InVivoPlus polyclonal Armenian hamster IgG

in vivo IFNγ neutralization

IFN-gamma dictates allograft fate via opposing effects on the graft and on recipient CD8 T cell responses PubMed

CD8 T cells are necessary for costimulation blockade-resistant rejection. However, the mechanism by which CD8 T cells mediate rejection in the absence of major costimulatory signals is poorly understood. IFN-gamma promotes CD8 T cell-mediated immune responses, but IFN-gamma-deficient mice show early graft loss despite costimulation blockade. In contrast, we found that IFN-gamma receptor knockout mice show dramatically prolonged graft survival under costimulation blockade. To investigate this paradox, we addressed the effects of IFN-gamma on T cell alloresponses in vivo independent of the effects of IFN-gamma on graft survival. We identified a donor-specific CD8 T cell breakthrough response temporally correlated with costimulation blockade-resistant rejection. Neither IFN-gamma receptor knockout recipients nor IFN-gamma-deficient recipients showed a CD8 breakthrough response. Graft death on IFN-gamma-deficient recipients despite costimulation blockade could be explained by the lack of IFN-gamma available to act on the graft. Indeed, the presence of IFN-gamma was necessary for graft survival on IFN-gamma receptor knockout recipients, as either IFN-gamma neutralization or the lack of the IFN-gamma receptor on the graft precipitated early graft loss. Thus, IFN-gamma is required both for the recipient to mount a donor-specific CD8 T cell response under costimulation blockade as well as for the graft to survive after allotransplantation.

in vivo CTLA-4 neutralization

Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy PubMed

Thermal ablation to destroy tumor tissue may help activate tumor-specific T cells by elevating the presentation of tumor antigens to the immune system. However, the antitumor activity of these T cells may be restrained by their expression of the inhibitory T-cell coreceptor CTLA-4, the target of the recently U.S. Food and Drug Administration-approved antibody drug ipilumimab. By relieving this restraint, CTLA-4-blocking antibodies such as ipilumimab can promote tumor rejection, but the full scope of their most suitable applications has yet to be fully determined. In this study, we offer a preclinical proof-of-concept in the TRAMP C2 mouse model of prostate cancer that CTLA-4 blockade cooperates with cryoablation of a primary tumor to prevent the outgrowth of secondary tumors seeded by challenge at a distant site. Although growth of secondary tumors was unaffected by cryoablation alone, the combination treatment was sufficient to slow growth or trigger rejection. In addition, secondary tumors were highly infiltrated by CD4(+) T cells and CD8(+) T cells, and there was a significant increase in the ratio of intratumoral T effector cells to CD4(+)FoxP3(+) T regulatory cells, compared with monotherapy. These findings documented for the first time an effect of this immunotherapeutic intervention on the intratumoral accumulation and systemic expansion of CD8(+) T cells specific for the TRAMP C2-specific antigen SPAS-1. Although cryoablation is currently used to treat a targeted tumor nodule, our results suggest that combination therapy with CTLA-4 blockade will augment antitumor immunity and rejection of tumor metastases in this setting.

Long-lived bone marrow plasma cells are induced early in response to T cell-independent or T cell-dependent antigens PubMed

The signals required to generate long-lived plasma cells remain unresolved. One widely cited model posits that long-lived plasma cells derive from germinal centers (GCs) in response to T cell-dependent (TD) Ags. Thus, T cell-independent (TI) Ags, which fail to sustain GCs, are considered ineffective at generating long-lived plasma cells. However, we show that long-lived hapten-specific plasma cells are readily induced without formation of GCs. Long-lived plasma cells developed in T cell-deficient mice after a single immunization with haptenated LPS, a widely used TI Ag. Long-lived plasma cells also formed in response to TD Ag when the GC response was experimentally prevented. These observations establish that long-lived plasma cells are induced in both TI and TD responses, and can arise independently of B cell maturation in GCs.

Combination immunotherapy with 4-1BBL and CTLA-4 blockade for the treatment of prostate cancer PubMed

Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via activation of 4-1BB or cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) blockade appears to be a promising strategy for cancer immunotherapy. In this study, we examined the effectiveness of 4-1BBL-expressing tumor cell vaccine in combination with CTLA-4 blockade on rejection of murine prostate cancer RM-1. We found that the combination of both a vaccine consisting of 4-1BBL-expressing RM-1 cells and CTLA-4 blockade resulted in regression of RM-1 tumors and a significant increase in survival of the tumour cell recipients, compared to that of either treatment alone. The combined vaccination resulted in higher CTL against RM-1 cells and increased secretion of IFN-gamma, TNF-alpha, and IL-2 in the mix-cultured supernatant. These results suggest that combining activation of 4-1BB and blockade of CTLA-4 may offer a new strategy for prostate cancer immunotherapy.

