Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.

Germinal Center T follicular helper (GC-Tfh) cell impairment in chronic HIV infection involves c-Maf signaling.

We have recently demonstrated that the function of T follicular helper (Tfh) cells from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating GC-Tfh cells from HIV-infected subjects were transcriptionally different than their HIV-uninfected counterparts, and displayed a significant downregulation of immune- and GC-Tfh-associated pathways and genes. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh cell impairment during HIV infection. Understanding how GC-Tfh cell function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies.

Interpretation of T cell states from single-cell transcriptomics data using reference atlases.

Single-cell RNA sequencing (scRNA-seq) has revealed an unprecedented degree of immune cell diversity. However, consistent definition of cell subtypes and cell states across studies and diseases remains a major challenge. Here we generate reference T cell atlases for cancer and viral infection by multi-study integration, and develop ProjecTILs, an algorithm for reference atlas projection. In contrast to other methods, ProjecTILs allows not only accurate embedding of new scRNA-seq data into a reference without altering its structure, but also characterizing previously unknown cell states that "deviate" from the reference. ProjecTILs accurately predicts the effects of cell perturbations and identifies gene programs that are altered in different conditions and tissues. A meta-analysis of tumor-infiltrating T cells from several cohorts reveals a strong conservation of T cell subtypes between human and mouse, providing a consistent basis to describe T cell heterogeneity across studies, diseases, and species.

Autoimmunity linked protein phosphatase PTPN22 as a target for cancer immunotherapy.

BACKGROUND: Cancer immunotherapy has evolved from interferon-alpha (IFNα) and interleukin-2 in the 1980s to CTLA-4 and PD-1/PD-L1 checkpoint inhibitors (CPIs), the latter highlighting the importance of enhancing T-cell functions. While the search for novel immunomodulatory pathways continues, combination therapies augmenting multiple pathways can also increase efficacy. The association of autoimmune-related adverse events with clinical efficacy following CPI treatment has been inferred and suggests that breaking tolerance thresholds associated with autoimmunity may affect host immune responses for effective cancer immunotherapy. RESULTS: Here, we show that loss of autoimmune associated PTPN22, a key desensitization node for multiple signaling pathways, including IFNα receptor (IFNAR) and T-cell receptor, can augment tumor responses. Implantation of syngeneic tumors in Ptpn22-/- mice led to expansion and activation of peripheral and intratumoral T cells and, in turn, spontaneous tumor regression as well as enhanced responses in combination with anti-PD-L1 treatment. Using genetically modified mice expressing a catalytically inactive PTPN22 or the autoimmunity-associated human single-nucleotide polymorphism variant, augmentation of antitumor immunity was dependent on PTPN22 phosphatase activity and partially on its adaptor functions. Further, antitumor responses were dependent on both CD4+ and CD8+T cells and, in part, IFNAR function. Finally, we demonstrate that the autoimmune susceptibility Ptpn22(C1858T) variant is associated with lower risk of developing non-melanoma skin cancers, improved overall survival and increased risk for development of hyperthyroidism or hypothyroidism following atezolizumab (anti-PD-L1) treatment. CONCLUSIONS: Together, these data suggest that inhibition of PTPN22 phosphatase activity may provide an effective therapeutic option for cancer immunotherapy and that exploring genetic variants that shift immune tolerance thresholds may serve as a paradigm for finding new cancer immunotherapy targets.

Immunization associated with primary tumor growth leads to rejection of commonly used syngeneic tumors upon tumor rechallenge.

The recent success of multiple immunomodulating drugs in oncology highlights the potential of relieving immunosuppression by directly engaging the immune system in the tumor bed to target cancer cells. Durable responses to immune checkpoint inhibitors experienced by some patients may be indicative of the formation of a T cell memory response. This has prompted the search for preclinical evidence of therapy-induced long-term immunity as part of the evaluation of novel therapeutics. A common preclinical method used to document long-term immunity is the use of tumor rechallenge experiments in which tumor growth is assessed in mice that have previously rejected tumors in response to therapy. Failure of rechallenge engraftment, typically alongside successful engraftment of the same tumor in naive animals as a control, is often presented as evidence of therapy-induced tumor immunity. Here, we present evidence that formation of tumor immunity often develops independent of therapy. We observed elevated rates of rechallenge rejection following surgical resection of primary tumors for four of five commonly used models and that such postexcision immunity could be adoptively transferred to treatment-naïve mice. We also show that tumor-specific cytolytic T cells are induced on primary tumor challenge independent of therapeutic intervention. Taken together these data call into question the utility of tumor rechallenge studies and the use of naïve animals as controls to demonstrate therapy-induced formation of long-term tumor immunity.

