Tissue-resident Memory T Cells Research Articles

Evaluation of adenoviral vector Ad19a encoding RSV-F as novel vaccine against respiratory syncytial virus

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants and toddlers. Since natural infections do not induce persistent immunity, there is the need of vaccines providing long-term protection. Here, we evaluated a new adenoviral vector (rAd) vaccine based on the rare serotype rAd19a and compared the immunogenicity and efficacy to the highly immunogenic rAd5. Given as an intranasal boost in DNA primed mice, both vectors encoding the F protein provided efficient protection against a subsequent RSV infection. However, intramuscular immunization with rAd19a vectors provoked vaccine-enhanced disease after RSV infection compared to non-vaccinated animals. While mucosal IgA antibodies and tissue-resident memory T-cells in intranasally vaccinated mice rapidly control RSV replication, a strong anamnestic systemic T-cell response in absence of local immunity might be the reason for immune-mediated enhanced disease. Our study highlighted the potential benefits of developing effective mucosal against respiratory pathogens.

Epidemic influenza virus nucleoprotein gene incorporated into vaccine influenza virus strain genome to optimize systemic and local T-cell immune response against live attenuated influenza vaccine

Introduction. Optimization of the vaccine-induced T-cell repertoire is one of the strategies to expand the spectrum of protective potential for live attenuated influenza vaccine (LAIV). LAIV cross-protective properties can be improved by introducing the nucleoprotein (NP) gene derived from epidemic parental virus into vaccine strain genome, i.e. by replacing the classical 6:2 genome formula with 5:3. The main objective of the present study was to detail evaluation for virus-specific systemic and tissue-resident memory T-cells subsets in mice immunized with seasonal H1N1 LAIV of the genome formula 6:2 and 5:3. Materials and methods. Two H1N1 LAIV strains with varying NP genes (LAIV 6:2 and LAIV 5:3) were generated using reverse genetics techniques. C57BL/6J mice were immunized intranasally with the vaccine candidates, twice, 3 weeks apart. Cells from the spleen and lung tissues were isolated 7 days after booster immunization to be stimulated with whole H1N1 influenza virus for assessing cytokine-producing memory CD44+CD62L– T-cells as well as expression of CD69 and CD103 surface markers using flow cytometry. Humoral murine serum immunity against H1N1 virus was assessed by ELISA. Results. The LAIV 5:3 vs classical 6:2 vaccine strain carrying the epidemic parental NP gene induced significantly more pronounced humoral immune response against recent influenza virus. The group of mice immunized with LAIV 5:3 demonstrated higher levels of virus-specific CD4+ and CD8+ effector memory T cells (TEM) in the spleen, including a subset of polyfunctional (IFNγ+TNFα+IL-2+) CD4+ TEM, compared to LAIV 6:2 group. Virus-specific memory T cell levels in lung tissues after immunization with LAIV 5:3 vs LAIV 6:2 also tended to increase, but no significant difference in stimulated tissue-resident CD69+CD103– and CD69+CD103+ T cells between the groups were found. Conclusion. Modification of the seasonal LAIV strain genome for updating its epitope composition allowed to enhance the virus-specific T-cell immune response both at systemic level and in lung tissues, thereby shoeing that the effectiveness of the vaccine against circulating influenza viruses can be potentially increased.

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.

Genetic predisposition to early mycosis fungoides: investigating genetic polymorphisms in tissue-resident memory T-cell genes.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma; it arises from tissue-resident memory T-cells (TRM). In the present study, we investigated potential functional genetic variations that may predispose MF development. A case-control study was conducted using whole-exome sequencing, with a focus on genes that are essential to TRM function. We included 21 patients and 19 healthy subjects in the study. Single nucleotide polymorphisms in the following genes were significantly more common in patients than in healthy subjects: GZMB, HLA-DRB1, CD103, and NOTCH1. Moreover, the number of patients carrying single nucleotide polymorphisms in LAG3, NR4A2, and CD26L was significantly greater in the patient group than in the control group. The presence of genetic variations in one or more TRM functional gene may predispose patients to develop MF. Further studies involving a larger patient population and a comparative analysis of protein expression will be necessary to validate these findings.

From PsO to PsA: the role of TRM and Tregs in psoriatic disease, a systematic review of the literature.

