Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment

BACKGROUND: Adoptive immunotherapy with cytotoxic T lymphocytes (CTL) represents an alternative approach to treating solid tumors. Ideally, this would confer long-term protection against tumor. We previously demonstrated that in vitro-generated tumor-specific CTL from the ovalbumin (OVA)-specific OT-I T cell receptor transgenic mouse persisted long after adoptive transfer as memory T cells. When recipient mice were challenged with the OVA-expressing E.G7 thymoma, tumor growth was delayed and sometimes prevented. The reasons for therapeutic failures were not clear. METHODS: OT-I CTL were adoptively transferred to C57BL/6 mice 21 – 28 days prior to tumor challenge. At this time, the donor cells had the phenotypical and functional characteristics of memory CD8+ T cells. Recipients which developed tumor despite adoptive immunotherapy were analyzed to evaluate the reason(s) for therapeutic failure. RESULTS: Dose-response studies demonstrated that the degree of tumor protection was directly proportional to the number of OT-I CTL adoptively transferred. At a low dose of OT-I CTL, therapeutic failure was attributed to insufficient numbers of OT-I T cells that persisted in vivo, rather than mechanisms that actively suppressed or anergized the OT-I T cells. In recipients of high numbers of OT-I CTL, the E.G7 tumor that developed was shown to be resistant to fresh OT-I CTL when examined ex vivo. Furthermore, these same tumor cells no longer secreted a detectable level of OVA. In this case, resistance to immunotherapy was secondary to selection of clones of E.G7 that expressed a lower level of tumor antigen. CONCLUSIONS: Memory engraftment with tumor-specific CTL provides long-term protection against tumor. However, there are several limitations to this immunotherapeutic strategy, especially when targeting a single antigen. This study illustrates the importance of administering large numbers of effectors to engraft sufficiently efficacious immunologic memory. It also demonstrates the importance of targeting several antigens when developing vaccine strategies for cancer

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8+ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8+ T cells require IL-2 provided by activated CD4+ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4+ cells is mediated through the IL-2Rβγ of CD8+ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8+ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8+ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8+ T-cell response is elicited by a critical temporal function of IL-2 released from CD4+ T cells, providing mechanistic insights into the regulation of CD8+ T cell activation and differentiation

CD40-Activated B Cells Can Efficiently Prime Antigen-Specific Naïve CD8+ T Cells to Generate Effector but Not Memory T cells

Background: The identification of the signals that should be provided by antigen-presenting cells (APCs) to induce a CD8 + T cell response in vivo is essential to improve vaccination strategies using antigen-loaded APCs. Although dendritic cells have been extensively studied, the ability of other APC types, such as B cells, to induce a CD8 + T cell response have not been thoroughly evaluated. Methodology/Principal Findings: In this manuscript, we have characterized the ability of CD40-activated B cells, stimulated or not with Toll-like receptor (TLR) agonists (CpG or lipopolysaccharide) to induce the response of mouse naïve CD8 + T cells in vivo. Our results show that CD40-activated B cells can directly present antigen to naïve CD8 + T cells to induce the generation of potent effectors able to secrete cytokines, kill target cells and control a Listeria monocytogenes infection. However, CD40-activated B cell immunization did not lead to the proper formation of CD8 + memory T cells and further maturation of CD40-activated B cells with TLR agonists did not promote the development of CD8 + memory T cells. Our results also suggest that inefficient generation of CD8 + memory T cells with CD40-activated B cell immunization is a consequence of reduced Bcl-6 expression by effectors and enhanced contraction of the CD8 + T cell response. Conclusions: Understanding why CD40-activated B cell immunization is defective for the generation of memory T cells and gaining new insights about signals that should be provided by APCs are key steps before translating the use of CD40-B cel

Small Interfering RNA Targeted to IGF-IR Delays Tumor Growth and Induces Proinflammatory Cytokines in a Mouse Breast Cancer Model

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2′-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2′-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines

The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain.

We investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1 degrees, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPDeltaknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPDeltaknob and for fiber protein deletion mutants lacking the beta-stranded region F of the knob (residues Leu-485-Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiDeltaHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in viv

The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain.

We investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1 degrees, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPDeltaknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPDeltaknob and for fiber protein deletion mutants lacking the beta-stranded region F of the knob (residues Leu-485-Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiDeltaHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in viv

Sustained Type I interferon signaling as a mechanism of resistance to PD-1 blockade

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy. Delineating mechanisms of secondary resistance to this strategy is increasingly important. Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb). Sustained IFNβ transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC). Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation. Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature. Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy

Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion.

T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFβ and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFβ/IL-6-mediated induction of Maf