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Patients with metastatic brain melanomas (MBM) experience shorter-lasting survival than patients with extracranial metastases, and this is associated with a higher fraction of dysfunctional CD8 T cells. The goal of this study was to understand the underlying cause of T cell dysfunction in MBM. To accomplish this, we compared murine B16 melanomas implanted intracranially (IC) or subcutaneously (SC). CD8 T cell activation was not altered, but representation in IC tumors was lower. Transferred activated or naïve CD8 T cells accumulated in similar numbers in both tumors, suggesting that the vasculature does not differentially impair T cell presence. Surprisingly, we found no evidence for T cell activation in draining lymph nodes of SC or IC tumor-bearing mice, consistent with the fact that dendritic cells (DC) that had acquired tumor antigen showed an immature phenotype. Instead, T cell activation occurred within both tumors, where the majority of tumor antigen + myeloid cells were found. While, the numbers of intratumoral DC were comparable, those in IC tumors acquired less tumor antigen, and were alternatively matured based on upregulation of MHCII without upregulation of CD86. Additionally, in IC tumors, the largest population of tumor antigen + myeloid cells were microglia. However, their presence did not influence either antigen acquisition or the phenotype of other myeloid cell populations. Overall, our data suggest that diminished representation of CD8 T cells in IC tumors is a consequence of alternatively matured DC and/or microglia that induce distinctly activated T cells, which ultimately fail to continue to accumulate inside the tumor., (© 2024. The Author(s).)

Tertiary lymphoid structures (TLS) are ectopic lymphoid structures that can arise in human cancers and are associated with improved overall survival (OS) and response to immune checkpoint blockade (ICB) in several cancers, including non-desmoplastic metastatic melanoma (NDMM). Desmoplastic melanoma (DM) has one of the highest response rates to ICB, and we previously identified that primary DM (PDM) contains TLS. Despite the association of TLS with survival and ICB response, it is unknown whether TLS or associated markers of immune activity can differ between PDM and NDMM. We hypothesized that PDM would contain higher frequencies of TLS than NDMM, that T and B-cell densities and proliferation would be greater in TLS of PDM than TLS of NDMM, and that proliferation rates of T and B-cells in PDM TLS would be concordant with those of intratumoral lymphocytes. We found that four features of TLS in PDM distinguish them from TLS in NDMM. TLS were peritumoral in NDMM but intratumoral in PDM. CD8 + T-cell and CD20 + B-cell densities and proliferative fractions were higher in PDM TLS than NDMM TLS. Additionally, the proliferative fractions of T- and B-cells were concordant between the TLS and tumor site in PDM and discordant in NDMM. Collectively, these data suggest that TLS and associated immune markers can differ across melanoma subsets and suggest that PDM TLS may be more immunologically active and have enhanced immune cell trafficking between tumor and TLS compared to NDMM., Competing Interests: Dr. Slingluff has the following disclosures, none of which conflict with the present manuscript: Research support to the University of Virginia from Celldex (funding, drug), Glaxo-Smith Kline (funding), Merck (funding, drug), 3M (drug), Theraclion (device staff support); Funding to the University of Virginia from Polynoma for PI role on the MAVIS Clinical Trial; Funding to the University of Virginia for roles on Scientific Advisory Boards for CureVac. Also Dr. Slingluff receives licensing fee payments through the UVA Licensing and Ventures Group for patents for peptides used in cancer vaccines. The other authors do not have any financial conflicts to disclose relevant to this manuscript., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

CD8 + T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8 + T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8 + T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8 + T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8 + T-cell dysfunction., (©2021 American Association for Cancer Research.)

Although CD8 + T cells are critical for controlling tumors, how they are recruited and home to primary and metastatic lesions is incompletely understood. We characterized the homing receptor (HR) ligands on tumor vasculature to determine what drives their expression and their role in T-cell entry. The anatomic location of B16-OVA tumors affected the expression of E-selectin, MadCAM-1, and VCAM-1, whereas the HR ligands CXCL9 and ICAM-1 were expressed on the vasculature regardless of location. VCAM-1 and CXCL9 expression was induced by IFNγ-secreting adaptive immune cells. VCAM-1 and CXCL9/10 enabled CD8 + T-cell effectors expressing α 4 β 1 integrin and CXCR3 to enter both subcutaneous and peritoneal tumors, whereas E-selectin enabled E-selectin ligand + effectors to enter subcutaneous tumors. However, MadCAM-1 did not mediate α 4 β 7 + effector entry into peritoneal tumors due to an unexpected lack of luminal expression. These data establish the relative importance of certain HRs expressed on activated effectors and certain HR ligands expressed on tumor vasculature in the effective immune control of tumors. Cancer Immunol Res; 5(12); 1062-73. ©2017 AACR ., (©2017 American Association for Cancer Research.)

