Low Ki-67 marks immunogenic CD8⁺ tissue-resident memory T cell enrichment in vitiligo skin.

A subset of melanoma patients treated with immune checkpoint inhibitors develops vitiligo, a CD8 T cell-mediated autoimmune disease associated with improved patient survival. Using a melanoma-associated vitiligo (MAV) mouse model, CD8 tissue resident memory (TRM) T cells in vitiligo skin were found to be necessary and sufficient for durable tumor immunity. Using immunofluorescence microscopy, we found that skin TRM cells formed large aggregates with CD11c myeloid cells proximal to hair follicles. CD11c depletion resulted in TRM cell loss, revealing an unexpected requirement for continued T cell-dendritic cell (DC) interactions in TRM cell maintenance. We hypothesize that DCs provide instructive signals that are required for continued in situ maturation of CD8 TRM cells. To identify these signals, we disrupted Toll Like Receptor (TLR) signaling in DCs by ablating the MyD88 adapter protein and observed a reduction in skin TRM cell accumulation. This prompted us to explore a role for the microbiota in CD8 TRM formation. Treatment with broad spectrum antibiotics resulted in a 50% reduction in vitiligo incidence. B6 mice from vendors with microbiota differences, exhibited divergent MAV incidence, but upon cohousing or fecal transplantation, mice from both sources exhibited high MAV incidence. Collectively, these findings indicates that the gut-skin microbiota axis plays a critical role in generating tumor protective TRM cells. Future studies seek to identify microbial antigens responsible for promoting TRM differentiation. Supported by T32-AI007363 P30-CA023108

Tissue resident memory T cells (Trm) form in the skin in vitiligo and persist to maintain disease, as white spots often recur rapidly after discontinuing therapy. We and others have recently described melanocyte-specific autoreactive Trm in vitiligo lesions. Here, we characterize the functional relationship between Trm and recirculating memory T cells (Tcm) in our vitiligo mouse model. We found that both Trm and Tcm sensed autoantigen in the skin long after stabilization of disease, producing IFN-γ, CXCL9, and CXCL10. Blockade of Tcm recruitment to the skin with FTY720 or depletion of Tcm with low-dose Thy1.1 antibody reversed disease, indicating that Trm cooperate with Tcm to maintain disease. Taken together, our data provide characterization of skin memory T cells in vitiligo, demonstrate that Trm and Tcm work together during disease, and indicate that targeting their survival or function may provide novel, durable treatment options for patients.

Tissue‐resident memory T (TRM) cells are abundant in the memory T cell pool and remain resident in peripheral tissues, such as the skin, where they act as alarm sensors or cytotoxic killers. TRM cells persist long after the pathogen is eliminated and can respond rapidly upon reinfection with the same antigen. When aberrantly activated, skin‐located TRM cells have a profound role in various skin disorders, including vitiligo and melanoma. Autoreactive TRM cells are present in human lesional vitiligo skin and mouse models of vitiligo, which suggests that targeting these cells could be effective as a durable treatment strategy for vitiligo. Furthermore, emerging evidence indicates that induction of melanoma‐reactive TRM cells is needed to achieve effective protection against tumor growth. This review highlights seminal reports about skin‐resident T cells, focusing mainly on their role in the context of vitiligo and melanoma, as well as their potential as therapeutic targets in both diseases.

Immune checkpoint blockade (ICB) has revolutionized the treatment of advanced malignancies such as melanoma. However, a significant proportion of patients fail to respond, owing to mechanisms that remain poorly understood. Here, we developed murine melanoma cell lines (R1 and R2) derived from MO4 tumors and that are resistant to anti-PD−1 therapy. These resistant cells exhibited increased tumor initiation capacity in vivo correlating with enhanced spheroid formation in vitro, and greater invasiveness and metastatic potential in a lung macrometastasis model. Mechanistically, both R1- and R2- derived tumors showed enrichment of a cancer stem cell (CSC)-like population overexpressing Wnt3a, which correlated with reduced infiltration and impaired function of CD8+ tissue-resident memory (TRMs) T cells. This suggested a functional link between Wnt-driven cancer stemness and TRMs dysfunction. Pharmacological inhibition of Wnt/β-catenin signaling in vitro enhanced CD8+ T cell proliferation and expression of functional markers. Notably, Wnt/β-catenin inhibition in vivo reversed anti-PD−1 resistance in resistant tumors, coinciding with reduced OCT4+Wnt3a+ CSC-like cells and restoration of TNFα+CD8+ TRMs. Finally, transcriptomic analysis of publicly available melanoma cohorts showed that enrichment of the Wnt pathway correlates with poor prognosis and ICB resistance, highlighting this pathway as a potential druggable negative regulator of ICB efficacy in melanoma.

