Tertiary lymphoid structures in desmoplastic melanoma have increased lymphocyte density, lymphocyte proliferation, and immune cross talk with tumor when compared to non-desmoplastic melanomas.

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.

NK cells reduce anergic T cell development in early-stage tumors by promoting myeloid cell maturation.

Introduction: Studies of NK cells in tumors have primarily focused on their direct actions towards tumor cells. We evaluated the impact of NK cells on expression of homing receptor ligands on tumor vasculature, intratumoral T cell number and function, and T cell activation in tumor draining lymph node. Methods: Using an implantable mouse model of melanoma, T cell responses and homing receptor ligand expression on the vasculature were evaluated with and without NK cells present during the early stages of the tumor response by flow cytometry. Results: NK cells in early-stage tumors are one source of IFNγ that augments homing receptor ligand expression. More significantly, NK cell depletion resulted in increased numbers of intratumoral T cells with an anergic phenotype. Anergic T cell development in tumor draining lymph node was associated with increased T-cell receptor signaling but decreased proliferation and effector cell activity, and an incomplete maturation phenotype of antigen presenting cells. These effects of NK depletion were similar to those of blocking CD40L stimulation. Discussion: We conclude that an important function of NK cells is to drive proper APC maturation via CD40L during responses to early-stage tumors, reducing development of anergic T cells. The reduced development of anergic T cells resulting in improved tumor control and T cell responses when NK cells were present.

Tumor necrosis factor receptor regulation of peripheral node addressin biosynthetic components in tumor endothelial cells.

Tumor-associated tertiary lymphoid structures are ectopic lymphoid aggregates that have considerable morphological, cellular, and molecular similarity to secondary lymphoid organs, particularly lymph nodes. Tumor vessels expressing peripheral node addressin (PNAd) are hallmark features of these structures. Previous work from our laboratory demonstrated that PNAd is displayed on intratumoral vasculature of murine tumors, and its expression is controlled by the engagement of lymphotoxin-α3, secreted by effector CD8 T cells, with tumor necrosis factor receptors (TNFR) on tumor endothelial cells (TEC). The goals of the present work were: 1) to identify differences in expression of genes encoding the scaffolding proteins and glycosyl transferases associated with PNAd biosynthesis in TEC and lymph node blood endothelial cells (LN BEC); and 2) to determine which of these PNAd associated components are regulated by TNFR signaling. We found that the same genes encoding scaffolding proteins and glycosyl transferases were upregulated in PNAd+ LN BEC and PNAd+ TEC relative to their PNAdneg counterparts. The lower level of PNAd expression on TEC vs LN BEC was associated with relatively lower expression of these genes, particularly the carbohydrate sulfotransferase Chst4. Loss of PNAd on TEC in the absence of TNFR signaling was associated with lack of upregulation of these same genes. A small subset of PNAd+ TEC remaining in the absence of TNFR signaling showed normal upregulation of a subset of these genes, but reduced upregulation of genes encoding the scaffolding proteins podocalyxin and nepmucin, and carbohydrate sulfotransferase Chst2. Lastly, we found that checkpoint immunotherapy augmented both the fraction of TEC expressing PNAd and their surface level of this ligand. This work points to strong similarities in the regulation of PNAd expression on TEC by TNFR signaling and on LN BEC by lymphotoxin-ß receptor signaling, and provides a platform for the development of novel strategies that manipulate PNAd expression on tumor vasculature as an element of cancer immunotherapy.

An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes.

There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

Characteristics of Immune Memory and Effector Activity to Cancer-Expressed MHC Class I Phosphopeptides Differ in Healthy Donors and Ovarian Cancer Patients.

Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

Associations of immune cell homing gene signatures and infiltrates of lymphocyte subsets in human melanomas: discordance with CD163+ myeloid cell infiltrates.