CD8 T-cells are a critical brake on the initial development of tumors. In established tumors, the presence of CD8 T-cells is correlated with a positive patient prognosis, although immunosuppressive mechanisms limit their effectiveness and they are rarely curative without manipulation. Cancer immunotherapies aim to shift the balance back to dominant antitumor immunity through antibody blockade of immunosuppressive signaling pathways, vaccination, and adoptive transfer of activated or engineered T-cells. These approaches have yielded striking responses in small subsets of patients with solid tumors, most notably those with melanoma. Importantly, the subset of patients who respond to vaccination or immunosuppression blockade therapies are those with CD8 T-cells present in the tumor prior to initiating therapy. While current adoptive cell therapy approaches can be dramatically effective, they require infusion of extremely large numbers of T-cells, but the number that actually infiltrates the tumor is very small. Thus, poor representation of CD8 T-cells in tumors is a fundamental hurdle to successful immunotherapy, over and above the well-established barrier of immunosuppression. In this review, we discuss the factors that determine whether immune cells are present in tumors, with a focus on the representation of cytotoxic CD8 T-cells. We emphasize the critically important role of tumor-associated vasculature as a gateway that enables the active infiltration of both effector and naïve CD8 T-cells that exert antitumor activity. We also discuss strategies to enhance the gateway function and extend the effectiveness of immunotherapies to a broader set of cancer patients., (© 2015 Elsevier Inc. All rights reserved.)

Peripheral tissue homing receptors enable T cells to access inflamed nonlymphoid tissues. In this study, we show that two such molecules, E-selectin ligand and α4β1 integrin, enable activated and memory T cells to enter lymph nodes (LN) as well. This affects the quantitative and qualitative distribution of these cells among regional LN beds. CD8 memory T cells in LN that express these molecules were mostly CD62L(lo) and would normally be classified as effector memory cells. However, similar to central memory cells, they expanded upon Ag re-encounter. This led to differences in the magnitude of the recall response that depended on the route of immunization. These novel cells share properties of both central and effector memory cells and reside in LN based on previously undescribed mechanisms of entry.

Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (T(CD8)). We demonstrate that LECs mediate deletion only via programmed cell death-1 (PD-1) ligand 1, despite expressing ligands for the CD160, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 inhibitory pathways. LECs induce activation and proliferation of T(CD8), but lack of costimulation through 4-1BB leads to rapid high-level expression of PD-1, which in turn inhibits up-regulation of the high-affinity IL-2 receptor that is necessary for T(CD8) survival. Rescue of tyrosinase-specific T(CD8) by interference with PD-1 or provision of costimulation results in autoimmune vitiligo, demonstrating that LECs are significant, albeit suboptimal, antigen-presenting cells. Because LECs express numerous peripheral tissue antigens, lack of costimulation coupled to rapid high-level up-regulation of inhibitory receptors may be generally important in systemic peripheral tolerance.

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4-5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

Previous work from this laboratory showed that generation of memory CD8 T cells by different immunization routes correlates with control of tumors growing in distinct sites. We hypothesized that effector CD8 T cell expression of adhesion proteins and chemokine receptors would be influenced by activation in different secondary lymphoid organs. In this report, CD8 T cells were activated by immunization with bone marrow-derived dendritic cells via i.p., i.v., or s.c. routes. Three distinct populations of activated CD8 T cells arise in mesenteric, axillary/brachial, and mediastinal lymph nodes and spleen based on differential expression of alpha4beta7 integrin, E-selectin ligand, and alpha4beta1 integrin, respectively. In contrast, three subsets of CD8 T cells defined by differential expression of P-selectin ligand and chemokine receptors were induced irrespective of activation site. The majority of activated CD8 T cells expressed CXCR3, with one subset additionally expressing P-selectin ligand, and another subset additionally expressing CCR3, CCR4, CCR5, CCR6, and CCR9. In the mesenteric lymph node, a fourth subset expressed CCR9 and CXCR3 in the absence of CCR5. Similar homing receptor profiles were induced in the same sites after localized vaccinia immunization. Homing receptor expression on CD8 T cells activated in vitro was distinct, revealing influences of both dendritic cells and the lymphoid microenvironment. Collectively, these results identify previously undescribed populations of activated CD8 T cells based on adhesion protein expression and coexpression of chemokine receptors that arise after activation in distinct secondary lymphoid organs.

Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory precursors from terminal effectors.