HER2-positive cancers present challenges of drug resistance, toxicity, and immune tolerance. HER2-targeted peptide vaccines have failed clinically, while nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccines show promise for robust antitumor immunity. This study utilized heterologous immunity (rat HER2 extracellular domain, rHER2 ECD) to circumvent HER2 tolerance, developing a safe mRNA-LNP vaccine and exploring its synergy with anti-PD-1 therapy. We synthesized N1-methyl-pseudouridine (m1ψ)-modified mRNAs, encoding either rHER2 ECD alone (rHER2 ECD mRNA-LNP) or rHER2 ECD fused to murine IFNγ (rHER2 ECD-IFNγ mRNA-LNP), and then encapsulated them in LNPs via microfluidic mixing. BALB/c mice were immunized on days 1, 15, 29, then challenged with HER2-overexpressing 4T1 (4T1-HER2) cells 2 weeks post-final immunization. Efficacy was assessed by tumor growth, immune responses via flow cytometry, ELISA, ELISpot and cytotoxicity assays, safety via body/organ weight and serum markers. HER2 ECD mRNA-LNPs exhibited >80% encapsulation efficiency and narrow particle size distribution. As monotherapy, rHER2 ECD mRNA-LNPs induced high anti-HER2 antibody titers, polyfunctional CD8+ T cells (IFNγ+/TNFα+), and durable memory T cells, achieving 87.0% tumor inhibition. This inhibition rate was significantly higher than that of the rHER2 ECD-IFNγ mRNA-LNP variant, which achieved 21.9% tumor inhibition and induced anti-IFNγ neutralizing antibodies. Combination with anti-PD-1 monoclonal antibody (mAb) further enhanced tumor inhibition, with 2/6 tumors failing to establish. This synergy was driven by tissue-resident memory T (TRM) cell enrichment in the tumor microenvironment (TME), which reached 15.2% in CD4+ T cells and 12.9% in CD8+ T cells. No systemic toxicity was observed. The rHER2 ECD mRNA-LNP effectively circumvents HER2-specific immune tolerance. As a potent and safe platform, it induces robust coordinated humoral and cellular immunity and significantly enhances checkpoint blockade efficacy via the enrichment of TRM cells in the TME, which underscores its promising potential for the treatment of HER2-positive cancers.

Description Renal cell carcinoma (RCC) is a prevalent and deadly type of kidney cancer that responds to immune checkpoint blockade immunotherapy. However, tumor infiltration of CD8+ T cells has shown opposing effects on patient survival, which may be explained by functional heterogeneity among tumor-specific CD8+ T cells. In this study we observed that RCC tumors are infiltrated by circulating (Tcirc) and tissue-resident (Trm) memory CD8+ T cells that specifically recognize autologous RCC cells in a HLA class I-dependent manner. Trm cell populations displayed higher tumor reactivity than Tcirc cells. Trm cells exhibited reduced stemness potential and a higher exhausted state. In contrast, Tcirc cells showed a high stemness and cytotoxic potential. Single-cell transcriptomics revealed a rather heterogenous composition of memory populations, including cytotoxic and progenitor Tcirc subsets, as well as multiple Trm subsets, including exhausted Trm cells. TCR and trajectory analyses indicate that circulating progenitors lose their circulation and stemness potential in the tumor while progressively acquiring a tissue-resident differentiation program followed by a terminal differentiation state. Interestingly, tumor enrichment of Trm and cytotoxic Tcirc cells predicts better survival, while exhausted Trm and total CD8+ T cells predict worse survival. Our study delineates the differentiation pathway of tumor-specific memory CD8+ T cells infiltrating human renal cell carcinoma tumors Funding Sources This work was funded by Centro Científico y Tecnológico de Excelencia Ciencia & Vida FB210008 and FONDECYT grants 1212070 (A.L.), 11190897 (V.B.), 3220788 (S.H.), 3220741 (A.H.O.) and 3230650 (F.S.) from Agencia Nacional de Investigación y Desarrollo (ANID). Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Irreversible electroporation (IRE) is a relatively new, non-thermal ablation technology for cancer treatment that requires further investigation to optimize its therapeutic efficacy. To improve IRE-ablation, we developed an IRE+Combo-treatment regimen that included the Combo adjuvants poly-I:C (pIC)/CpG, anti-PD-L1 antibody (PD-L1-Ab) and the 41BB-agonist, and investigated its anti-tumor immunity in a 3LLOVA lung cancer model. We demonstrated that inclusion of the 41BB-agonist in the IRE+Combo-ablation stimulated a more efficient CD8+ T cell response (5.3%) than that observed in the absence of 41BB-agonist (3.0%) or upon IRE ablation alone (0.4%), leading to eradication of subcutaneous 3LLOVA cancer in 75% of 3LLOVA-bearing mice. We further showed that the IRE+Combo-treatment regimen resulted in the eradication of both 3LLOVA cancer and lung tumor metastases. Interestingly, our flow cytometry analyses argued that addition of the 41BB-agonist to the IRE+Combo-ablation stimulated a higher frequency of novel CD8+CD103+ conventional type-1 dendritic cells (cDC1) (14.4%) in tumor-drainage lymph-nodes (TDLNs) relative to control IRE+CpG/pIC/PD-L1-Ab- (7.5%) and IRE- (4.0%) treatment groups. This novel cDC1 subpopulation exhibited the most robust expression of DC maturation markers and costimulatory 41BBL and 41BB of all cDC1 subsets. The 41BB-agonist also stimulated a higher frequency of 41BB+CD103+TCF-1+ tissue-resident memory T (TRM) cells (14.5%) in TDLNs when compared with the two control (2.6% and 0.3%) treatment groups. Importantly, the IRE+Combo-treatment regimen was more efficient than the two control groups at converting the immunosuppressive tumor microenvironment (TME), an effect that was mitigated by reducing the frequency of inhibitory myeloid-derived suppressive cells while increasing that of immunogenic cDC1 and CD8+ T cells and rescuing T cell exhaustion. Taken together, our data establish that the 41BB-agonist potentiates the efficacy of IRE+Combo-therapy for lung cancer treatment by promoting unexpected cDC1 and TRM cell responses, and emphasize the importance of targeting this promising molecular signal to improve current cancer IRE-ablation protocols.