CD96 functions as a co-stimulatory receptor to enhance CD8+ T cell activation and effector responses.

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.

PD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancer.

Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.

Tetravalent biepitopic targeting enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members.

Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.

Coexpression of Inhibitory Receptors Enriches for Activated and Functional CD8+ T Cells in Murine Syngeneic Tumor Models.

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8+ T cells in commonly used syngeneic tumor models, the coexpression of inhibitory receptors PD-1, LAG3, and TIM3 defined a group of highly activated and functional effector cells. Coexpression of these receptors further enriched for antigen-specific cells with increased T-cell receptor clonality. Anti-PD-L1 treatment increased the number and activation of these triple-positive CD8+ T cells without affecting the density of PD-1- cells. The intratumoral density of CD8+ T cells coexpressing inhibitory receptors negatively correlated with tumor burden. The density ratio and pretreatment phenotype of CD8+ T cells coexpressing inhibitory receptors was positively correlated with response across a variety of tumor models. Our results demonstrate that coexpression of inhibitory receptors is not a signifier of exhausted T cells, but rather can define a group of activated and functional effector cells in syngeneic tumor models. In the cancer setting, these cells could represent a heterogeneous population of not only exhausted but also highly activated cells responsive to treatment.

Adenosine deaminase-1 delineates human follicular helper T cell function and is altered with HIV.

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.

Anti-PD-L1 Treatment Results in Functional Remodeling of the Macrophage Compartment.

Checkpoint inhibitors like anti-PD1/PD-L1 have demonstrated significant therapeutic efficacy in a subset of patients partly through reinvigoration of CD8 T cells. However, their impact on myeloid cells remains largely unknown. Here, we report that anti-PD-L1 treatment favorably impacts the phenotype and function of tumor macrophages by polarizing the macrophage compartment toward a more proinflammatory phenotype. This phenotype was characterized by a decrease in Arginase-I (ARG1) expression and an increase in iNOS, MHCII, and CD40 expression. Whole-transcriptome profiling further confirmed extensive polarization of both tumor monocytes and macrophages from a suppressive to a proinflammatory, immunostimulatory phenotype. This polarization was driven mainly through IFNγ and was associated with enhanced T-cell activity. Transfer of monocytes into anti-PD-L1-treated tumor-bearing mice led to macrophage differentiation into a more proinflammatory phenotype, with an increase in CD8 T cells expressing granzyme-B and an increase in the CD8/Treg ratio compared with control-treated mice. Although in responsive tumor models, anti-PD-L1 treatment remodeled the macrophage compartment with beneficial effects on T cells, both macrophage reprogramming and depletion were needed to maximize anti-PD-L1 responses in a tumor immune contexture with high macrophage burden. Our results demonstrate that anti-PD-L1 treatment can favorably remodel the macrophage compartment in responsive tumor models toward a more proinflammatory phenotype, mainly through increased IFNγ levels. They also suggest that directly targeting these cells with reprogramming and depleting agents may further augment the breadth and depth of response to anti-PD-L1 treatment in less responsive or more macrophage-dense tumor microenvironments. SIGNIFICANCE: This work demonstrates that increased IFNγ signaling following anti-PD-L1 treatment can remodel the macrophage compartment to enhance T-cell responses.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1493/F1.large.jpg.

CD226 regulates natural killer cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1.

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.

Chemotherapy Combines Effectively with Anti-PD-L1 Treatment and Can Augment Antitumor Responses.

Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with chemotherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune response generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti-PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity mediated by anti-PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various chemotherapies were also agent specific, and synergy with anti-PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor.