The ability of CD8+ T cells to recognize melanoma tumors has led to the development of immunotherapeutic approaches that use the antigens CD8+ T cells recognize. However, clinical response rates have been disappointing. Here we summarize our work to understand the mechanisms of self-tolerance that limit responses to currently utilized antigens and our approach to identify new antigens directly tied to malignancy. We also explore several aspects of the anti-tumor immune response induced by peptide-pulsed dendritic cells (DCs). DCs differentially augment the avidity of recall T cells specific for self-antigens and overcome a process of aberrant CD8+ T-cell differentiation that occurs in tumor-draining lymph nodes. DC migration is constrained by injection route, resulting in immune responses in localized lymphoid tissue, and differential control of tumors depending on their location in the body. We demonstrate that CD8+ T-cell differentiation in different lymphoid compartments alters the expression of homing receptor molecules and leads to the presence of systemic central memory cells. Our studies highlight several issues that must be addressed to improve the efficacy of tumor immunotherapy.

The growth of immunogenic tumors in immunocompetent individuals is one of the oldest conundrums in tumor immunology. Although the ability of mouse CD8+ T cells to control transplanted tumors is well documented, little is known about their impact on autochthonous tumors. To gain insight into the role of CD8+ T cells during the course of cancer development, we produced a novel model of spontaneous melanoma. The metallothionein (MT)-ret/AAD mouse is transgenic for the RET oncogene and the chimeric MHC molecule AAD (alpha1-alpha2 domains of HLA-A2 linked to alpha3 domain of H2-Dd). This model recapitulates the natural history of human melanoma, and expression of the AAD molecule makes it suitable for analyzing CD8+ T cell responses directed against peptide Ags that have been previously identified in HLA-A2+ melanoma patients. We found that, as tumors grow, mice develop a broad melanoma-specific CD8+ T cell response. Occurrence of cutaneous nodules is not affected by CD8+ T cell depletion, showing that although CD8+ T cells are functional, they have no effect on established cutaneous tumors. However, depleted mice die from visceral disease much earlier than controls, showing that CD8+ T cells control metastasis spreading and disease progression. Antigenic modulation is observed in visceral metastases, suggesting that visceral nodules may be subject to immunoediting. Our data demonstrate that growth of melanoma in the MT-ret/AAD model involves several tolerance mechanisms sequentially. They also reveal a different role for CD8+ T cells toward early stage of cutaneous tumors and late visceral metastatic stage of the disease.

Exogenous dendritic cells (bone marrow-derived dendritic cell (BMDC)) display restricted trafficking in vivo after injection into mice, but the route(s) by which they generate gut-homing effector cells is unclear. Mesenteric lymph nodes (LN) and spleen were differentially targeted by i.p. and i.v. administration of BMDC, respectively, whereas mediastinal LN were targeted by both routes. BMDC injected by either route activated CD8(+) T cells to up-regulate both alpha(4)beta(1) and alpha(4)beta(7) integrins. However, the lymphoid compartment in which activation occurred determined their expression kinetics, magnitude, and population distribution. Only T cells activated in mesenteric LN after i.p. immunization expressed high levels of alpha(4)beta(7), which also correlated with localization to small intestine. These alpha(4)beta(7)(high) cells also redistributed to mediastinal LN in a manner sensitive to treatment with alpha(4)beta(7) blocking Abs, but not to mucosal addressin cell adhesion molecule-1 blocking Abs. Our results demonstrate the importance of lymphoid compartment, as dictated by immunization route, in determining integrin expression on activated T cells and their distribution in lymphoid and nonlymphoid tissues.

CD8 T cells lacking effector activity have been recovered from lymphoid organs of mice and patients with progressing tumors. We explored the basis for lack of effector activity in tumor-bearing mice by evaluating Ag presentation and CD8 T cell function in lymphoid organs over the course of tumor outgrowth. Early after tumor injection, cross-presentation by bone marrow-derived APC was necessary for T cell activation, inducing proliferation and differentiation into IFN-gamma-producing, cytolytic effectors. At later stages of outgrowth, tumor metastasized to draining lymph nodes. Both cross- and direct presentation occurred, but T cell differentiation induced by either modality was incomplete (proliferation without cytokine production). T cells within tumor-infiltrated nodes differentiated appropriately if Ag was presented by activated, exogenous dendritic cells. Thus, activated T cells lacking effector function develop through incomplete differentiation in the lymph nodes of late-stage tumor-bearing mice, rather than through suppression of previously differentiated cells.

We studied the size and avidity of primary and recall CD8(+) T cell responses in vivo in mice immunized with dendritic cells presenting different densities of a MHC class I-restricted peptide. Increasing the epitope density on a fixed number of dendritic cells increased the size of the primary response, yet had no influence on the avidity of the effector cells. However, epitope density-based selection of cells with different avidities was evident in the subsequent memory population, and in recall responses. Additionally, mice primed with different peptide densities had similarly sized quiescent memory and recall responses. Our findings provide evidence for an important role for epitope density in the selection of T cells in vivo.