Tissue resident memory T (TRM) cells reside in peripheral, non-lymphoid tissues such as the skin, where they act as alarm-sensor cells or cytotoxic cells. Physiologically, skin TRM cells persist for a long term and can be reactivated upon reinfection with the same antigen, thus serving as peripheral sentinels in the immune surveillance network. CD8+CD69+CD103+ TRM cells are the well-characterized subtype that develops in the epidermis. The local mediators such as interleukin (IL)-15 and transforming growth factor (TGF)-β are required for the formation of long-lived TRM cell population in skin. Skin TRM cells engage virus-infected cells, proliferate in situ in response to local antigens and do not migrate out of the epidermis. Secondary TRM cell populations are derived from pre-existing TRM cells and newly recruited TRM precursors from the circulation. In addition to microbial pathogens, topical application of chemical allergen to skin causes delayed-type hypersensitivity and amplifies the number of antigen-specific CD8+ TRM cells at challenged site. Skin TRM cells are also involved in the pathological conditions, including vitiligo, psoriasis, fixed drug eruption and cutaneous T-cell lymphoma (CTCL). The functions of these TRM cells seem to be different, depending on each pathology. Psoriasis plaques are seen in a recurrent manner especially at the originally affected sites. Upon stimulation of the skin of psoriasis patients, the CD8+CD103+CD49a- TRM cells in the epidermis seem to be reactivated and initiate IL-17A production. Meanwhile, autoreactive CD8+CD103+CD49a+ TRM cells secreting interferon-γ are present in lesional vitiligo skin. Fixed drug eruption is another disease where skin TRM cells evoke its characteristic clinical appearance upon administration of a causative drug. Intraepidermal CD8+ TRM cells with an effector-memory phenotype resident in the skin lesions of fixed drug eruption play a major contributing role in the development of localized tissue damage. CTCL develops primarily in the skin by a clonal expansion of a transformed TRM cells. CD8+ CTCL with the pagetoid epidermotropic histology is considered to originate from epidermal CD8+ TRM cells. This review will discuss the current understanding of skin TRM biology and their contribution to skin homeostasis and diseases.

Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease that can progress to liver fibrosis. Recent clinical advance suggests a reversibility of liver fibrosis, but the cellular and molecular mechanisms underlying NASH resolution remain unclarified. Here, using a murine diet-induced NASH and the subsequent resolution model, we demonstrate direct roles of CD8+ tissue-resident memory CD8+ T (CD8+ Trm) cells in resolving liver fibrosis. Single-cell transcriptome analysis and FACS analysis revealed CD69+CD103−CD8+ Trm cell enrichment in NASH resolution livers. The reduction of liver CD8+ Trm cells, maintained by tissue IL-15, significantly delayed fibrosis resolution, while adoptive transfer of these cells protected mice from fibrosis progression. During resolution, CD8+ Trm cells attracted hepatic stellate cells (HSCs) in a CCR5-dependent manner, and predisposed activated HSCs to FasL-Fas-mediated apoptosis. Histological assessment of patients with NASH revealed CD69+CD8+ Trm abundance in fibrotic areas, further supporting their roles in humans. These results highlight the undefined role of liver CD8+ Trm in fibrosis resolution.

