Resident memory CD8+ T cells in regional lymph nodes mediate immunity to metastatic melanoma.

The nature of the anti-tumor immune response changes as primary tumors progress and metastasize. We investigated the role of resident memory (Trm) and circulating memory (Tcirm) cells in anti-tumor responses at metastatic locations using a mouse model of melanoma-associated vitiligo. We found that the transcriptional characteristics of tumor-specific CD8+ T cells were defined by the tissue of occupancy. Parabiosis revealed that tumor-specific Trm and Tcirm compartments persisted throughout visceral organs, but Trm cells dominated lymph nodes (LNs). Single-cell RNA-sequencing profiles of Trm cells in LN and skin were distinct, and T cell clonotypes that occupied both tissues were overwhelmingly maintained as Trm in LNs. Whereas Tcirm cells prevented melanoma growth in the lungs, Trm afforded long-lived protection against melanoma seeding in LNs. Expanded Trm populations were also present in melanoma-involved LNs from patients, and their transcriptional signature predicted better survival. Thus, tumor-specific Trm cells persist in LNs, restricting metastatic cancer.

4PD Functionalized Dendrimers: A Flexible Tool for In Vivo Gene Silencing of Tumor-Educated Myeloid Cells.

Myeloid cells play a key role in tumor progression and metastasis by providing nourishment and immune protection, as well as facilitating cancer invasion and seeding to distal sites. Although advances have been made in understanding the biology of these tumor-educated myeloid cells (TEMCs), their intrinsic plasticity challenges our further understanding of their biology. Indeed, in vitro experiments only mimic the in vivo setting, and current gene-knockout technologies do not allow the simultaneous, temporally controlled, and cell-specific silencing of multiple genes or pathways. In this article, we describe the 4PD nanoplatform, which allows the in vivo preferential transfection and in vivo tracking of TEMCs with the desired RNAs. This platform is based on the conjugation of CD124/IL-4Rα-targeting peptide with G5 PAMAM dendrimers as the loading surface and can convey therapeutic or experimental RNAs of interest. When injected i.v. in mice bearing CT26 colon carcinoma or B16 melanoma, the 4PD nanoparticles predominantly accumulate at the tumor site, transfecting intratumoral myeloid cells. The use of 4PD to deliver a combination of STAT3- and C/EBPß-specific short hairpin RNA or miR-142-3p confirmed the importance of these genes and microRNAs in TEMC biology and indicates that silencing of both genes is necessary to increase the efficacy of immune interventions. Thus, the 4PD nanoparticle can rapidly and cost effectively modulate and assess the in vivo function of microRNAs and mRNAs in TEMCs.

Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration.

Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration.

Reciprocal relationship of T regulatory cells and monocytic myeloid-derived suppressor cells in LP-BM5 murine retrovirus-induced immunodeficiency.

Immunomodulatory cellular subsets, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumour microenvironment and are targets of immunotherapy, but their role in retroviral-associated immunosuppression is less well understood. Due to known crosstalk between Tregs and MDSCs in the tumour microenvironment, and also their hypothesized involvement during human immunodeficiency virus/simian immunodeficiency virus infection, studying the interplay between these immune cells during LP-BM5 retrovirus-induced murine AIDS is of interest. IL-10-producing FoxP3+ Tregs expanded after LP-BM5 infection. Following in vivo adoptive transfer of natural Treg (nTreg)-depleted CD4+T-cells, and subsequent LP-BM5 retroviral infection, enriched monocytic MDSCs (M-MDSCs) from these nTreg-depleted mice displayed altered phenotypic subsets. In addition, M-MDSCs from LP-BM5-infected nTreg-depleted mice exhibited increased suppression of T-cell, but not B-cell, responses, compared with M-MDSCs derived from non-depleted LP-BM5-infected controls. Additionally, LP-BM5-induced M-MDSCs modulated the production of IL-10 by FoxP3+ Tregs in vitro. These collective data highlight in vitro and for the first time, to the best of our knowledge, in vivo reciprocal modulation between retroviral-induced M-MDSCs and Tregs, and may provide insight into the immunotherapeutic targeting of such regulatory cells during retroviral infection.

Dendritic cells maintain anti-tumor immunity by positioning CD8 skin-resident memory T cells.

Tissue-resident memory (TRM) T cells are emerging as critical components of the immune response to cancer; yet, requirements for their ongoing function and maintenance remain unclear. APCs promote TRM cell differentiation and re-activation but have not been implicated in sustaining TRM cell responses. Here, we identified a novel role for dendritic cells in supporting TRM to melanoma. We showed that CD8 TRM cells remain in close proximity to dendritic cells in the skin. Depletion of CD11c+ cells results in rapid disaggregation and eventual loss of melanoma-specific TRM cells. In addition, we determined that TRM migration and/or persistence requires chemotaxis and adhesion mediated by the CXCR6/CXCL16 axis. The interaction between CXCR6-expressing TRM cells and CXCL16-expressing APCs was found to be critical for sustaining TRM cell-mediated tumor protection. These findings substantially expand our knowledge of APC functions in TRM T-cell homeostasis and longevity.