Psoriasis (PsO) is a chronic skin condition driven by immune mediators like TNFα, INFγ, IL-17, and IL-23. Psoriatic arthritis (PsA) can develop in PsO patients. Although psoriatic lesions may apparently resolve with therapy, subclinical cutaneous inflammation may persist. The role of tissue-resident memory T-cells (TRM), and regulatory T cells (Tregs) that also contribute to chronic inflammation are being explored in this context. This systematic review explores TRM and Tregs in psoriatic disease (PsD) and its progression. A systematic review, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was performed using Pubmed® and Web of Science™ databases on June 3rd 2023, using patient/population, intervention, comparison, and outcomes (PICO) criteria limited to the English language. A total of 62 reports were identified and included. In PsO, chronic inflammation is driven by cytokines including IL-17 and IL-23, and cellular mediators such as CD8+ and CD4+ T cells. TRM contributes to local inflammation, while Tregs may be dysfunctional in psoriatic skin lesions. Secukinumab and guselkumab, which target IL-17A and the IL-23p19 subunit, respectively, have different effects on CD8+ TRM and Tregs during PsO treatment. Inhibition of IL-23 may provide better long-term results due to its impact on the Treg to CD8+ TRM ratio. IL-23 may contribute to inflammation persisting even after treatment. In PsA, subclinical enthesitis is perceived as an early occurence, and Th17 cells are involved in this pathogenic process. Recent EULAR guidelines highlight the importance of early diagnosis and treatment to intercept PsA. In PsA, CD8+ TRM cells are present in synovial fluid and Tregs are reduced in peripheral blood. The progression from PsO to PsA is marked by a shift in immune profiles, with specific T-cells subsets playing key roles in perpetuating inflammation. Early intervention targeting TRM cells may hold promising, but clinical studies are limited. Ongoing studies such as IVEPSA and PAMPA aim to improve our knowledge regarding PsA interception in high-risk PsO patients, emphasizing the need for further research in this area. Early intervention is crucial for PsO patients at high risk of PsA; T cells, particularly type 17 helper T cells, and CD8+ cells are key in the progression from PsO-to-PsA. Early targeting of TRM in PsD shows promise but more research is needed.

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.

Tertiary Lymphoid Structure-Associated B Cells Enhance CXCL13+CD103+CD8+ Tissue-Resident Memory T-Cell Response to Programmed Cell Death Protein 1 Blockade in Cancer Immunotherapy

BACKGROUND & AIMSAlthough the presence of tertiary lymphoid structures (TLS) correlates with positive responses to immunotherapy in many solid malignancies, the mechanism by which TLS enhances anti-tumor immunity is not well understood. The present study aimed to investigate the underlying cross-talk circuits between B cells and tissue-resident memory T (Trm) cells within the TLS and to understand their role in the context of immunotherapy. METHODSImmunostaining and hematoxylin and eosin staining of TLS and CXCL13+CD103+CD8+Trm cells were performed on tumor sections from patients with gastric cancer (GC). The mechanism of communication between B cells and CXCL13+CD103+CD8+Trm cells was determined both in vitro and in vivo. The effect of CXCL13+CD103+CD8+Trm cells in suppressing tumor growth was evaluated through anti-PD-1 therapy. RESULTSThe presence of TLS and CXCL13+CD103+CD8+Trm cells in tumor tissues favored a superior response to anti-PD-1 therapy in GC patients. Additionally, our research identified that activated B cells enhanced CXCL13 and granzyme B secretion by CD103+CD8+Trm cells. Mechanistically, B cells facilitated the glycolysis of CD103+CD8+Trm cells through the Lymphotoxin Alpha (LTα)/Tumor necrosis factor receptor 2 (TNFR2) axis, and the mTOR signaling pathway played a critical role in CD103+CD8+Trm cells glycolysis during this process. Moreover, the presence of TLS and CXCL13+CD103+CD8+Trm cells correlated with potent responsiveness to anti-PD-1 therapy in a TNFR2 dependent manner. CONCLUSIONSThis study further reveals a crucial role for cellular communication between TLS-associated B cell and CXCL13+CD103+CD8+Trm cells in anti-tumor immunity, providing valuable insights into the potential utilization of the LTα/TNFR2 axis within CXCL13+CD103+CD8+Trm cells for advancing immunotherapy strategies in GC.

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.

CD8+ tissue-resident memory T-cell development depends on infection-matching regulatory T-cell types

Immunological memory is critical for immune protection, particularly at epithelial sites, which are under constant risk of pathogen invasions. To counter invading pathogens, CD8+ memory T cells develop at the location of infection: tissue-resident memory T cells (TRM). CD8+ T-cell responses are associated with type-1 infections and type-1 regulatory T cells (TREG) are important for CD8+ T-cell development, however, if CD8+ TRM cells develop under other infection types and require immune type-specific TREG cells is unknown. We used three distinct lung infection models, to show that type-2 helminth infection does not establish CD8+ TRM cells. Intracellular (type-1) and extracellular (type-3) infections do and rely on the recruitment of response type-matching TREG population contributing transforming growth factor-β. Nevertheless, type-1 TREG cells remain the most important population for TRM cell development. Once established, TRM cells maintain their immune type profile. These results may have implications in the development of vaccines inducing CD8+ TRM cells.

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.

