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.

CD103 and periplakin are potential biomarkers for response of metastatic melanoma to pembrolizumab.

This study was designed to screen for preliminary evidence of predictive markers of melanoma response to PD-1 blockade. We hypothesized that the following immune markers would be positive predictors of response: increased densities of CD103 + CD8 + T cells or Th1 lineage T-bet + T cells, high expression of CXCL9-11 and presence of tertiary lymphoid structures. Conversely, we hypothesized that the high expression of barrier molecules would be a negative predictor of response. Patients with advanced melanoma treated with pembrolizumab were identified, and clinical response as well as overall survival data were collected. Tumor samples were evaluated by multiplex immunofluorescence histology. All statistical analyses were performed in R Studio and Microsoft Excel using the Mann-Whitney U test, chi-square test, Spearman's rank correlation and Kaplan-Meier survival curves. Sixty-five advanced melanoma patients were identified, of whom 46 met inclusion criteria and were included in this study. Increased densities ( P = 0.04) and proportions ( P = 0.02) of CD8 + T cells expressing CD103 + were associated with complete response (CR) to pembrolizumab. Improved survival was associated with increased proportions of CD8 + cells expressing CD103 ( P = 0.0085) as well as decreased density of periplakin + cells ( P = 0.012) and periplakin + SOX10 + cells ( P = 0.0012). The density and proportion of CD8 + T cells expressing CD103 + positively correlated with PD-L1 expression, though PD-L1 expression was not significantly correlated with outcomes. This screening study found that increased density and proportion of CD8 + T cells expressing CD103 and decreased density of periplakin were associated with positive outcomes in patients with melanoma metastases treated with pembrolizumab and may warrant further study.

Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-Associated Tertiary Lymphoid Structures.

The presence of tertiary lymphoid structures (TLS) is correlated with prolonged patient survival in a variety of solid cancers, including melanoma. However, few methods have been described that could enable a more comprehensive understanding of the organization and functionality of TLS in solid cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying TLS and intra-tumoral immune infiltrates in melanoma. The described methods are not limited to melanoma alone and could be used to evaluate tertiary lymphoid structures in a wide variety of human cancers.

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.

Proliferating CD8+ T Cell Infiltrates Are Associated with Improved Survival in Glioblastoma.

BACKGROUND: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. METHODS: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. RESULTS: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). CONCLUSIONS: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.

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.

Deconvolution of the immunological contexture of mouse tumors with multiplexed immunohistochemistry.

In a variety of solid tumors, the presence of higher densities of tumor-infiltrating lymphocytes or tertiary lymphoid structures (TLS) are correlated with prolonged patient survival. Murine studies are usually required to define mechanisms that govern immunologic infiltrate in tumors. However, few methods have been described that could enable a more comprehensive understanding of the functionality of intratumoral immune infiltrate and TLS in solid murine cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying intratumoral immune infiltrate and TLS in murine tumor models.

Incomplete Freund's adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment.

BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).