in vivo CD4+ T cell depletion

Tumor-specific CD4+ T cells maintain effector and memory tumor-specific CD8+ T cells PubMed

Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor-specific CD4(+) T cells enhance CD8(+) T-cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase-related protein 1-specific CD4(+) transgenic T cells-CD4(+) T cells and pmel-CD8(+) T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p = 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8(+) T cells with tumor-specific cytokine expression. When combined with CD4(+) T cells, transfer of total (naive and effector) or effector CD8(+) T cells were highly effective, suggesting CD4(+) T cells can help mediate therapeutic effects by maintaining function of activated CD8(+) T cells. In addition, CD4(+) T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8(+) T cells recovered from mice treated with both CD8(+) and CD4(+) T cells had decreased expression of PD-1 and PD-1-blockade enhanced the therapeutic efficacy of pmel-CD8 alone, suggesting that CD4(+) T cells help reduce CD8(+) T-cell exhaustion. These data support combining immunotherapies that elicit both tumor-specific CD4(+) and CD8(+) T cells for treatment of patients with cancer.

in vivo IL-1β neutralization, in vivo TNFα neutralization

Role of disease-associated tolerance in infectious superspreaders PubMed

Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

Flow Cytometry, in vivo depletion of Gr-1+ myeloid cells, in vivo IL-17A neutralization, in vivo IL-1β neutralization, in vivo IL-6 neutralization, in vivo TNFα neutralization

Mannan induces ROS-regulated, IL-17A-dependent psoriasis arthritis-like disease in mice PubMed

Psoriasis (Ps) and psoriasis arthritis (PsA) are poorly understood common diseases, induced by unknown environmental factors, affecting skin and articular joints. A single i.p. exposure to mannan from Saccharomyces cerevisiae induced an acute inflammation in inbred mouse strains resembling human Ps and PsA-like disease, whereas multiple injections induced a relapsing disease. Exacerbation of disease severity was observed in mice deficient for generation of reactive oxygen species (ROS). Interestingly, restoration of ROS production, specifically in macrophages, ameliorated both skin and joint disease. Neutralization of IL-17A, mainly produced by gammadelta T cells, completely blocked disease symptoms. Furthermore, mice depleted of granulocytes were resistant to disease development. In contrast, certain acute inflammatory mediators (C5, Fcgamma receptor III, mast cells, and histamine) and adaptive immune players (alphabeta T and B cells) were redundant in disease induction. Hence, we propose that mannan-induced activation of macrophages leads to TNF-alpha secretion and stimulation of local gammadelta T cells secreting IL-17A. The combined action of activated macrophages and IL-17A produced in situ drives neutrophil infiltration in the epidermis and dermis of the skin, leading to disease manifestations. Thus, our finding suggests a new mechanism triggered by exposure to exogenous microbial components, such as mannan, that can induce and exacerbate Ps and PsA.

in vivo blocking of PD-1/PD-L signaling, in vivo CD8+ T cell depletion

Intratumoral administration of mRNA encoding a fusokine consisting of IFN-beta and the ectodomain of the TGF-beta receptor II potentiates antitumor immunity PubMed

It is generally accepted that the success of immunotherapy depends on the presence of tumor-specific CD8(+) cytotoxic T cells and the modulation of the tumor environment. In this study, we validated mRNA encoding soluble factors as a tool to modulate the tumor microenvironment to potentiate infiltration of tumor-specific T cells. Intratumoral delivery of mRNA encoding a fusion protein consisting of interferon-beta and the ectodomain of the transforming growth factor-beta receptor II, referred to as Fbeta(2), showed therapeutic potential. The treatment efficacy was dependent on CD8(+) T cells and could be improved through blockade of PD-1/PD-L1 interactions. In vitro studies revealed that administration of Fbeta(2) to tumor cells resulted in a reduced proliferation and increased expression of MHC I but also PD-L1. Importantly, Fbeta(2) enhanced the antigen presenting capacity of dendritic cells, whilst reducing the suppressive activity of myeloid-derived suppressor cells. In conclusion, these data suggest that intratumoral delivery of mRNA encoding soluble proteins, such as Fbeta(2), can modulate the tumor microenvironment, leading to effective antitumor T cell responses, which can be further potentiated through combination therapy.

in vivo CTLA-4 neutralization

Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival PubMed

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete alphaSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.