Resident memory T cells in the skin mediate durable immunity to melanoma.

Tissue-resident memory T (TRM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen-specific TRM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein-1) or LAG-3 (lymphocyte activation gene-3), and were capable of making IFN-γ (interferon-γ). CD103 expression on CD8 T cells was required for the establishment of TRM cells in the skin but was dispensable for vitiligo development. CD103+ CD8 TRM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating antitumor immunity.

Tadalafil reduces myeloid-derived suppressor cells and regulatory T cells and promotes tumor immunity in patients with head and neck squamous cell carcinoma.

PURPOSE: Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play a key role in the progression of head and neck squamous cell carcinoma (HNSCC). On the basis of our preclinical data demonstrating that phosphodiesterase-5 (PDE5) inhibition can modulate these cell populations, we evaluated whether the PDE5 inhibitor tadalafil can revert tumor-induced immunosuppression and promote tumor immunity in patients with HNSCC. EXPERIMENTAL DESIGN: First, we functionally and phenotypically characterized MDSCs in HNSCCs and determined, retrospectively, whether their presence at the tumor site correlates with recurrence. Then, we performed a prospective single-center, double-blinded, randomized, three-arm study in which patients with HNSCC undergoing definitive surgical resection of oral and oropharyngeal tumors were treated with tadalafil 10 mg/day, 20 mg/day, or placebo for at least 20 days preoperatively. Blood and tumor MDSC and Treg presence and CD8(+) T-cell reactivity to tumor antigens were evaluated before and after treatment. RESULTS: MDSCs were characterized in HNSCC and their intratumoral presence significantly correlates with recurrence. Tadalafil treatment was well tolerated and significantly reduced both MDSCs and Treg concentrations in the blood and in the tumor (P < 0.05). In addition, the concentration of blood CD8(+) T cells reactive to autologous tumor antigens significantly increased after treatment (P < 0.05). Tadalafil immunomodulatory activity was maximized at an intermediate dose but not at higher doses. Mechanistic analysis suggests a possible off-target effect on PDE11 at high dosages that, by increasing intracellular cAMP, may negatively affect antitumor immunity. CONCLUSIONS: Tadalafil seems to beneficially modulate the tumor micro- and macro-environment in patients with HNSCC by lowering MDSCs and Tregs and increasing tumor-specific CD8(+) T cells in a dose-dependent fashion.

FOXP3 subcellular localization predicts recurrence in oral squamous cell carcinoma.

Forkhead box protein P3 (FOXP3) expression in tumor infiltrating CD4(+)T cells is generally associated with an intrinsic capacity to suppress tumor immunity. Based on this notion, different studies have evaluated the prognostic value of this maker in cancer but contradictory results have been found. Indeed, even within the same cancer population, the presence of CD4(+)FOXP3(+)T cells has been associated,with either a poor or a good prognosis, or no correlation has beenfound. Here, we demonstrate,in patients with oral squamous cell carcinoma (OSCC), that what really represents a prognostic parameter is not the overall expression of FOXP3 but its intracellular localization.While overallFOXP3 expression in tumor infiltrating CD4(+)T cells does not correlate with tumor recurrence, its intracellular localization within the CD4 cells does: nuclear FOXP3 (nFOXP3) is associated with tumor recurrence within 3 years, while cytoplasmicFOXP3 (cFOXP3) is associated with a lower likelihood of recurrence. Thus, we propose elevated levels of the cFOXP3/nFOXP3 ratio within tumor infiltrating CD4(+) T cells as a predictor of OSCC recurrence.

Aptamer-mediated blockade of IL4Rα triggers apoptosis of MDSCs and limits tumor progression.

In addition to promoting tumor progression and metastasis by enhancing angiogenesis and invasion, myeloid-derived suppressor cells (MDSC) and tumor-associated macrophage (TAM) also inhibit antitumor T-cell functions and limit the efficacy of immunotherapeutic interventions. Despite the importance of these leukocyte populations, a simple method for their specific depletion has not been developed. In this study, we generated an RNA aptamer that blocks the murine or human IL-4 receptor-α (IL4Rα or CD124) that is critical for MDSC suppression function. In tumor-bearing mice, this anti-IL4Rα aptamer preferentially targeted MDSCs and TAM and unexpectedly promoted their elimination, an effect that was associated with an increased number of tumor-infiltrating T cells and a reduction in tumor growth. Mechanistic investigations of aptamer-triggered apoptosis in MDSCs confirmed the importance of IL4Ra-STAT6 pathway activation in MDSC survival. Our findings define a straightforward strategy to deplete MDSCs and TAMs in vivo, and they strengthen the concept that IL4Rα signaling is pivotal for MDSC survival. More broadly, these findings suggest therapeutic strategies based on IL4Rα signaling blockades to arrest an important cellular mechanism of tumoral immune escape mediated by MDSCs and TAM in cancer.