Depletion Of Treg Cells Research Articles

Abstract 1014: Selective impairment of intratumoral regulatory T cells by targeting Ezh2 enhances cancer immunity

Abstract High numbers of tumor infiltrating regulatory T (Treg) cells are indicative of poor outcome in several malignancies, including ovarian cancer, colorectal cancer, and melanoma. Thus, selectively abrogating intratumoral Treg cell function while maintaining systemic immune tolerance remains an attractive, albeit elusive, strategy for cancer immunotherapy. We have identified the epigenetic enzyme Ezh2, an H3K27 methyltransferase, to be a critical mediator of lineage stability and function in activated Treg cells. Consequently, deficiency of Ezh2 in Treg cells strongly impairs their function in non-lymphoid tissues. We hypothesized that this phenomenon might translate to a similar defect that is restricted to the tumor microenvironment. Here we tested this hypothesis in three transplantable syngeneic tumor models in mice (MC38 colon carcinoma, TRAMP-C2 prostate cancer, and B16F10 melanoma). We observed that constitutive deletion of Ezh2 in Treg cells resulted in potent anti-tumor activity, significantly impaired tumor outgrowth, and in many instances, complete tumor rejection. These results were also recapitulated when Ezh2 was temporally deleted in the vast majority of Treg cells at the time of tumor inoculation. Functional analyses at early time points after Ezh2 deletion revealed enhanced functionality of effector CD4 and CD8 populations, as evidenced by increased IFN-γ production. These effects were evident without reduction of intratumoral Treg cell frequencies and despite presence of residual wild type Treg cells. Mechanistic studies showed that Ezh2-deficient Treg cells were destabilized and produced pro-inflammatory cytokines IL-2 and IFN-γ selectively within the tumor microenvironment, suggesting a direct contribution of Ezh2-deficient Treg cells to tumor clearance. To more directly address this hypothesis, we generated mice that harbored both Ezh2-deficient and wild type Treg cells and observed anti-tumor activity similar to mice that exclusively harbored Ezh2-deficient Treg cells. In stark contrast to Ezh2 deficiency, mice whose Treg cells were completely depleted, using a Foxp3-driven diphtheria toxin receptor allele, were unable to reject tumors, further supporting a dominant role of Ezh2-deficient Treg cells in driving anti-tumor immunity. Finally, tumor protection in the presence of Ezh2-deficient Treg cells occurred without significant morbidity due to autoimmune pathologies, which were prevalent in mice with systemic Treg cell depletion. These results suggest that targeted deletion of Ezh2 in Treg cells drives a unique phenotype among tumor infiltrating Treg cells, reprogramming the tumor microenvironment and selectively augmenting the anti-tumor immune response. Cancer therapies that pharmacologically inhibit Ezh2 to target tumor cells are under investigation, going forward it will also be important to assess the potential for beneficial effects via modulating the immune response. Citation Format: David Q. Wang, Jason R. Quiros, Chien-Chun S. Pai, Lawrence H. Fong, Jeffrey A. Bluestone, Michel J. DuPage. Selective impairment of intratumoral regulatory T cells by targeting Ezh2 enhances cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2017-1014