BACKGROUND: Immune cells in the tumor microenvironment have prognostic value. In preclinical models, recruitment and infiltration of these cells depends on immune cell homing (ICH) genes such as chemokines, cell adhesion molecules, and integrins. We hypothesized ICH ligands CXCL9-11 and CCL2-5 would be associated with intratumoral T-cells, while CXCL13 would be more associated with B-cell infiltrates. METHODS: Samples of human melanoma were submitted for gene expression analysis and immune cells identified by immunohistochemistry. Associations between the two were evaluated with unsupervised hierarchical clustering using correlation matrices from Spearman rank tests. Univariate analysis performed Mann-Whitney tests. RESULTS: For 119 melanoma specimens, analysis of 78 ICH genes revealed association among genes with nonspecific increase of multiple immune cell subsets: CD45+, CD8+ and CD4+ T-cells, CD20+ B-cells, CD138+ plasma cells, and CD56+ NK-cells. ICH genes most associated with these infiltrates included ITGB2, ITGAL, CCL19, CXCL13, plus receptor/ligand pairs CXCL9 and CXCL10 with CXCR3; CCL4 and CCL5 with CCR5. This top ICH gene expression signature was also associated with genes representing immune-activation and effector function. In contrast, CD163+ M2-macrophages was weakly associated with a different ICH gene signature. CONCLUSION: These data do not support our hypothesis that each immune cell subset is uniquely associated with specific ICH genes. Instead, a larger set of ICH genes identifies melanomas with concordant infiltration of B-cell and T-cell lineages, while CD163+ M2-macrophage infiltration suggesting alternate mechanisms for their recruitment. Future studies should explore the extent ICH gene signature contributes to tertiary lymphoid structures or cross-talk between homing pathways.

Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts.

Tumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-α1ß2. Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.

Heterogeneity in tertiary lymphoid structure B-cells correlates with patient survival in metastatic melanoma.

BACKGROUND: Tertiary lymphoid structures (TLSs) are immune aggregates in peripheral tissues that may support adaptive immune responses. Their presence has been associated with clinical response to checkpoint blockade therapy (CBT), but it is unknown whether TLS have prognostic significance independent of CBT in melanoma. We hypothesized that TLS in melanoma metastases would be associated with increased intratumoral lymphocyte infiltration, but that the intra-TLS immunological milieu would be distinct from the intratumoral immunological milieu. We also hypothesized that the presence of TLS would be associated with improved survival, and that TLS maturation or intra-TLS lymphocyte activity would also correlate with survival. METHODS: Cutaneous melanoma metastases (CMM) from 64 patients were evaluated by multiplex immunofluorescence for the presence and maturation status of TLS. Intra-TLS lymphocyte density, proliferation and B-cell Ig somatic hypermutation (AID+) were analyzed, as were markers of T-cell exhaustion and Th1/Tc1 differentiation. Associations between TLS maturation and intra-TLS immunologic activity were assessed, as well as associations with intratumoral immune cell infiltration. Independent associations with overall survival (OS) were assessed using log-rank tests and Cox proportional hazards models. RESULTS: TLS were identified in 30 (47%) of 64 CMM (TLS+) and were associated with increased intratumoral lymphocyte infiltration. However, proliferation of intra-TLS lymphocytes did not correlate with intratumoral lymphocyte proliferation. Most were early TLS; however, subsets of primary or secondary follicle-like TLS were also present. TLS+ lesions were associated with lower risk of tumor recurrence after metastasectomy and with improved OS in multivariate analyses (HR 0.51, p=0.04). OS was longer for TLS with low fractions of CD21+ B-cells (HR 0.29, p=0.02) and shorter for those with low AID+ fraction of B-cells (HR 2.74, p=0.03). CONCLUSIONS: The presence of TLS in CMMs is associated with improved OS in patients treated with surgery before CBT, but TLS vary widely in maturation state, in proportions of proliferating T and B cells, and in markers of B cell function, including AID and CD21. Importantly, these features have additional prognostic significance, which suggest that some TLS may have regulatory function, while others functioning to support antigen-driven immune responses, depending on the cellular composition and activation status.

Phospho-ß-catenin expression in primary and metastatic melanomas and in tumor-free visceral tissues, and associations with expression of PD-L1 and PD-L2.

ß-catenin (ßcat) is an important downstream effector in the Wnt signaling pathway and plays important roles in the development and progression of many cancers including melanoma. ßcat expression is regulated by GSK-3ß-mediated phosphorylation at positions 33, 37 and 41. In normal cells, phosphorylation at these sites triggers proteasomal degradation, which prevents accumulation of free cytoplasmic ßcat. In cancer cells, stabilized ß-catenin translocates into the nucleus, where it associates with TCF/Lef proteins to activate transcription of genes that promote tumorigenesis and metastasis, including PD-L1. It has been suggested that nuclear phospho-ßcat (pßcat) staining may be diagnostically useful in differentiating primary from metastatic melanoma. Also, a pßcat peptide (residues 30-39, with only S33 phosphorylated) is naturally presented by melanoma cells as a T-cell target. We evaluated expression of pS33-ßcat in primary and metastatic melanomas by immunohistochemistry and found its expression varied widely but was most commonly cytoplasmic. Nuclear staining was identified in only 18% of metastatic melanomas. Staining with antibodies to pS33-ßcat and pS33/37/T41-ßcat was most intense in mitotic melanoma cells; however, pS33-ßcat intensity was not significantly associated with AJCC stage, tumor location, BRAF mutation status, or immune infiltrates. Yet, PD-L1 and PD-L2 expression by tumor cells were significantly higher in tumors with high pS33-ßcat expression. The low rate of nuclear pS33-ßcat expression suggests that pS33-ßcat may have limited utility for identifying metastatic melanomas. However, high expression in dividing cells and strong associations with PD-L1 and PD-L2 expression may inform future personalized therapies for tumors with high pS33-ßcat expression.

Differential Expression of CD49a and CD49b Determines Localization and Function of Tumor-Infiltrating CD8+ T Cells.

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.

Insights into Tumor-Associated Tertiary Lymphoid Structures: Novel Targets for Antitumor Immunity and Cancer Immunotherapy.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that phenotypically resemble conventional secondary lymphoid organs and are commonly found at sites of chronic inflammation. They are also found in a wide variety of primary and metastatic human tumors. The presence of tumor-associated TLS (TA-TLS) is associated with prolonged patient survival, higher rates of disease-free survival, and a favorable response to current cancer therapies. However, the immune responses that occur in these structures, and how they contribute to improved clinical outcomes, remain incompletely understood. In addition, it is unknown how heterogeneity in TA-TLS cellular composition, structural organization, and anatomic location influences their functionality and prognostic significance. Understanding more about TA-TLS development, formation, and function may offer new therapeutic options to modulate antitumor immunity.

Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound.

Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.

MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma.

BACKGROUND: Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides. METHODS: Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3126-134) was determined by 51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA-IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund's adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay. RESULTS: pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3126-134 controlled outgrowth of a tumor xenograft. The pIRS21097-1105 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3-4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS21097-1105 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% to 44%). CONCLUSION: This study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3126-134 and pIRS21097-1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses. TRIAL REGISTRATION NUMBER: NCT01846143.

Antibiotics Drive Microbial Imbalance and Vitiligo Development in Mice.

Vitiligo is impacted by environmental triggers. We studied the contribution of the microbiome in FH mice, in which depigmentation is mediated by tyrosinase-reactive T cells. The mice received oral antibiotics and were monitored for depigmentation. The microbiome was studied in fecal and skin samples using 16S rRNA analysis. The resulting T-cell distributions were evaluated. In untreated mice, pigment loss did not expand to the pelage, whereas mice in the ampicillin group were approximately 1/3 depigmented at 30 weeks. In contrast to models of autoimmunity that are less dependent on IFN-γ, ampicillin but not neomycin treatment correlated with accelerated disease and reduced bacteria in the fecal pellets. Modified cytokine patterns in the tissue and serum suggest a response that transcends the gut. Ampicillin-induced depigmentation was accompanied by gut but not skin dysbiosis, and reduced T cell numbers in both sites. Neomycin induced a redistribution of gut T cells and an accumulation of skin regulatory T cells. This treatment spurred a Bacteroides-dominated population of fecal bacteria. Reduced diversity is prominent particularly after ampicillin treatment, when the gut is dominated by Pseudomonas species. In line with current concepts relating the microbiome and the immune system, we predict that dietary measures might promote skin health and delay vitiligo onset.

The Barrier Molecules Junction Plakoglobin, Filaggrin, and Dystonin Play Roles in Melanoma Growth and Angiogenesis.

OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.

Comparative Transcriptomic Analysis Identifies a Range of Immunologically Related Functional Elaborations of Lymph Node Associated Lymphatic and Blood Endothelial Cells.

Lymphatic and blood vessels are formed by specialized lymphatic endothelial cells (LEC) and blood endothelial cells (BEC), respectively. These endothelial populations not only form peripheral tissue vessels, but also critical supporting structures in secondary lymphoid organs, particularly the lymph node (LN). Lymph node LEC (LN-LEC) also have been shown to have important immunological functions that are not observed in LEC from tissue lymphatics. LN-LEC can maintain peripheral tolerance through direct presentation of self-antigen via MHC-I, leading to CD8 T cell deletion; and through transfer of self-antigen to dendritic cells for presentation via MHC-II, resulting in CD4 T cell anergy. LN-LEC also can capture and archive foreign antigens, transferring them to dendritic cells for maintenance of memory CD8 T cells. The molecular basis for these functional elaborations in LN-LEC remain largely unexplored, and it is also unclear whether blood endothelial cells in LN (LN-BEC) might express similar enhanced immunologic functionality. Here, we used RNA-Seq to compare the transcriptomic profiles of freshly isolated murine LEC and BEC from LN with one another and with freshly isolated LEC from the periphery (diaphragm). We show that LN-LEC, LN-BEC, and diaphragm LEC (D-LEC) are transcriptionally distinct from one another, demonstrating both lineage and tissue-specific functional specializations. Surprisingly, tissue microenvironment differences in gene expression profiles were more numerous than those determined by endothelial cell lineage specification. In this regard, both LN-localized endothelial cell populations show a variety of functional elaborations that suggest how they may function as antigen presenting cells, and also point to as yet unexplored roles in both positive and negative regulation of innate and adaptive immune responses. The present work has defined in depth gene expression differences that point to functional specializations of endothelial cell populations in different anatomical locations, but especially the LN. Beyond the analyses provided here, these data are a resource for future work to uncover mechanisms of endothelial cell functionality.