Description Increasing emergence of pathogens affecting the brain has made nervous system disorders the leading cause of global disease burden, highlighting the need to study immune-CNS interactions. We have shown that peripheral pathogen exposure induces tissue-resident memory (TRM) CD8 T cells in the brain, but the impact of these newly resident immune cells on brain function remains unclear. Adult neurogenesis, crucial for memory and cognition, occurs in the subgranular zone (SGZ) and subventricular zone (SVZ) and declines in aged Specific Pathogen Free (SPF) mice, correlating with brain CD8 TRM accumulation. To introduce T cells into the young adult mouse brain and mimic human pathogen exposures, we employed a model of “dirty” mice, Specific Pathogen Exposed (SPExp) mice. We found significant CD8 TRM cell enrichment at neurogenic borders in young SPExp brains. Neural stem cells (NSCs) increased in SPExp mice, but their proliferative capacity was lost, indicating quiescence. Total CD8 depletion resulted in reduced NSC numbers but restored proliferative capacity. Notably, CD8 depletion resulted in increased CD4 residency and increased MHC Class II expression on microglia. Depletion of only CD8 TRM subset enhanced NSC numbers and proliferation, suggesting their potent role in suppressing neurogenesis. These findings provide fundamental knowledge about pathogen induced CD8 TRM function in the brain and novel insights into their localized effects on CNS cell function and cognition. Funding Sources NIH/NIAID R21AI185067; NIH/NIAID R21AI178159; T32AI007260. Topic Categories Neuroimmunology (NEUR)

Objective Antigen specific CD8+ T cells were isolated from primary tumor tissue and analyzed for on the single cell level to determine tumor-specific signatures for further study. Methods Primary tumor, metastatic lesions, adjacent healthy tissue and peripheral blood were from collected from patients at Dell Seton Medical Center at the University of Texas. Matched tumor and healthy samples underwent exome and RNA sequencing to allow prediction of tumor-specific epitopes. We then synthesized DNA-barcoded tetramers for all predicted tumor associated-epitopes and an HLA-matched library of exogenous epitopes. Tetramer stained CD8+ T cells were then sorted and analyzed by TetTCR-SeqHD, a technology that we recently developed to link TCR antigen specificity, TCR sequence, gene expression, and phenotype of single T cells. Results We analyzed ~2900 tumor infiltrating lymphocytes (TILs) and T cells from adjacent healthy tissue and observed enrichment for cytotoxic effector and tissue resident memory cells within the tumor versus the healthy tissues. We also note an enrichment of bystander cells, specifically those reactive to Epstein Barr Virus epitopes, within the cytotoxic cluster. Finally, we analyze the distribution and differential expression between tumor-specific lymphocytes and bystander T cells, noting reduced expression of CD57 and CD49d in the tumor-specific TILs, as previously described. Conclusions We validate the use of TetTCR-SeqHD for gaining insight into phenotypic and gene expression differences between bystander and tumor-specific TILs. We identified markers for future exploration and demonstrate key differences between shared clonal populations within healthy and cancerous tissues.

The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.

Despite being a standard treatment option in breast cancer, immune checkpoint inhibitors (ICIs) are only efficacious for a subset of patients. To gain a better understanding of the antitumor immune response in breast cancer, we examined the heterogeneity of CD8+ T cells in tumors, metastatic lymph nodes (mLNs), and peripheral blood from patients with early breast cancer (n=131). Among tissue-resident memory CD8+ T (TRM) cells, including virus- and tumor-specific CD8+ T cells, CD39 expression was observed in a tumor-specific and exhausted subpopulation in both tumors and mLNs. CD39+ TRM cells from tumors and mLNs exhibited a phenotypic similarity and clonally overlapped with each other. Moreover, tumor or mLN CD39+ TRM cells clonally overlapped with CD39- TRM and non-TRM cells in the same compartment, implying a tissue-specific differentiation process. These inter-subpopulationally overlapping CD39+ TRM clonotypes were frequently detected among effector memory CD8+ T cells in peripheral blood, suggesting a systemic clonal overlap. CD39+ TRM cell enrichment was heterogeneous among molecular subtypes of breast cancer, which is associated with the different role of antitumor immune responses in each subtype. In vitro blockade of PD-1 and/or CTLA-4 effectively restored proliferation of CD39+ TRM cells and enhanced cytokine production by CD8+ T cells from tumors or mLNs, particularly in the presence of CD39+ TRM enrichment. This suggests that CD39+ TRM cells have a capacity for functional restoration upon ICI treatment. Thus, our study indicates that CD39+ TRM cells with a clonal overlap across compartments are key players in antitumor immunity in breast cancer.