TGFß attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

Circulating CXCR5+CXCR3+PD-1lo Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth.

HIV-1-specific broadly neutralizing antibodies (bnAbs) typically develop in individuals with continuous high-level viral replication and increased immune activation, conditions that cannot be reproduced during prophylactic immunization. Understanding mechanisms supporting bnAb development in the absence of high-level viremia may be important for designing bnAb-inducing immunogens. Here, we show that the breadth of neutralizing antibody responses in HIV-1 controllers was associated with a relative enrichment of circulating CXCR5+CXCR3+PD-1lo CD4+ T cells. These CXCR3+PD-1lo Tfh-like cells were preferentially induced in vitro by functionally superior dendritic cells from controller neutralizers, and able to secrete IL-21 and support B cells. In addition, these CXCR3+PD-1lo Tfh-like cells contained higher proportions of stem cell-like memory T cells, and upon antigenic stimulation differentiated into PD-1hi Tfh-like cells in a Notch-dependent manner. Together, these data suggest that CXCR5+CXCR3+PD-1lo cells represent a dendritic cell-primed precursor cell population for PD-1hi Tfh-like cells that may contribute to the generation of bnAbs in the absence of high-level viremia.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

The dysfunction of T follicular helper cells.

PURPOSE OF REVIEW: T follicular helper (Tfh) cells play a critical role as providers of B-cell help and dysfunction in Tfh/B-cell interactions can lead to autoimmunity or immunodeficiency. These observations have generated a great deal of interest in understanding how these cells are affected during HIV infection and how their functional changes might affect antibody responses. RECENT FINDINGS: Recent studies have shown that HIV/simian immunodeficiency virus (SIV) infection affects both Tfh-cell frequency and function and suggest that Tfh-cell perturbations might contribute to the relative inefficiency of HIV-infected individuals to generate broadly neutralizing antibodies (bNAbs). SUMMARY: The present review will highlight these recent findings addressing the role of Tfh cells in HIV infection as well as the impact HIV infection has on Tfh and circulating memory Tfh (cTfh) cell frequency and function.

Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.

The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4⁺ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1⁺CXCR5⁺CD4⁺ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV⁺ individuals.

Mesothelin virus-like particle immunization controls pancreatic cancer growth through CD8+ T cell induction and reduction in the frequency of CD4+ foxp3+ ICOS- regulatory T cells.

Our previous study has shown that mesothelin (MSLN) is a potential immunotherapeutic target for pancreatic cancer. Here, we further studied the immunogenicity of chimeric murine MSLN-virus-like particles (mMSLN-VLPs), their ability to break tolerance to mMSLN, a self-antigen, and deciphered the mechanism of immune responses elicited by mMSLN-VLP immunization using a pancreatic cancer (PC) mouse model. In addition to what we have found with xenogeneic human MSLN-VLP (hMSLN-VLP), mMSLN-VLP immunization was able to break the tolerance to intrinsic MSLN and mount mMSLN-specific, cytotoxic CD8(+) T cells which led to a significant reduction in tumor volume and prolonged survival in an orthotopic PC mouse model. Furthermore, CD4(+)foxp3(+) regulatory T cells (Tregs) were progressively decreased in both spleen and tumor tissues following mMSLN-VLP immunization and this was at least partly due to elevated levels of IL-6 production from activated plasmocytoid dendritic cell (pDC)-like cells following mMSLN-VLP immunization. Moreover, mMSLN-VLP treatment mainly reduced the frequency of the CD4(+)foxp3(+)ICOS(-) Treg subset. However, mMSLN-VLP induced IL-6 production also increased ICOSL expression on pDC-like cells which supported the proliferation of immunosuppressive CD4(+)foxp3(+)ICOS(+) Treg cells. This study reveals that mMSLN-VLP immunization is capable of controlling PC progression by effectively mounting an immune response against mMSLN, a tumor self-antigen, and altering the immunosuppressive tumor microenvironment via activation of pDCs-like cells and reduction in the frequency of CD4(+)foxp3(+)ICOS(-) Treg cells. However, combination therapies will likely need to be used in order to target residual CD4(+)foxp3(+)ICOS(+) Treg cells.