Flow Cytometry, in vivo blocking of CD40/CD40L signaling, In vivo CD70 blockade

Epitope-specific regulation of memory programming by differential duration of antigen presentation to influenza-specific CD8(+) T cells PubMed

Memory CD8(+) T cells are programmed during the primary response for robust secondary responsiveness. Here we show that CD8(+) T cells responding to different epitopes of influenza virus received qualitatively different signals during the primary response that altered their secondary responsiveness. Nucleoprotein (NP)-specific CD8(+) T cells encountered antigen on CD40-licensed, CD70-expressing, CD103(-)CD11b(hi) dendritic cells (DCs) at later times in the primary response. As a consequence, they maintained CD25 expression and responded to interleukin-2 (IL-2) and CD27, which together programmed their robust secondary proliferative capacity and interferon-gamma (IFN-gamma)-producing ability. In contrast, polymerase (PA)-specific CD8(+) T cells did not encounter antigen-bearing, CD40-activated DCs at later times in the primary response, did not receive CD27 and CD25 signals, and were not programmed to become memory CD8(+) T cells with strong proliferative and cytokine-producing ability. As a result, CD8(+) T cells responding to abundant antigens, like NP, dominated the secondary response.

in vitro T cell stimulation/activation, in vivo blocking of CD40/CD40L signaling

PU.1 Expression in T Follicular Helper Cells Limits CD40L-Dependent Germinal Center B Cell Development PubMed

PU.1 is an ETS family transcription factor that is important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development and in limiting Th2 cytokine production. Whether PU.1 has functions in other Th lineages is not clear. In this study, we examined the effects of ectopic expression of PU.1 in CD4+ T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1lck-/-) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1lck-/- mice had increased numbers of Tfh cells, increased germinal center B cells (GCB cells), and increased Ab production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1lck-/- mice compared with control mice. Finally, although blockade of IL-21 did not affect GCB cells in Sfpi1lck-/- mice, anti-CD40L treatment of immunized Sfpi1lck-/- mice decreased GCB cell numbers and Ag-specific Ig concentrations. Together, these data indicate an inhibitory role for PU.1 in the function of Tfh cells, germinal centers, and Tfh-dependent humoral immunity.

in vivo blocking of PD-1/PD-L signaling

Cutting Edge: PD-1 Regulates Imiquimod-Induced Psoriasiform Dermatitis through Inhibition of IL-17A Expression by Innate gammadelta-Low T Cells PubMed

Programmed cell death 1 (PD-1) is a key regulatory molecule that has been targeted in human cancers, including melanoma. In clinical testing, Abs against PD-1 have resulted in psoriasiform dermatitis (PsD). To determine whether PD-1 regulates PsD, we compared skin responses of PD-1-deficient (PD-1KO) mice and wild-type (WT) controls in an imiquimod (IMQ)-induced murine model of psoriasis. PD-1KO mice showed severe epidermal hyperplasia, greater neutrophilic infiltration, and higher expression of Th17 cytokines (versus WT mice). IMQ exposure increased PD-1 expression by skin gammadelta-low (GDL) T cells and enhanced expression of PD-L1 by keratinocytes. Three-fold increases in the percentage of IL-17A(+) GDL T cells were observed in skin cell suspensions derived from IMQ-treated PD-1KO mice (versus WT controls), suggesting that the lack of PD-1 has a functional effect not only on alphabeta T cells, but also on GDL T cells, and that PD-1 may play a regulatory role in PsD.

in vivo blocking of PD-1/PD-L signaling

ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy PubMed

PD-1 negatively regulates CD8(+) cytotoxic T lymphocytes (CTL) cytotoxicity and anti-tumor immunity. However, it is not fully understood how PD-1 expression on CD8(+) CTL is regulated during anti-tumor immunotherapy. In this study, we have identified that the ADAP-SKAP55 signaling module reduced CD8(+) CTL cytotoxicity and enhanced PD-1 expression in a Fyn-, Ca(2+)-, and NFATc1-dependent manner. In DC vaccine-based tumor prevention and therapeutic models, knockout of SKAP55 or ADAP showed a heightened protection from tumor formation or metastases in mice and reduced PD-1 expression in CD8(+) effector cells. Interestingly, CTLA-4 levels and the percentages of tumor infiltrating CD4(+)Foxp3(+) Tregs remained unchanged. Furthermore, adoptive transfer of SKAP55-deficient or ADAP-deficient CD8(+) CTLs significantly blocked tumor growth and increased anti-tumor immunity. Pretreatment of wild-type CD8(+) CTLs with the NFATc1 inhibitor CsA could also downregulate PD-1 expression and enhance anti-tumor therapeutic efficacy. Together, we propose that targeting the unrecognized ADAP-SKAP55-NFATc1-PD-1 pathway might increase efficacy of anti-tumor immunotherapy.

in vivo CTLA-4 neutralization

Robust Antitumor Responses Result from Local Chemotherapy and CTLA-4 Blockade PubMed

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8(+) and CD4(+) T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy.