Conventional type 1 dendritic cells (DC1) contribute to the development of pathogenic T helper type 1 (Th1) cells in part via the production of the proinflammatory cytokine interleukin-12. Thus, depletion of DC1 has the potential to dampen autoimmune responses. Here, we developed X-C motif chemokine receptor 1 (XCR1)-specific chimeric antigen receptor (CAR)-T cells and CAR-Tregs that specifically targeted DC1. XCR1 CAR-T cells were successfully generated as CD4+ and CD8+ T cells, expressed XCR1 CAR efficiently, and induced XCR1-dependent activation, cytokine production and proliferation. XCR1 CAR-T cells selectively depleted DC1 when transferred into RAG2−/− mice with a compensatory increase in conventional type 2 DC (DC2) and plasmacytoid DC (pDC). XCR1 CAR-T cell-mediated depletion of DC1 modestly suppressed the onset of Th1-driven experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Diphtheria toxin-mediated DC1 depletion in XCR1-diphtheria toxin receptor mice also suppressed EAE, suggesting that DC1 depletion was responsible for EAE suppression. XCR1 CAR-Tregs were successfully generated and suppressed effector T cells in the presence of XCR1+ cells. Therapeutic treatment with XCR1 CAR-Tregs suppressed Th1-driven EAE. Therefore, we conclude that depletion of DC1 with XCR1 CAR-T cells or immune suppression with XCR1 CAR-Tregs can modestly suppress Th1-driven EAE.

Conventional type 1 dendritic cells (cDC1s) are superior in antigen cross-presentation and priming CD8+ T cell anti-tumor immunity and thus, are a target of high interest for cancer immunotherapy. Type I interferon (IFN) is a potent inducer of antigen cross-presentation, but, unfortunately, shows only modest results in the clinic given the short half-life and high toxicity of current type I IFN therapies, which limit IFN exposure in the tumor. CD8+ T cell immunity is dependent on IFN signaling in cDC1s and preclinical studies suggest targeting IFN directly to cDC1s may be sufficient to drive anti-tumor immunity. Here, we engineered an anti-XCR1 antibody (Ab) and IFN mutein (IFNmut) fusion protein (XCR1Ab-IFNmut) to determine whether systemic delivery could drive selective and sustained type I IFN signaling in cDC1s leading to anti-tumor activity and, in parallel, reduced systemic toxicity. We found that the XCR1Ab-IFNmut fusion specifically enhanced cDC1 activation in the tumor and spleen compared to an untargeted control IFN. However, multiple treatments with the XCR1Ab-IFNmut fusion resulted in robust anti-drug antibodies (ADA) and loss of drug exposure. Using other cDC1-targeting Ab-IFNmut fusions, we found that localizing IFN directly to cDC1s activates their ability to promote ADA responses, regardless of the cDC1 targeting antigen. The development of ADA remains a major hurdle in immunotherapy drug development and the cellular and molecular mechanisms governing the development of ADA responses in humans is not well understood. Our results reveal a role of cDC1s in ADA generation and highlight the potential ADA challenges with targeting immunostimulatory agents to this cellular compartment.

Autoimmune diseases affect roughly 5-10% of the total population, with women affected more than men. The standard treatment for autoimmune or autoinflammatory diseases had long been immunosuppressive agents until the advent of immunomodulatory biologic drugs, which aimed at blocking inflammatory mediators, including proinflammatory cytokines. At the frontier of these biologic drugs are TNF-α blockers. These therapies inhibit the proinflammatory action of TNF-α in common autoimmune diseases such as rheumatoid arthritis, psoriasis, ulcerative colitis, and Crohn's disease. TNF-α blockade quickly became the "standard of care" for these autoimmune diseases due to their effectiveness in controlling disease and decreasing patient's adverse risk profiles compared to broad-spectrum immunosuppressive agents. However, anti-TNF-α therapies have limitations, including known adverse safety risk, loss of therapeutic efficacy due to drug resistance, and lack of efficacy in numerous autoimmune diseases, including multiple sclerosis. The next wave of truly transformative therapeutics should aspire to provide a cure by selectively suppressing pathogenic autoantigen-specific immune responses while leaving the rest of the immune system intact to control infectious diseases and malignancies. In this review, we will focus on three main areas of active research in immune tolerance. First, tolerogenic vaccines aiming at robust, lasting autoantigen-specific immune tolerance. Second, T cell therapies using Tregs (either polyclonal, antigen-specific, or genetically engineered to express chimeric antigen receptors) to establish active dominant immune tolerance or T cells (engineered to express chimeric antigen receptors) to delete pathogenic immune cells. Third, IL-2 therapies aiming at expanding immunosuppressive regulatory T cells in vivo.

