SUMMARY Oral tolerance prevents oral sensitization to dietary antigens (Ags), including proteins and haptens, and development of delayed-type hypersensitivity (DTH) responses. We showed here that plasmacytoid dendritic cells (pDCs) prevented oral T cell priming and were responsible for systemic tolerance to CD4+ and CD8+ T cell-mediated DTH responses induced by Ag feeding. Systemic depletion of pDCs prevented induction of tolerance by antigen feeding. Transfer of oral Ag-loaded liver pDCs to naive recipient mice induced Ag-specific suppression of CD4+ and CD8+ T cell responses to protein and hapten, respectively. Liver is a site of oral Ag presentation, and pDCs appeared to induce anergy or deletion of Ag-specific T cells in the liver relatively rapidly via a CD4+ T cell-independent mechanism. These data demonstrate that oral tolerance relies on Ag presentation by pDC to T cells and suggest that pDC could represent a key therapeutic target for intestinal and systemic inflammatory diseases.

The nature of dendritic cell(s) (DC[s]) that conditions efficient in vivo priming of CD8+ CTL after immunization via epithelial tissues remains largely unknown. Here, we show that myeloid DCs rapidly recruited by adjuvants into the buccal mucosa or skin are essential for CD8+ T cell crosspriming. Recruitment of circulating DC precursors, including Gr1+ monocytes, precedes the sequential accumulation of CD11c+ MHC class II+ DCs in dermis and epithelium via a CCR6/CCL20-dependent mechanism. Remarkably, a defect in CCR6, local neutralization of CCL20, or depletion of monocytes prevents in vivo priming of CD8+ CTL against an innocuous protein antigen administered with adjuvant. In addition, transfer of CCR6-sufficient Gr1+ monocytes restores CD8+ T cell priming in CCR6( degrees / degrees ) mice via a direct Ag presentation mechanism. Thus, newly recruited DCs likely derived from circulating monocytes are responsible for efficient crosspriming of CD8+ CTL after mucosal or skin immunization.

IntroductionOral tolerance has long been recognized as a physiologic mechanism of immune unresponsiveness to dietary antigens and bacterial microflora antigens, which maintain tissue integrity by preventing harmful delayed-type hypersensitivity (DTH) responses in the intestine and may also limit the efficiency of oral vaccination. Indeed, antigen encounter in the intestine triggers an active inhibitory process preventing the onset of CD4 ϩ and CD8 ϩ T-cell antigen-specific immune responses to subsequent systemic immunization with the same antigen (reviewed in Garside and Mowat 1 ). Several mechanisms have been proposed to explain peripheral tolerance induced by antigen feeding. These include anergy 2,3 or deletion of antigen-specific T cells, 4,5 immune deviation to Th2-biased immune response, and induction of regulatory Th3 (transforming growth factor ␤ [TGF␤]-producing) cells. 6,7 The naturally occurring regulatory subset of CD4 ϩ CD25 ϩ T cells accounting for 5% to 10% of peripheral CD4 ϩ T cells has been extensively reported to exert potent immunosuppressive function in vivo and in vitro toward CD4 ϩ T-cell effectors 8 and may represent regulatory T cells responsible for orally induced peripheral tolerance. Indeed, CD4 ϩ CD25 ϩ T cells, which arise from the thymus as early as day 3 of life, 9 are characterized by a memory phenotype; low proliferative capacity and interleukin-2 (IL-2) production; secretion of high levels of the immunosuppressive cytokines IL-10 and TGF-␤; and expression of cytotoxic Tlymphocyte antigen 4 (CTLA-4), 10-13 a molecule that contributes to orally induced tolerance. 14 These cells have been described in a variety of experimental models to protect from autoimmune diseases, as well as colitis and allograft rejection. 8 Reminiscent to these cells, IL-10-producing ovalbumin (OVA)-specific CD4 ϩ T clones (Tr1) generated in vitro after repeated stimulation with antigen in the presence of IL-10 were shown to prevent colitis when cotransferred with naive CD4 ϩ CD45RB high T cells in OVAfed immunocompromised severe combine immunodeficiency (SCID) or Nude mice. 15 Interestingly, mice genetically deficient for either IL-2, IL-2R, T-cell receptor ␣␤ (TcR␣␤), TGF␤, IL-10, or major histocompatibility complex (MHC) class II were shown to develop spontaneous colitis, [16][17][18][19][20] compatible with a shared physical or functional defect in the regulatory CD4 ϩ CD25 ϩ subset. Although recent studies in TcR transgenic models have reported that oral antigen delivery can induce activation and/or differentiation of regulatory CD4 ϩ CD25 ϩ T cells, 21,22 evidence that they are instrumental for in vivo induction of oral tolerance has not been provided. Moreover, evidence as to whether CD4 ϩ CD25 ϩ cells are responsible for peripheral suppression of antigen-specific CD8 ϩ cytotoxic T-lymphocyte (CTL) responses is still sparse. 23 In this study we examined whether CD4 ϩ CD25 ϩ T cells contribute to oral tolerance in normal nonlymphopenic hosts, using a pathophysiologic model of antigen-specific skin in...