Abstract 4586: Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models

Abstract T regulatory (Treg) cells play an important role in maintaining immunological tolerance to self-antigens, thus limiting immune responses to tumor antigens. Therefore, depleting or suppressing Tregs is one strategy by which anti-tumor immunity can be restored. The immunotoxin ONTAK® is an IL-2-diphteria toxin fusion protein that has been shown to diminish Tregs in patients and animal models of cancer in peripheral blood using a systemic intravenous (i.v.) administration route. E7777 is a new version of ONTAK®. In this study we tested the hypothesis that locally-diminished Tregs by intratumoral (i.t.) administration of E7777 generate effective anti-tumor immune response at both local and systemic levels. First, we showed superior anti-tumor activity and safety of E7777 i.t. over E7777 i.v., where i.t. administration resulted in complete tumor regressions in both moderately immunogenic CT26 and non-immunogenic B16F10 tumors with minimal animal body weight loss. In contrast, only tumor growth delay was observed for E7777 i.v. with dose-limiting animal body weight reduction in the same models. Immune phenotyping showed a 4 fold reduction of intratumoral Tregs in treated CT-26 tumors without significant change of Tregs in the spleens of treated animals, confirming a local Treg-depleting effect of E7777 i.t. In contrast, intratumoral CD8+ T cells were not reduced. Second, E7777 i.t. enhanced overall anti-tumor immune response, manifested by significantly increased numbers of CD45+ hematopoietic cells, Granzyme B+ CD8+ cytotoxic T cells, and ratios of cytotoxic T cells/Tregs in the treated tumors. Importantly, E7777 i.t. also resulted in distant effects in the spleen characterized by increased ratio of T lymphocytes to myeloid cells and increased frequencies of both effector memory CD8+ T cells (CD8+CD62L-CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+) indicative of systemic immune activation. Consistent with the generation of immunological memory, 60% of the tumor-free animals treated with E7777 i.t. rejected completely and 40% displayed delayed tumor growth of B16F10 cell challenge while all naïve control animals grew tumors. Taken together, our results demonstrate that intratumoral Treg depletion by local administration of E7777 leads to an effective local and memory anti-tumor response in preclinical models and support further evaluation of local E7777 delivery as a cancer immunotherapy. Citation Format: Diana I. Albu, Christy Ingersoll, Kuan-Chun Huang, Mary Woodall-Jappe, Xingfeng Bao. Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4586. doi:10.1158/1538-7445.AM2017-4586

Abstract LB-059: Neoadjuvant immunotherapy with androgen deprivation therapy (ADT) prior to radiation in prostate cancer: Impact on multiparametric prostate MRI and immune responses

Abstract Background: There is increasing interest in using combination immunotherapy in the neoadjuvant setting in prostate cancer, however, endpoints for such studies remain elusive. We have conducted a clinical trial evaluating immunotherapy with ADT in patients with high risk prostate cancer. Patients were assessed for immune responses and changes in endorectal (er) MRI which can be used to assess intraprostatic tumors. Methods: Treatment-naïve high-risk (Gleason 8-10, PSA>20, or stage T3) prostate cancer patients (pts) were randomized to standard ADT+Radiation + an immunotherapy targeting MUC1 (tecemotide, aka L-BLP25) in this trial (NCT01496131). ADT consisted of gonadotropin-releasing hormone therapy. Immunotherapy included low dose (300 mg/m2, maximum 600 mg) pre-treatment cyclophosphamide for regulatory T-cell depletion. erMRI was done at baseline and after 2 months of immunotherapy including multiparametric MRI evaluation of apparent diffusion coefficient (ADC) maps from diffusion-weighted MRI. Monthly peripheral blood assessments analyzed immune cell subsets using flow cytometry and intracellular cytokine (ICC) staining for MUC-1 specific responses. Results: 28 pts with high risk prostate cancer were enrolled (n=14/arm). As expected, PSA declined in all pts 2 months after ADT. erMRI after 2 months of treatment suggested greater improvements in ADC values in pts receiving immunotherapy+ADT vs. ADT alone. Improved ADC on MRI indicates increased intratumoral diffusion and has been associated with decreased tumor density. The improvements in ADC were seen when one dominant tumor per patient was evaluated (p=0.17) but were more pronounced when up to 3 lesions were evaluated per pt (n=44 lesions; p=0.031). Compared to baseline, there were trends to increases in CTLA4+ CD8+ T-cells consistent with immune activation and decreases in myeloid derived suppressor cells (MDSCs) in pts receiving immunotherapy+ADT coinciding with the erMRI changes. These immune findings were not seen in the ADT alone group. 3 of 14 pts had MUC1 specific immune response by ICC. 2 of these patients had the greatest changes in ADC noted on erMRI over a 2-year period. Conclusions: Based on assessments by erMRI, pts who received ADT+immunotherapy had greater improvements in ADC than pts receiving ADT alone. Given that ADC improvements are associated with decreased tumor density, this suggests a possible greater anti-tumor effect of the ADT-immunotherapy combination vs. ADT alone. These findings were associated with trends to increased activated CD8+ T-cells and decreased MDSCs in pts receiving immunotherapy+ADT, with 3/14 pts having MUC1 specific immune responses. Further studies are required to confirm the potential to use ADC on erMRI as a potential (bio)marker of anti-tumor effect of immune combinations including ADT. Citation Format: Ravi A. Madan, Baris Turkbey, Lauren M. Lepone, Renee N. Donahue, Italia Grenga, Samuel Samuel Borofsky, Peter A. Pinto, Deborah Citrin, Aradhana Kaushal, Andra Krauze, Sheri McMahon, Myrna Rauchhorst, Anna Couvillon, Martin H. Falk, S Peter Eggleton, Stephen C. Greco, Peter L. Choyke, William L. Dahut, Jeffrey Schlom, James L. Gulley. Neoadjuvant immunotherapy with androgen deprivation therapy (ADT) prior to radiation in prostate cancer: Impact on multiparametric prostate MRI and immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-059. doi:10.1158/1538-7445.AM2017-LB-059

Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells

Interleukin-2 (IL-2) is an established therapeutic agent used for cancer immunotherapy. Since treatment efficacy is mediated by CD8+ and NK cell activity at the tumour site, considerable efforts have focused on generating variants that expand these subsets systemically, as exemplified by IL-2/antibody complexes and ‘superkines’. Here we describe a novel determinant of antitumour activity using fusion proteins consisting of IL-2 and the antibody fragment crystallizable (Fc) region. Generation of long-lived IL-2-Fc variants in which CD25 binding is abolished through mutation effectively prevents unwanted activation of CD25+ regulatory T-cells (Tregs) and results in strong expansion of CD25− cytotoxic subsets. Surprisingly, however, such variants are less effective than wild-type IL-2-Fc in mediating tumour rejection. Instead, we report that efficacy is crucially dependent on depletion of Tregs through Fc-mediated immune effector functions. Our results underpin an unexpected mechanism of action and provide important guidance for the development of next generation IL-2 therapeutics.

Efficient activation of latent HIV reservoir in human memory T cells by depletion of regulatory T cells in humanized mice in vivo

Abstract Regulatory T (Treg) cells play an important role in modulating immune activation and human immunodeficiency virus type 1 (HIV-1) infection. However, the role of Treg cells in HIV-1 latency establishment or maintenance in vivo remains unknown because of the lack of a robust model. We have established a humanized mouse model that supports long-term stable HIV-1 replication. When treated with combination antiretroviral therapy (cART), HIV-1 infection was stably suppressed to undetectable levels. Upon cART interruption, HIV-1 replication rebounded rapidly to pre-treatment levels. Interestingly, HIV-1 viral replication was significantly increased upon Treg cell depletion under effective cART. The HIV-1 reactivation during cART was mainly through increased gene expression from T cells. The rebound viruses during cART did not acquire drug resistance mutations. We found that most activated HIV-1 reservoir target cells were CD4 memory T cells in all lymphoid organs. These findings demonstrate that Treg cells suppress HIV-1 replication and its reactivation from latently infected T cells in vivo. Treg cell depletion provides an efficient way to reverse latency and reactivate HIV-1 reservoirs in vivo.

Depletion of Treg cells induces an increased activation of T-CD4+ and T-CD8+ cells during colorectal cancer

Abstract Colorectal Cancer (CAC) is the second most commonly diagnosed cancer in women and the third in men in North America and Europe. CAC is associated with inflammatory responses where intestinal pathology is caused by a T-cell dysregulation that concludes in an imbalance between T activated (Tact) and T regulatory (Treg) cells. Treg cells are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, contributing to the maintenance of immune homeostasis. It has been suggested a tumor-promoting function for Treg cells in CAC, but the kinetics of Treg cells during CAC development is poorly known. Thereby we used a mouse model of CAC induced by azoxymethane and three cycles of dextran sodium sulfate (DSS, 2%). We analyzed the percentage of Treg cells in blood, spleen and mesenteric lymph nodes (MLN) in each cycle. A significant reduction of Treg cells in blood and spleen during the first cycle, but increased percentage of Treg cells during the second and third cycle was observed. We did not observe reduction on Treg cells in the first DSS cycle in MLN, but a significant increase in percentage was detected during the second and third cycle of DSS. A Higher percentage of Treg cells-expressing PD1, CD127 and Tim-3 during the second and third DSS cycle in spleen and MLN was observed, whereas CD25 had no changes. However, we found increasing percentages of CD4+CD25+Foxp3− Tact cells only in the first DSS cycle in blood, spleen, and MLN. These results suggest that a gradually increased number of Treg cells suppress Tact cells during the second and third DSS cycle. Depletion of Treg cells with anti-CD25 PC61 antibody in the second DSS cycle increased percentages of CD4+CD25+Foxp3− Tact and TCD8+ cells, with an impact in tumorigenesis.