Background Recent advances in the field of cancer immunotherapy have identified CD8 + T cell responses against tumor-specific mutations as a key driver of tumor regression and overall survival. ADXS-NEO is a personalized Listeria monocytogenes ( Lm )-based immunotherapy designed to target a patient’s mutation-derived tumor-specific neoantigens. The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations and control tumor growth by generating neoantigen-specific T cell responses using a pre-clinical mouse tumor model. Methods Whole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique non-synonymous mutations. The netMHCcons algorithm was used to predict 137 potential neoantigens. We validated 20 immunogenic neoantigens either by peptide immunization followed by ELISPOT or by the presence of CD8 + T cells recognizing the neoantigen peptide following checkpoint inhibitor treatment. Two ADXS-NEO vectors were constructed; Lm20, targeting 20 validated immunogenic neoantigens, and Lm19, targeting most of the non-validated NSMs. Results Both Lm19 & Lm20 significantly slowed tumor growth in C57BL/6 mice compared to control. An accumulation of ADXS-NEO-specific TILs was observed in tumor bearing mice treated with either Lm19 or Lm20. Examination of the tumor microenvironment in Lm19 or Lm20 treated mice revealed a decrease in the frequency and absolute number of Tregs, TAMs, MDSCs, and PD1 high exhausted CD8 + T cells as well as an increase in the frequency and absolute number of effector CD8 + T cells, relative to control. Conclusion ADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control in mice.

Single-cell RNA-seq analysis reveals that IL-2 mutein treatment expands multiple sub-states of regulatory T cells with superior suppressive function in mice. The therapeutic expansion of Foxp3+ regulatory T cells (Tregs) shows promise for treating autoimmune and inflammatory disorders. Yet, how this treatment affects the heterogeneity and function of Tregs is not clear. Using single-cell RNA-seq analysis, we characterized 31,908 Tregs from the mice treated with a half-life extended mutant form of murine IL-2 (IL-2 mutein, IL-2M) that preferentially expanded Tregs, or mouse IgG Fc as a control. Cell clustering analysis revealed that IL-2M specifically expands multiple sub-states of Tregs with distinct expression profiles. TCR profiling with single-cell analysis uncovered Treg migration across tissues and transcriptional changes between clonally related Tregs after IL-2M treatment. Finally, we identified IL-2M–expanded Tnfrsf9+Il1rl1+ Tregs with superior suppressive function, highlighting the potential of IL-2M to expand highly suppressive Foxp3+ Tregs.

Cancer vaccines using synthetic long peptides (SLP) targeting tumor antigens have been tested in the clinic but the outcomes have been unimpressive, perhaps because these peptides elicit predominantly CD4 + T cell responses. We hypothesized that enhanced delivery of peptide antigens to, and uptake in, secondary lymphoid tissues should elicit more robust CD8 + and CD4 + T cell responses and improved anti-tumor responses. Here, we have designed SLP-containing cationic lipoplexes (SLP–Lpx) that improve delivery of peptides to myeloid cells in the spleen and lymphatics. Using the G12D KRAS mutations as neoantigens, we found that vaccination of mice with naked synthetic peptides harboring the G12D mutation with CpG adjuvant stimulated mainly CD4 + T cell responses with limited tumor growth inhibition. On the other hand, immunization with SLP–Lpx stimulated both CD4 + and CD8 + T cells and suppressed tumor growth in a CD8 + T cell-dependent manner. Combination of the SLP–Lpx vaccines with a checkpoint inhibitor led to profound growth suppression of established tumors. These studies suggest that preferential targeting of peptides derived from neoantigens to the spleen via lipoplexes elicits potent CD4 + and CD8 + T cell responses that inhibit tumor growth.