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Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

The Wilms' tumor 1 (WT1) antigen is expressed in solid and hematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA-A2/WT1126-134 (RMFPNAPYL) (HLA-A2/RMFPNAPYL epitope (WT1A)) and its modified variant YMFPNAPYL (HLA-A2/YMFPNAPYL epitope (WT1B)), can induce WT1-specific CD8+ T cells, although WT1B is more stably bound to HLA-A*02:01. Here, to further determine the benefits of those two targets, we assessed the naive precursor frequencies; immunogenicity and cross-reactivity of CD8+ T cells directed toward these two WT1 epitopes. Ex vivo naive WT1A- and WT1B-specific CD8+ T cells were detected in healthy HLA-A*02:01+ individuals with comparable precursor frequencies (1 in 105–106) to other naive CD8+ T-cell pools (for example, A2/HIV-Gag77-85), but as expected, ~100 × lower than those found in memory populations (influenza, A2/M158-66; EBV, A2/BMLF1280-288). Importantly, only WT1A-specific naive precursors were detected in HLA-A2.1 mice. To further assess the immunogenicity and recruitment of CD8+ T cells responding to WT1A and WT1B, we immunized HLA-A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8+ T-cell responses to the native tumor epitope following re-stimulation, although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly, however, WT1B immunization generated cross-reactive CD8+ T-cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single- and cross-reactive WT1A+CD8+ T cells with unique T-cell receptor-αβ gene signatures. Therefore, although both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.

Purpose: Women infected with human papillomavirus (HPV) with normal cytology to mild abnormalities currently have no treatment options other than watchful waiting or surgery if highgrade cervical lesions or cancer develop. A therapeutic vaccine would offer the possibility of preventing high-grade lesions in HPV-infected women. GTL001 is a therapeutic vaccine composed of recombinant HPV16 and HPV18 E7 proteins fused to catalytically inactive Bordetella pertussis CyaA. This study examined the tolerability and immunogenicity of GTL001 in women infected with HPV16 or HPV18 with normal cytology.Experimental Design: This was a phase I trial (EudraCT No. 2010-018629-21). In an open-label part, subjects received two intradermal vaccinations 6 weeks apart of 100 or 600 mg GTL001 þ topical 5% imiquimod cream at the injection site. In a doubleblind part, subjects were randomized 2:1:1 to two vaccinations 6 weeks apart of 600 mg GTL001 þ imiquimod, 600 mg GTL001 þ placebo cream, or placebo þ imiquimod.Results: Forty-seven women were included. No dropouts, treatment-related serious adverse events, or dose-limiting toxicities occurred. Local reactions were transient and mostly mild or moderate. HPV16/18 viral load decreased the most in the 600 mg GTL001 þ imiquimod group. In post hoc analyses, the 600 mg GTL001 þ imiquimod group had the highest rates of initial and sustained HPV16/18 clearance. Imiquimod increased antigenspecific T-cell response rates but not rates of solicited reactions. All subjects seroconverted to CyaA.Conclusions: For women infected with HPV16 or HPV18 with normal cervical cytology, GTL001 was immunogenic and had acceptable safety profile. Clin Cancer Res; 22(13); 3238-48.Ó2016 AACR.

Despite the progress that has been made in other forms of cancer therapy, Provenge® (Sipuleucel-T) is the only FDA-approved vaccine for the treatment of cancer. To understand the current landscape of therapeutic oncology vaccines we performed a quantitative analysis of phase 2 and phase 3 therapeutic cancer vaccine trials. We highlight shifts in trends for the vaccine platforms examined, common adjuvant use, target indications, antibody or treatment combinations between past and recent trials as well as discuss the relationship between these trends and ratio between the number of phase 3: phase 2 for different vaccine platforms. Despite the poor success rate in vaccine approvals, registration of phase 3 trials between 2010 and 2014 were stable indicating continued investment and efforts towards development of immunotherapeutic vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0093-x) contains supplementary material, which is available to authorized users.