Vitamin D supplementation modulates Th2 immune response by inducing T regulatory cells in allergic conjunctivitis

Abstract Allergic conjunctivitis (AC) is one of the most common diseases encountered in eye clinics. Recently, Vitamin D deficiency has been shown to be associated with allergic AC. This present study was designed to evaluate the efficacy of vitamin D to suppress ovalbumin (OVA)-sensitized AC in a murine model. BALB/c mice were sensitized with OVA and aluminum hydroxide. Two weeks later, mice were challenged by OVA eyedrops for 12 days with intraperitoneal injection of vehicle or 1,25(OH)2D3. We evaluated clinical signs, the infiltration of inflammatory cells, regulatory T cells (Treg), serum IgE, and Th2 cytokines through flow cytometry and ELISA. In addition, to evaluate inhibitory function of Treg cells, anti–CD25 blocking antibody or isotype control antibody was injected intravenously during topical OVA challenge. AC development and conjunctival infiltration of eosinophils and mast cells were significantly impaired with 1,25(OH)2D3 treatment. In addition, 1,25(OH)2D3 suppressed production of OVA-specific IgE in serum and Th2 cytokines in vitro T cell assays, such as IL-4 and IL-13, compared to vehicle group. Interestingly, 1,25(OH)2D3 led to increase Treg cells population in draining LNs and suppressive levels of clinical signs and inflammatory cells infiltration into conjunctivae were reversed by depleting Treg cells. Our results suggest that vitamin D supplement alleviate allergic conjunctivitis, indicated by suppressing Th2 response in draining LNs and inflammatory cells infiltration into conjunctiva through increasing population of Treg cells in draining LNs. Therefore our results demonstrated the therapeutic potential of vitamin D for allergic diseases including asthma or atopic dermatitis.

Suppression of vaccination-induced antigen-specific regulatory T cells enhances the antiviral immunity against influenza virus infection

Abstract T cell immunity targeting the conserved epitopes of influenza virus has the potential to provide cross-protection against distinctly-related strains. Peptide vaccines are an ideal strategy to induce specific antiviral T cell immunity, but subimmunogenic stimulation by peptide vaccines tend to induce regulatory T (Treg) cells. However, it remains unclear about the roles of vaccine-induced antigen-specific Treg cells in vivo. Here, we aimed to investigate how vaccine-induced antigen-specific Treg cells respond during acute influenza virus infection, and the effects of adjuvants on them. By adoptive transfer experiments with OT-II TCR transgenic T cells, we found that the OVA OT-II peptide vaccine induced Treg cells and secondary vaccination further expanded them. Infection with influenza virus containing the OVA OT-II peptide also drove the expansion of pre-existing vaccine-induced Treg cells. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells enhanced the antiviral immunity. Vaccination combined with adjuvants, especially with CpG, suppressed the development of vaccine-induced antigen-specific Treg cells. Finally, CpG-adjuvanted OVA protein or whole inactivated influenza vaccine by the subcutaneous-prime-intranasal-boost strategy reduced the ratio of antigen-specific Treg cells in lung and protected mice from the heterosubtypic influenza infection. In conclusion, unadjuvanted peptide vaccines promoted antigen-specific Treg cells, which were further expanded upon acute influenza infection. The CpG adjuvant restricted the development of vaccine-induced antigen-specific Treg cells, and enhanced the antiviral T cell immunity against heterosubtypic influenza infection.