Rationale: The use of eosinophils (eos) as a biomarker in management of COPD has been validated but a standardized approach for the interpretation of sampling methods has not been established. Appreciating that there are differences between sputum and blood eosinophil count (BEC) analysis, we sought to compare the two sampling methods. Methods: In an interim analysis of an ongoing observational study at UCLA, 108 subjects with stable moderate/severe COPD and 14 individuals having exacerbations were evaluated on 2 occasions over a 3-month interval, each time completing extensive clinical and biomarker characterization. Results: Agreement between sputum and blood eos values was poor (κ=0.05). In contrast to sputum analysis, BEC was more consistently captured (94.9 v 48.7%), was more repeatable (76.7%, κ=0.53 for BEC v 72.7%, κ=0.29 for sputum) and showed better cell count correlation between cytology and flow cytometry (ρ=0.89 v ρ=0.06, respectively). Using cutoff values ≥150 cells/mL for BEC, and ≥2% for sputum, we classified more individuals with eosinophilic COPD using blood (58.8%) v sputum (31.7%). High sputum eos were associated with history of exacerbations, lower FEV1, and symptoms in COPD individuals, but this was not observed with high BEC (Figure 1). Conclusions: Routine testing for sputum eos is less practical in daily management of COPD but, compared to BEC cutoff ≥150, sputum Eos ≥2% may better distinguish eosinophilic COPD.

Abstract Introduction: Neoantigens derived from tumor-specific mutations have been shown to drive tumor specific CD8+ T cell responses leading to tumor regression and extending overall survival. Frameshift mutations are estimated to generate up to nine times more neoantigens per mutation compared to in-frame mutations. However, it is not clear if vaccination against frameshift mutations induces neoantigen-specific CD8+ T cell responses that result in control of tumor growth. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target mutation-derived tumor-specific neoantigens. Advaxis' Lm-based immunotherapies consist of live attenuated bacterial vectors that are bioengineered to secrete an antigen-adjuvant fusion protein consisting of a truncated non-hemolytic fragment of listeriolysin O, which has adjuvant properties, and tumor-specific antigens. Here, we demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific frameshift mutations in order to generate neoantigen-specific T cells that control tumor growth. Results: Whole-exome sequencing of the CT26 and MC38 mouse tumor cell lines identified 30 and 31 unique frameshift mutations respectively. Individual frameshift mutations ranged in size from 12 to as many as 150 amino acids (aa). Lm vectors targeting the two longest frameshift mutations were constructed for each tumor model. The therapeutic efficacy of Lm vectors expressing either a single 57 aa (Lm-57) or a single 150 aa (Lm-150) MC38 frameshift mutation were evaluated in C57BL/6J mice. Both Lm vectors generated multiple unique frameshift-specific TILs and slowed tumor growth. Furthermore, we evaluated the tumor microenvironment following Lm-57 or Lm-150 treatment and observed a decrease in the frequency and absolute number of Tregs, TAMs, and MDSCs and an increase in the frequency and absolute number of total cytotoxic granzyme A+ effector CD8+ T cells. Similarly, Lm vectors expressing either a 64 aa (Lm-64) or a 93 aa (Lm-93) CT26 frameshift mutation were evaluated in the CT26 tumor model. Both Lm-64 and Lm-93 significantly controlled tumor growth. Additionally, an influx of neoantigen-specific TILs and a significant decrease in the frequency of intratumoral Tregs was observed. Conclusion: ADXS-NEO induced potent immune responses against tumor-specific frameshift mutations and controlled tumor growth. Advaxis' Lm platform is able to target frameshift mutations ≥150 aa and generate multiple neoantigen-specific T cells per frameshift. ADXS-NEO controls tumor growth via multiple mechanisms, including the generation of tumor-specific cytotoxic TILs, by secreting tumor-derived neoantigens directly into dendritic cells and by attenuating the suppressive tumor microenvironment. Citation Format: Brandon Coder, Daniel O. Villarreal, Susan Armington, Elena Filippova, Andrew L'Huillier, Dipti Kelkar, Xiaoming Ju, Cristina Mottershead, David Balli, Kim Ramos, Hyewon Phee, Jim Johnston, Robert Petit, Michael Princiotta. Targeting frameshift mutations with a Listeria monocytogenes immunotherapy drives neoantigen-specific antitumor immunity in the MC38 and CT26 mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-148.