Strengthened EPR effect of AIEgen-Poly(I:C) composite nanoparticles to facilitate antigen cross-presentation and improve melanoma prognosis by evoking immunity

Nanoparticles offer a promising platform for drug delivery due to their long circulation time, enhanced solubility, low toxicity and excellent targeting of lesions. Herein, we report an AIEgen-Poly (I:C) composite nanoparticle (P@AP) for dual-modality tumor photodynamic-immunotherapy. Its specific size and capacity for partial degradation under acidic conditions determine its enhanced EPR effect. By complexing with the immunoadjuvant Poly(I:C), the immune silencing mechanism induced by apoptosis can be overcome and ICD is evoked efficiently at the cellular level. In the process of antigen presentation, P@AP, as an exogenous antigen, on the one hand, strengthens the expression of antigen presentation signal molecule MHC II and activates CD4+ T cells, on the other hand, enhancing pAPC infiltration by the increase of endogenous antigens represented by damage-associated molecular patterns while expressing MHC I via antigenic cross-presentation remarkably induces activation of CD8+ T cells, directly enhancing the killing of target cells. Massive immune arousal results in sustained tissue-resident memory T-cell residency in melanoma and reduces expression of CD4+ subsets of Treg cells, thereby prolonging the survival of subcutaneous melanoma mice and ridding of the poor prognosis of malignant melanoma stemming from its auto-immunosuppressive properties.

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.

Intranasal delivery of an adenovirus-vector vaccine co-expressing a modified spike protein and a genetic adjuvant confers lasting mucosal immunity against SARS-CoV-2

The ongoing COVID-19 pandemic caused by SARS-CoV-2 infection has threatened global health. Since the first case of infection was reported in December 2019, SARS-CoV-2 has rapidly spread worldwide and caused millions of deaths. As vaccination is the best way to protect the host from invading pathogens, several vaccines have been developed to prevent the infection of SARS-CoV-2, saving numerous lives thus far. However, SARS-CoV-2 constantly changes its antigens, resulting in escape from vaccine-induced protection, and the longevity of immunity induced by vaccines remains an issue. Additionally, traditional intramuscular COVID-19 vaccines are insufficient at evoking mucosal-specific immune responses. Because the respiratory tract is the primary route of SARS-CoV-2 entry, the need for mucosal vaccines is strong. Using an adenoviral (Ad) vector platform, we generated Ad5-S.Mod, a recombinant COVID-19 vaccine that encodes modified-spike (S) antigen and the genetic adjuvant human CXCL9. Intranasal delivery of Ad5-S.Mod elicited superior airway humoral and T-cell responses over traditional intramuscular vaccines and protected mice from lethal SARS-CoV-2 infection. cDC1 cells were required for the generation of antigen-specific CD8+ T-cell responses and CD8+ tissue-resident memory T-cell development in intranasal Ad5-S.Mod vaccinated mice. Furthermore, we confirmed the efficacy of the intranasal Ad5-S.Mod vaccine in terms of transcriptional changes and identified lung macrophages as a key supporter of maintenance of lung-resident memory T and B cells. Our study demonstrates Ad5-S.Mod has the potential to confer protective immunity against SARS-CoV-2 and that lung macrophages support the maintenance of vaccine-induced tissue-resident memory lymphocytes.

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.

Tissue-Resident Memory T Cells Shape Tumor-Immune Evolution in Lung Cancer.

Tissue-resident memory T-cell immunosurveillance is associated with cigarette smoking before tumor onset.

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.

Mucosal vaccination with an adenoviral vector vaccine protects against pulmonary breast cancer metastasis by robust induction of tissue-resident memory T-cells in the lung

Abstract The presence of tissue-resident memory T-cells (TRM) in solid tumors positively correlates with an improved overall prognosis and responsiveness to checkpoint inhibitor therapy. Rationally designed vaccine approaches capable of inducing antitumor TRM at cancer sites are needed to harness the full potential of localized immunity in tumor therapy. Our project aims to establish tumor antigen-specific lung TRM by mucosal vaccination with an adenoviral (Ad) vaccine to prevent and treat lung metastasis in the murine 4T1 breast cancer model. We employ Ad serotype 5 vectors encoding for 4T1 tumor-associated antigens from Mage-b and MuLV gp70 for intranasal vaccination, supplemented with an Interleukin-1β-encoding vector to specifically boost lung TRM induction. Indeed, particularly if Ad-IL-1β was co-delivered, mucosal vaccination established high numbers of tumor antigen-specific CD69 +CD103 +lung TRM as shown by phenotypic and functional analyses in combination with intravascular staining. Vaccine efficacy was evaluated in a 4T1 pseudometastasis model mimicking a prophylactic vaccination against lung metastasis in breast cancer patients. Local immunization led to a significant protection against pulmonary metastasis and improved survival compared to non-vaccinated and, notably, intramuscularly immunized mice. By using FTY720 to inhibit T-cell circulation upon tumor challenge, we confirmed that TRM are crucial contributors to the vaccine efficacy. In conclusion, induction of tumor-antigen specific lung TRM are a key point in local protection against pulmonary metastasis. Ongoing studies analyze the therapeutic use of this vaccination strategy as well as its potential in combination with immune checkpoint inhibition. Supported by grants from 'Anschubfinanzierung (ELAN) im IZKF' and 'Doktor Robert Pfleger-Stiftung'.

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