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection.

Bifidobacterium animalis subsp. lactis 420 mitigates the pathological impact of myocardial infarction in the mouse.

There is a growing appreciation that our microbial environment in the gut plays a critical role in the maintenance of health and the pathogenesis of disease. Probiotic, beneficial gut microbes, administration can directly attenuate cardiac injury and post-myocardial infarction (MI) remodelling, yet the mechanisms of cardioprotection are unknown. We hypothesised that administration of Bifidobacterium animalis subsp. lactis 420 (B420), a probiotic with known anti-inflammatory properties, to mice will mitigate the pathological impact of MI, and that anti-inflammatory T regulatory (Treg) immune cells are necessary to impart protection against MI as a result of B420 administration. Wild-type male mice were administered B420, saline or Lactobacillus salivarius 33 (Ls-33) by gavage daily for 14 or 35 days, and underwent ischemia/reperfusion (I/R). Pretreatment with B420 for 10 or 28 days attenuated cardiac injury from I/R and reduced levels of inflammatory markers. Depletion of Treg cells by administration of anti-CD25 monoclonal antibodies eliminated B420-mediated cardio-protection. Further cytokine analysis revealed a shift from a pro-inflammatory to an anti-inflammatory environment in the probiotic treated post-MI hearts compared to controls. To summarise, B420 administration mitigates the pathological impact of MI. Next, we show that Treg immune cells are necessary to mediate B420-mediated protection against MI. Finally, we identify putative cellular, epigenetic and/or post-translational mechanisms of B420-mediated protection against MI.

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

SummaryCD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology.

Dual genetic absence of STAT6 and IL-10 does not abrogate anti-hyperglycemic effects of Schistosoma mansoni in streptozotocin-treated diabetic mice

Schistosoma mansoni (Sm) is known to exert protective effects against various allergic and autoimmune disorders. It has been reported that this parasite protects NOD mice from spontaneous type 1 diabetes (T1D) and ameliorates streptozotocin (STZ)-induced T1D in wild-type mice. Here, we tried to clarify the anti-diabetic mechanisms of Sm in the latter model. Sm infection partially prevented the degradation of pancreatic islets and hyperglycemia in multiple low-dose (MLD) STZ-treated mice. Neither Treg cell depletion nor genetic absences of IL-10 and/or STAT6 abrogated the anti-hyperglycemic effects of Sm. Among M2 macrophage markers, Arg-1 and Ym1, but not Retnla, remained up-regulated in the pancreatic lymph nodes and in the spleens of STAT6/IL-10 double deficient (DKO) mice. Collectively, it is suggested that Sm exerts anti-diabetic effects on this experimental T1D model via Treg/IL-4/IL-13/IL-10-independent mechanisms. Augmented expressions of Arg-1 and Ym1 in the lymphoid organs adjacent to pancreas may be relevant to the anti-diabetic effects of Sm.

Abstract PR07: Use of a novel mouse model to investigate immune related adverse events arising from immunotherapies

Abstract While immunotherapy is rapidly growing as an excellent treatment for many cancer types, most immunotherapies, particularly in combination can induce immune related adverse events (irAEs) which are generally caused by immune cell activation and expansion. Although most irAEs are low grade (1-2), higher grade events (3-4) can prevent patients from completing therapy and can also be life threatening. Thus, there is a strong need to develop pre-clinical mouse models to identify which immunotherapeutic combinations induce the best anti-tumor responses without inducing severe irAEs. To replicate irAEs associated with immunotherapies in humans, we utilized the FoxP3-DTR mouse model where regulatory T-cell (Treg) depletion can be achieved by injecting diphtheria toxin (DT), releasing Treg associated immune suppression. Transient Treg depletion by a single dose of DT induced mild T-cell activation and increased T-cell immune checkpoint expression, making mice more sensitive to irAEs development from clinically relevant immunotherapies. In three tumor models, we demonstrated that transient Treg depletion with anti-PD-1 or TIM-3 had a good anti-tumor efficacy and mild irAEs. Conversely, combining transient Treg depletion with an antibody targeting the co-stimulatory receptor CD137 had a lower therapeutic window due to increased irAEs. This combination was effective in suppressing established tumors, but induced severe irAEs including elevated serum cytokines and acute liver inflammation. These irAEs were similar to those seen in clinical trials of anti-CD137 antibodies where liver toxicity was a major concern and were driven by a rapid systemic CD8+ T-cell expansion. We also demonstrated that irAEs from anti-CD137 therapy could be mitigated by neutralising TNF without impacting on anti-tumor efficacy. We believe this model can provide valuable data on the efficacy and irAEs associated with novel combination immunotherapies which may aid clinicians and pharmaceutical companies in clinical trial design. We are currently utilizing this model to predict the therapeutic irAEs and efficacy of other novel immunotherapies that are in pre-clinical and early stage clinical trials. This abstract is also being presented as Poster B58. Citation Format: Stephen J. Blake, Jing Liu, Harjunpaa Heidi, Mark J. Smyth, Michele W.L. Teng. Use of a novel mouse model to investigate immune related adverse events arising from immunotherapies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR07.