Abstract Introduction: Recent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific neoantigens as a key driver of tumor regression and prolonged survival. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to generate immune responses against mutation-derived tumor-specific neoantigens. Advaxis' Lm-based immunotherapies consist of live highly-attenuated bacterial vectors that are bioengineered to secrete a fusion protein consisting of a truncated non-hemolytic fragment of listeriolysin O, which has adjuvant properties, and tumor-specific neoantigens that harbor nonsynonymous point mutations (NSMs). The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations to generate neoantigen-specific T cells and control tumor growth. Results: Whole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique NSMs. Among these, the IC50 of 138 NSMs were predicted to be less than 500 nM by the netMHCcons algorithm. We evaluated the immunogenicity of 37 NSMs, and found that 12 immunogenic NSMs elicited a CD8+ T cell response following peptide immunization. Moreover, we identified 10 additional immunogenic NSMs in MC38-bearing mice treated with a check point inhibitor. Altogether, we identified 22 immunogenic and 23 non-immunogenic NSMs. Two ADXS-NEO vectors were constructed, Lm-19 & Lm-20, targeting 19 non-immunogenic and 20 immunogenic NSMs respectively. The ability of Lm-19 and Lm-20 to control MC38 tumor growth was evaluated in C57BL/6J mice. We found that both Lm-19 & Lm-20 led to an accumulation of neoantigen-specific CD8+ TILs and significantly slowed tumor growth. Moreover, both Lm-19 and Lm-20 decreased the frequency and absolute number of intratumoral Tregs, TAMs, and MDSCs and increased the frequency and absolute number of effector CD8+ T cells. Interestingly, expression of PD-L1 was decreased in TAMs and MDSCs and the frequency and total number of granzyme A+ CD8+ effector T cells was increased. Furthermore, the proportion of phenotypically exhausted PD-1hiLAG3+ TILs was decreased. Together, these data suggest the tumor microenvironment in mice receiving Lm-19 and Lm-20 becomes more cytotoxic and less suppressive. Conclusion: ADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control. The effectiveness of the Lm platform is demonstrated by the generation of neoantigen-specific T cells to peptide sequences that were identified as “non-immunogenic” using a conventional peptide-adjuvant immunization. This study is a clear demonstration that T cell mediated anti-tumor responses can be generated by targeting tumor-derived NSMs with the ADXS-NEO Listeria monocytogenes vector. Citation Format: Brandon Coder, Hyewon Phee, Cristina Mottershead, Dipti Kelkar, Elena Filippova, Xiaoming Ju, Bryan Vander Lugt, Olga Pryshchep, Justin Lesch, Xian Liu, Jason DeVoss, Keegan Cooke, Claret Liu, Jinghui Zhan, Petia Mitchell, Kim Ramos, Daniel O. Villarreal, Jim Johnston, Robert Petit, Michael Princiotta. Neoantigens that fail to elicit measurable T cell responses following peptide immunization can control tumor growth when delivered using a Listeria-based immunotherapy platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-150.

The p21-activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus-derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T-cell subsets including Foxp3+ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3+ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3+ Treg cells upon T-cell receptor and interleukin-2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3+ Treg cells.

Foxp3, a key transcription factor that drives lineage differentiation of regulatory T cells (Tregs), was thought to imprint a unique and irreversible genetic signature within Tregs. Recent evidence, however, suggests that loss or attenuation of Foxp3 expression can cause Tregs to de-differentiate into effector T cells capable of producing proinflammatory cytokines. Herein, we report that the signaling kinase, p21-activated kinase 2 (Pak2), is essential for maintaining Treg stability and suppressive function. Loss of Pak2, specifically in Tregs, resulted in reduced expression of multiple Treg functional molecules, including Foxp3, CD25, Nrp-1 and CTLA-4, coupled with a loss of Treg suppressive function in vitro and in vivo . Interestingly, Pak2-deficient Tregs gained expression of Th2-associated cytokines and the transcription factor, Gata3, becoming Th2-like cells, explaining their inability to regulate immune responses. Collectively, these findings suggest Pak2 as an important signaling molecule for guarding against aberrant immune responses through regulating the stability of Foxp3 + Tregs and maintaining a suppressive Treg phenotype.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag −/ − mice. The CD8α + DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4 + T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4 + T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α + DCs and other cells of the immune system.

Although significant effort has been devoted to understanding the thymic development of Foxp3(+) regulatory T cells (Tregs), the precise signaling pathways that govern their lineage commitment still remain enigmatic. Our findings show a novel role for the actin cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), in Treg development and homeostasis. The absence of Pak2 in T cells resulted in a marked reduction in both thymus-and peripherally derived Tregs, accompanied by the development of spontaneous colitis in Pak2-deficient mice. Additionally, Pak2 was required for the proper differentiation of in vitro-induced Tregs as well as maintenance of Tregs. Interestingly, Pak2 was necessary for generating the high-affinity TCR- and IL-2-mediated signals that are required by developing Tregs for their lineage commitment. These findings provide novel insight into how developing thymocytes translate lineage-specific high-affinity TCR signals to adopt the Treg fate, and they further posit Pak2 as an essential regulator for this process.

NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression.

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation.