62Llo phenotype, characteristic of a TRM response, and well as CD69lo effector memory T (TEM) cells. DDLPS had 3-fold more TRM than WDLPS. DDLPS had a much higher level of activated PD-1+ CD69+ CD8+ resident memory T-cell population compared to WDLPS. Additionally, the population of TRM had higher PD-1 expression compared to TEM in both DDLPS and WDLPS. Conclusions: These data suggest that TRM subsets may contribute to the responsiveness to anti-PD1 therapy in patients with DDLPS. Further investigations may lead to the discovery of TRM-targeted T-cell therapies in DDLPS, which currently has limited treatment options. Citation Format: Christina V. Angeles, Jichang Han, Jodi Wilkoswki, Scott Bresler. Resident memory T cells express PD-1 in high grade liposarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A007.

Does Aging Activate T-cells to Reduce Bone Mass and Quality?

Aging leads to decline in bone mass and quality starting at age 30 in humans. All mammals undergo a basal age-dependent decline in bone mass. Osteoporosis is characterized by low bone mass and changes in bone microarchitecture that increases the risk of fracture. About a third of men over the age of 50 years are osteoporotic because they have higher than basal bone loss. In women, there is an additional acute decrement in bone mass, atop the basal rate, associated with loss of ovarian function (menopause) causing osteoporosis in about half of the women. Both genetics and environmental factors such as smoking, chronic infections, diet, microbiome, and metabolic disease can modulate basal age-dependent bone loss and eventual osteoporosis. Here, we review recent studies on the etiology of age-dependent decline in bone mass and propose a mechanism that integrates both genetic and environmental factors. Recent findings support that aging and menopause dysregulate the immune system leading to sterile low-grade inflammation. Both animal models and human studies demonstrate that certain kinds of inflammation, in both men and women, mediate bone loss. Senolytics, meant to block a wide array of age-induced effects by preventing cellular senescence, have been shown to improve bone mass in aged mice. Based on a synthesis of the recent data, we propose that aging activates long-lived tissue resident memory T-cells to become senescent and proinflammatory, leading to bone loss. Targeting this population may represent a promising osteoporosis therapy. Emerging data indicates that there are several mechanisms that lead to sterile low-grade chronic inflammation, inflammaging, that cause age- and estrogen-loss dependent osteoporosis in men and women.

Allergic sensitization impairs lung resident memory CD8 T-cell response and virus clearance

Patients with asthma often suffer from frequent respiratory viral infections and reduced virus clearance. Lung resident memory T cells provide rapid protection against viral reinfections. Because the development of resident memory T cells relies on the lung microenvironment, we investigated the impact of allergen sensitization on the development of virus-specific lung resident memory T cells and viral clearance. Mice were sensitized with house dust mite extract followed by priming with X47 and a subsequent secondary influenza infection. Antiviral memory T-cell response and protection to viral infection was assessed before and after secondary influenza infection, respectively. Gene set variation analysis was performed on data sets from the U-BIOPRED asthma cohort using an IFN-γ-induced epithelial cell signature and a tissue resident memory T-cell signature. Viral loads were higher in lungs of sensitized compared with nonsensitized mice after secondary infection, indicating reduced virus clearance. X47 priming induced fewer antiviral lung resident memory CD8 T cells and resulted in lower pulmonary IFN-γ levels in the lungs of sensitized as compared with nonsensitized mice. Using data from the U-BIOPRED cohort, we found that patients with enrichment of epithelial IFN-γ-induced genes in nasal brushings and bronchial biopsies were also enriched in resident memory T-cell-associated genes, had more epithelial CD8 T cells, and reported significantly fewer exacerbations. The allergen-sensitized lung microenvironment interferes with the formation of antiviral resident memory CD8 T cells in lungs and virus clearance. Defective antiviral memory response might contribute to increased susceptibility of patients with asthma to viral exacerbations.

Abstract 575: Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors

Abstract In this study, we evaluated the feasibility of expanding tumor infiltrating lymphocytes (TIL) from surgically resected renal cell carcinoma (RCC) tumors. Tumors were collected from 43 patients undergoing surgery to remove primary kidney tumors. Tumor types included clear cell RCC (86.0%), papillary RCC (11.6%) as well as chromophobe RCC (2.3%). Tumors were minced into fragments, placed in individual wells of a 24-well plate containing media with high dose IL-2 (6000 IU/mL), and cultured for four weeks. Successful expansion was considered when at least one fragment expanded to a minimum of 2 wells. Successfully expanded TIL were then evaluated for T-cell phenotypes as well as reactivity to the autologous tumor. Of all RCC tumors collected, TIL were successfully grown for 79% of the samples (34/43). The T-cell phenotype of these TIL was highly variable across samples, although there was a higher percentage of CD4+ T-cells relative to CD8+ T-cells. The reactivity of expanded TIL was then assessed by IFNγ release. We found that TIL secreted IFNγ in response to autologous tumor in 71% of the samples. Together, these results demonstrated the feasibility of expanding functional tumor reactive TIL from RCC. Given that hypoxia plays an important role in the development of RCC tumors and is associated with poor prognosis, we then evaluated the ability to expand TIL under hypoxic conditions. To this end, primary TIL were cultured at various O2 levels (20% O2, 5% O2 or 1% O2) and the cell yield as well as the T-cell memory phenotypes were evaluated. We found that the yield of TIL grown in hypoxic conditions was lower when compared to normoxic conditions (20% O2), suggesting a reduction of TIL proliferation in hypoxia. TIL grown in hypoxic conditions displayed increased percentage of central memory T-cells compared to TIL cultured at 20% O2, as evaluated by CD45RA and CCR7 expression (CD45RA-CCR7+). Primary TIL were then expanded to larger numbers using a rapid expansion protocol (REP) by stimulation with anti-CD3 antibody in the presence of allogenic feeder cells. REP of TIL was carried out at either atmospheric O2 (20% O2) or at a hypoxic O2 level (5% O2), and the T-cell phenotype as well as reactivity to the autologous tumor were assessed. Unlike pre-REP TIL, TIL that underwent REP were able to expand in hypoxic conditions (5% O2) and displayed increased percentage of tissue resident memory T-cells (CD69+CD103+; 19%) when compared to the starting TIL population (0.11%) or TIL expanded at atmospheric O2 (0.17%). When co-cultured with the autologous tumor, hypoxic TILs displayed increased IFNγ, TNFα as well as increased Granzyme B release (p<0.001) when compared to TIL expanded in normoxia (20% O2). Collectively, these results support the advantage of adapting TIL to hypoxic conditions in the production of tumor-reactive TIL. Citation Format: Mohammed Alkhouli, Veronica Martinez-Brockhus, Matthew S. Beatty, Robert J. Gillies, Shari Pilon-Thomas, Jad Chahoud. Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 575.

Leishmania Major Centrin Gene-Deleted Parasites Generate Skin Resident Memory T-Cell Immune Response Analogous to Leishmanization.

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene-deleted parasite strain (LmCen-/- ) that induced protection against homologous and heterologous challenges. We demonstrated that the protection is mediated by IFN (Interferon) γ-secreting CD4+ T-effector cells and multifunctional T cells, which is analogous to leishmanization. In addition, in a leishmanization model, skin tissue-resident memory T (TRM) cells were also shown to be crucial for host protection. In this study, we evaluated the generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. We show that immunization with LmCen-/- generated skin CD4+ TRM cells and is supported by the induction of cytokines and chemokines essential for their production and survival similar to leishmanization. Following challenge with wild-type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice. Furthermore, upon challenge, CD4+ TRM cells induce higher levels of IFNγ and Granzyme B in the immunized and leishmanized mice than in non-immunized mice. Taken together, our studies demonstrate that the genetically modified live attenuated LmCen -/- vaccine generates functional CD4+ skin TRM cells, similar to leishmanization, that may play a crucial role in host protection along with effector T cells as shown in our previous study.