Dual Roles for Regulatory T-cell Depletion and Costimulatory Signaling in Agonistic GITR Targeting for Tumor Immunotherapy.

Agonistic monoclonal antibodies (mAb) targeting the T-cell receptor coregulatory molecule GITR exert potent therapeutic activities in preclinical tumor models. Although anti-GITR mAb are thought to act by depleting and destabilizing the intratumoral T regulatory cell (Treg) population, the precise mechanism of action is obscure. Here, we addressed this issue using a Treg fate-mapping approach, which revealed that Treg loss was primarily due to cell depletion, with minimal evidence of Treg conversion to a non-Foxp3-expressing population. Further characterization of persisting Tregs following anti-GITR mAb treatment showed that a highly activated subpopulation of CD44hiICOShi intratumoral Tregs were preferentially targeted for elimination, with the remaining Tregs exhibiting a less suppressive phenotype. With these changes in the Treg population, intratumoral CD8+ T cells acquired a more functional phenotype characterized by downregulation of the exhaustion markers PD-1 and LAG-3. This reversal of CD8+ T-cell exhaustion was dependent on both agonistic GITR signaling and Treg depletion, as neither mechanism by itself could fully rescue the exhaustion phenotype. Tests of anti-human GITR antibody MK-4166 in a humanized mouse model of cancer mimicked many of the effects of anti-mouse GITR mAb in syngeneic tumor models, decreasing both Treg numbers and immune suppressor phenotype while enhancing effector responsiveness. Overall, our results show how anti-GITR mAb shifts Treg populations to enable immune attack on tumors, with clinical implications for molecular markers to modify emerging treatments. Cancer Res; 77(5); 1108-18. ©2016 AACR.

Changes in multiparametric prostate MRI and immune subsets in patients (Pts) receiving neoadjuvant immunotherapy and androgen deprivation therapy (ADT) prior to radiation.

30 Background: Endorectal(er) MRI is an emerging tool in assessing intraprostatic tumors. Immunotherapy development in prostate cancer has been limited due to the lack of intermediate (bio)markers of response. Methods: Untreatedpts with high-risk prostate cancer were randomized in a trial (NCT01496131) of standard ADT+Radiation + an immunotherapy targeting MUC1 (tecemotide, aka L-BLP25). Pts had erMRI at baseline and after 2 months of ADT+/- biweekly immunotherapy. Low dose (300 mg/m2, maximum 600 mg) cyclophosphamide for regulatory T-cell depletion preceded first immunotherapy. Multiparametric MRI included evaluation of apparent diffusion coefficient (ADC) maps from diffusion-weighted MRI. Monthly peripheral blood assessments utilized flow cytometry to evaluate immune cell subsets. This analysis focuses on the 2 month neoadjuvant period of ADT+/-immunotherapy before radiation. Results: 28 pts (n = 14/arm) with high risk prostate cancer (Gleason 8-10, PSA > 20, or stage T3) were enrolled. PSA declined in all pts 2 months after ADT. erMRI findings at 2 months indicated greater improvements in ADC values in pts receiving immunotherapy+ADT vs. ADT alone. Improved ADC on MRI suggests improvements in intratumoral diffusion and has been associated with decreased tumor density. This relative improvement between the groups occurred both per patient (p = 0.16) and per lesion (p = 0.031). Relative to baseline, pts receiving immunotherapy+ADT had increases in CTLA4+ CD8+ T-cells consistent with immune activation (p = 0.0134) and decreases in myeloid derived suppressor cells (MDSCs; p = 0.0353) during the neoadjuvant period corresponding to the erMRI changes. These immune findings were not seen in the ADT alone group. Conclusions: Pts who received immunotherapy+ADT for 2 months had greater improvements in ADC values on erMRI, consistent with decreased tumor density, relative to pts receiving ADT alone. Corresponding increases in activated CD8+ T-cells and decreases in MDSCs were seen in pts receiving vaccine+ADT. These preliminary findings suggest that ADC on MRI may be useful in assessing immunologic impact. Further study is warranted. Clinical trial information: NCT01496131.

Bee Venom Phospholipase A2 Ameliorates House Dust Mite Extract Induced Atopic Dermatitis Like Skin Lesions in Mice.

Atopic dermatitis (AD) is a biphasic inflammatory skin disease that is provoked by epidermal barrier defects, immune dysregulation, and increased skin infections. Previously, we have demonstrated that bvPLA2 evoked immune tolerance by inducing regulatory T cells (Treg), and thus alleviated Th2 dominant allergic asthma in mice. Here, we would like to determine whether treatment with bvPLA2 exacerbates the AD-like allergic inflammations induced by house dust mite extract (DFE) in a murine model. Epidermal thickness, immune cell infiltration, serum immunoglobulin, and cytokines were measured. Ear swelling, skin lesions, and the levels of total serum IgE and Th1/Th2 cytokines were elevated in DFE/DNCB-induced AD mice. Topical application of bvPLA2 elicited significant suppression of the increased AD symptoms, including ear thickness, serum IgE concentration, inflammatory cytokines, and histological changes. Furthermore, bvPLA2 treatment inhibited mast cell infiltration into the ear. On the other hand, Treg cell depletion abolished the anti-atopic effects of bvPLA2, suggesting that the effects of bvPLA2 depend on the existence of Tregs. Taken together, the results revealed that topical exposure to bvPLA2 aggravated atopic skin inflammation, suggesting that bvPLA2 might be a candidate for the treatment of AD.

T Regulatory Cells Support Plasma Cell Populations in the Bone Marrow

Long-lived plasma cells (PCs) in the bone marrow (BM) are a critical source of antibodies after infection or vaccination, but questions remain about the factors that control PCs. We found that systemic infection alters the BM, greatly reducing PCs and regulatory T (Treg) cells, a population that contributes to immune privilege in the BM. The use of intravital imaging revealed that BM Treg cells display a distinct behavior characterized by sustained co-localization with PCs and CD11c-YFP+ cells. Gene expression profiling indicated that BM Treg cells express high levels of Treg effector molecules, and CTLA-4 deletion in these cells resulted in elevated PCs. Furthermore, preservation of Treg cells during systemic infection prevents PC loss, while Treg cell depletion in uninfected mice reduced PC populations. These studies suggest a role for Treg cells in PC biology and provide a potential target for the modulation of PCs during vaccine-induced humoral responses or autoimmunity.

Characterisation of Immune and Neuroinflammatory Changes Associated with Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) and associated neuropathic pain is a debilitating adverse effect of cancer treatment. Current understanding of the mechanisms underpinning CIPN is limited and there are no effective treatment strategies. In this study, we treated male C57BL/6J mice with 4 cycles of either Paclitaxel (PTX) or Oxaliplatin (OXA) over a week and tested pain hypersensitivity and changes in peripheral immune responses and neuroinflammation on days 7 and 13 post 1st injection. We found that both PTX and OXA caused significant mechanical allodynia. In the periphery, PTX and OXA significantly increased circulating CD4+ and CD8+ T-cell populations. OXA caused a significant increase in the percentage of interleukin-4+ lymphocytes in the spleen and significant down-regulation of regulatory T (T-reg) cells in the inguinal lymph nodes. However, conditional depletion of T-reg cells in OXA-treated transgenic DEREG mice had no additional effect on pain sensitivity. Furthermore, there was no leukocyte infiltration into the nervous system of OXA- or PTX-treated mice. In the peripheral nervous system, PTX induced expression of the neuronal injury marker activating transcription factor-3 in IB4+ and NF200+ sensory neurons as well as an increase in the chemokines CCL2 and CCL3 in the lumbar dorsal root ganglion. In the central nervous system, PTX induced significant astrocyte activation in the spinal cord dorsal horn, and both PTX and OXA caused reduction of P2ry12+ homeostatic microglia, with no measurable changes in IBA-1+ microglia/macrophages in the dorsal and ventral horns. We also found that PTX induced up-regulation of several inflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL11, CCL4, CCL3, IL-12p70 and GM-CSF) in the spinal cord. Overall, these findings suggest that PTX and OXA cause distinct pathological changes in the periphery and nervous system, which may contribute to chemotherapy-induced neuropathic pain.

New Targeted Treatments for Cutaneous T-cell Lymphomas.

Cutaneous T-cell lymphomas (CTCLs) represent a group of rare and heterogeneous diseases that are very difficult to treat at advanced stages. The development of monoclonal antibodies is a new hope for the treatment of these diseases. Alemtuzumab (Campath) is a humanized IgG1 kappa monoclonal antibody specific for CD52, an antigen expressed by most T and B lymphocytes. Alemtuzumab may frequently induce long-term remissions in patients with Sezary syndrome but high-dose treatments lead to severe cytopenia, immune depletion, and opportunistic infections. This treatment is less efficient in mycosis fungoides (MF). Brentuximab vedotin is a chimeric anti-CD30 monoclonal antibody conjugated to monomethyl auristatin E, a cytotoxic antitubulin agent. Brentuximab vedotin is a very interesting new treatment for advanced tumor MF, Sezary syndrome, and primary cutaneous CD30+ lymphoproliferative disorders. The main limiting adverse event is neurosensitive peripheral neuropathy. Mogamulizumab is a humanized anti-C-C chemokine receptor Type 4 monoclonal antibody with a defucosylated Fc region leading to increased antibody-dependent cellular cytotoxicity. Mogamulizumab is very efficient on aggressive peripheral T-cell lymphomas, particularly adult T-cell leukemia/lymphoma and CTCLs, especially on the blood component of tumor cells. The main limiting events are related to the concomitant depletion of regulatory T-cells. IPH4102 is a humanized monoclonal antibody that targets the immune receptor KIR3DL2/CD158k. Preclinical results with this antibody offer proofs of concept for the clinical development of IPH4102 to treat patients with advanced CTCL.

Regulatory T cells in cancer immunotherapy.

FOXP3-expressing regulatory T (Treg) cells, which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. Infiltration of a large number of Treg cells into tumor tissues is often associated with poor prognosis. There is accumulating evidence that the removal of Treg cells is able to evoke and enhance anti-tumor immune response. However, systemic depletion of Treg cells may concurrently elicit deleterious autoimmunity. One strategy for evoking effective tumor immunity without autoimmunity is to specifically target terminally differentiated effector Treg cells rather than all FOXP3+ T cells, because effector Treg cells are the predominant cell type in tumor tissues. Various cell surface molecules, including chemokine receptors such as CCR4, that are specifically expressed by effector Treg cells can be the candidates for depleting effector Treg cells by specific cell-depleting monoclonal antibodies. In addition, other immunological characteristics of effector Treg cells, such as their high expression of CTLA-4, active proliferation, and apoptosis-prone tendency, can be exploited to control specifically their functions. For example, anti-CTLA-4 antibody may kill effector Treg cells or attenuate their suppressive activity. It is hoped that combination of Treg-cell targeting (e.g., by reducing Treg cells or attenuating their suppressive activity in tumor tissues) with the activation of tumor-specific effector T cells (e.g., by cancer vaccine or immune checkpoint blockade) will make the current cancer immunotherapy more effective.