The Antigen Processing and Presentation Machinery in Lymphatic Endothelial Cells.

Until a few years ago, lymphatic vessels and lymphatic endothelial cells (LEC) were viewed as part of a passive conduit for lymph and immune cells to reach lymph nodes (LN). However, recent work has shown that LEC are active immunological players whose interaction with dendritic cells and T cells is of important immunomodulatory relevance. While the immunological interaction between LEC and other immune cells has taken a center stage, molecular analysis of LEC antigen processing and presentation machinery is still lagging. Herein we review the current knowledge of LEC MHC I and MHC II antigen processing and presentation pathways, Including the role of LEC in antigen phagocytosis, classical, and non-classical MHC II presentation, proteasome processing and MHC I presentation, and cross-presentation. The ultimate goal is to provide an overview of the LEC antigen processing and presentation machinery that constitutes the molecular basis for their role in MHC I and MHC II-restricted immune responses.

Patterns of immune-cell infiltration in murine models of melanoma: roles of antigen and tissue site in creating inflamed tumors.

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.

Formation and phenotypic characterization of CD49a, CD49b and CD103 expressing CD8 T cell populations in human metastatic melanoma.

Integrins α1ß1 (CD49a), α2ß1 (CD49b) and αEß7 (CD103) mediate retention of lymphocytes in peripheral tissues, and their expression is upregulated on tumor infiltrating lymphocytes (TIL) compared to circulating lymphocytes. Little is known about what induces expression of these retention integrins (RI) nor whether RI define subsets in the tumor microenvironment (TME) with a specific phenotype. Human metastatic melanoma-derived CD8 TIL could be grouped into five subpopulations based on RI expression patterns: RIneg, CD49a+ only, CD49a+CD49b+, CD49a+CD103+, or positive for all three RI. A significantly larger fraction of the CD49a+ only subpopulation expressed multiple effector cytokines, whereas CD49a+CD103+ and CD49a+CD49b+ cells expressed IFNγ only. RIneg and CD49a+CD49b+CD103+ CD8 TIL subsets expressed significantly less effector cytokines overall. Interestingly, however, CD49a+CD49b+CD103+ CD8 expressed lowest CD127, and highest levels of perforin and exhaustion markers PD-1 and Tim3, suggesting selective exhaustion rather than conversion to memory. To gain insight into RI expression induction, normal donor PBMC were cultured with T cell receptor (TCR) stimulation and/or cytokines. TCR stimulation alone induced two RI+ cell populations: CD49a single positive and CD49a+CD49b+ cells. TNFα and IL-2 each were capable of inducing these populations. Addition of TGFß to TCR stimulation generated two additional populations; CD49a+CD49bnegCD103+ and CD49a+CD49b+CD103+. Taken together, our findings identify opportunities to modulate RI expression in the TME by cytokine therapies and to generate subsets with a specific RI repertoire in the interest of augmenting immune therapies for cancer or for modulating other immune-related diseases such as autoimmune diseases.

Identification and Characterization of Tertiary Lymphoid Structures in Murine Melanoma.

Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates that often share structural similarities to conventional secondary lymphoid organs. In a variety of solid cancers, the presence of these structures commonly correlates with high densities of tumor-infiltrating T lymphocytes and prolonged patient survival. These observations suggest that TLS act as sites for the development of beneficial antitumor immune responses. However, few murine tumor models have been described that could enable a more comprehensive understanding of the functionality of TLS in solid cancers. We previously reported that murine B16-F1 melanoma or Lewis lung carcinoma cells transfected to express the model antigen ovalbumin form intratumoral TLS after implantation into the peritoneal cavity of C57BL/6 mice. In this chapter, we describe immunofluorescent microscopy and flow cytometry approaches for identifying and characterizing intratumoral TLS. Additionally, we describe an adoptive transfer method for demonstrating the infiltration of naïve T cells into B16-OVA melanoma tumors via the lymph node-like vasculature, which is an essential functional feature of tumor-associated TLS.