Non-oncogenic Acute Viral Infections Disrupt Anti-cancer Responses and Lead to Accelerated Cancer-Specific Host Death.

In light of increased cancer prevalence and cancer-specific deaths in patients with infections, we investigated whether infections alter anti-tumor immune responses. We report that acute influenza infection of the lung promotes distal melanoma growth in the dermis and leads to accelerated cancer-specific host death. Furthermore, we show that during influenza infection, anti-melanoma CD8+ T cells are shunted from the tumor to the infection site, where they express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1). Immunotherapy to block PD-1 reverses this loss of anti-tumor CD8+ T cells from the tumor and decreases infection-induced tumor growth. Our findings show that acute non-oncogenic infection can promote cancer growth, raising concerns regarding acute viral illness sequelae. They also suggest an unexpected role for PD-1 blockade in cancer immunotherapy and provide insight into the immune response when faced with concomitant challenges.

HIV infection accelerates gastrointestinal tumor outgrowth in NSG-HuPBL mice.

HIV infection is a risk factor for the tumorigenesis including non-AIDS-defining cancers such as those of the gastrointestinal tract. However, the mechanisms underlying such cancer outgrowth are still unknown. Furthermore, combined HIV/cancer studies are difficult to evaluate using primate models or in the clinical patient setting. To understand the mechanisms of tumor outgrowth in the context of HIV infection, we adopted a humanized mouse model permissive to infection and cancer as well as an in vivo humanized mouse challenge with colon cancer in the context of HIV infection. Immunodeficient NOD SCID IL-2R(-/-) mice were immunologically reconstituted by adoptive transfer of 10(7) HIV-negative donor peripheral blood leukocytes and challenged with 10(6) HCT116 human colon cancer cells. A group of mice was treated with antiretroviral therapy. Tumor microenvironment and epithelial tissues in the context of HIV infection were analyzed using immunohistochemistry. We demonstrate that HIV-infected humanized mice develop significantly larger tumors than uninfected mice (p<0.05). Epithelial cell proliferation in HIV-infected mice is significantly enhanced in comparison to proliferation in uninfected mice (p<0.01). Moreover, the activation of ß-catenin, an important step in intestinal epithelial cell proliferation and tumorigenesis, is elevated in the tumors of HIV-infected mice (p<0.0001). Importantly, antiretroviral therapy reverses these pathological processes independently of CD4(+) T cell return. These findings model the ability of HIV infection to result in tumor outgrowth that is evident in HIV-positive patients and lend insight into previously unrecognized mechanisms that may underlie this pathology.

Antigenic peptide nanofibers elicit adjuvant-free CD8⁺ T cell responses.

Vaccines that elicit robust CD8⁺ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8⁺ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8⁺ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8⁺ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8⁺ T cell-mediated protection is needed.

Mutant HSP70 reverses autoimmune depigmentation in vitiligo.

Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell-mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

CD8(+) T cells sabotage their own memory potential through IFN-γ-dependent modification of the IL-12/IL-15 receptor α axis on dendritic cells.

CD8(+) T cell responses have been shown to be regulated by dendritic cells (DCs) and CD4(+) T cells, leading to the tenet that CD8(+) T cells play a passive role in their own differentiation. In contrast, by using a DNA vaccination model, to separate the events of vaccination from those of CD8(+) T cell priming, we demonstrate that CD8(+) T cells, themselves, actively limit their own memory potential through CD8(+) T cell-derived IFN-γ-dependent modification of the IL-12/IL-15Rα axis on DCs. Such CD8(+) T cell-driven cytokine alterations result in increased T-bet and decreased Bcl-2 expression, and thus decreased memory progenitor formation. These results identify an unrecognized role for CD8(+) T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation and memory formation.

NKG2D signaling on CD8⁺ T cells represses T-bet and rescues CD4-unhelped CD8⁺ T cell memory recall but not effector responses.

CD4-unhelped CD8(+) T cells are functionally defective T cells primed in the absence of CD4(+) T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8(+) T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8(+) T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8(+) T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4(+) T cells in a CD4-dependent influenza model and rescues HIV-specific CD8(+) T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8(+) T cells from their pathophysiological fate and may provide therapeutic benefits.

Engagement of NK receptor NKG2D, but not 2B4, results in self-reactive CD8+ T cells and autoimmune vitiligo.

In this study, we demonstrate that engagement of two different natural killer receptors (NKRs) can lead to contrasting effects in the development of self-reactive CD8+T cells and autoimmune vitiligo. Specifically, using a mouse model, we show that CD8+T-cell targeting of a melanocyte antigen, tyrosinase-related protein-1 (TRP-1) in combination with delivery of the NKG2D ligands (Rae-1ϵ or H60), results in strong CD8+T-cell responses against TRP-1 and in the development of autoimmune vitiligo. In contrast, targeting of TRP-1 in combination with delivery of CD48, the natural ligand for the NKR 2B4, leads to reduced formation of TRP-1-reactive CD8+T-cell responses and decreased development of vitiligo. These data indicate that autoimmune vitiligo is limited by insufficient signals, despite plentiful self-reactive T cells in the peripheral immune system. To our knowledge, this is the first experimental evidence supporting the role of NKRs in modulating CD8+T-cell autoimmune vitiligo. This study supports the utilization of NKR signaling as a therapeutic avenue toward prevention of vitiligo and other autoimmune diseases.

Priming with very low-affinity peptide ligands gives rise to CD8(+) T-cell effectors with enhanced function but with greater susceptibility to transforming growth factor (TGF)ß-mediated suppression.

While the effects of TCR affinity and TGFß on CD8(+) T-cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFß-mediated suppression remains unknown. To address this issue, we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8(+) T cells primed with lower-affinity TCR ligands are more effectively regulated by TGFß. Susceptibility to TGFß-mediated suppression is associated with downregulation of RGS3, a recently recognized negative regulator of TGFß signaling, but not expression of TGFß receptors I/II. These results suggest a novel tolerance mechanism whereby CD8(+) T cells are discriminately regulated by TGFß according to the affinity of the ligand on which they were initially primed. In addition, because of the major role played by TGFß in tumor-induced immune suppression, these results identify the affinity of the priming ligand as a primary concern in CD8(+) T-cell-mediated cancer immunotherapeutic strategies.

Development of tumor-infiltrating CD8+ T cell memory precursor effector cells and antimelanoma memory responses are the result of vaccination and TGF-ß blockade during the perioperative period of tumor resection.

A main goal of cancer immunology research is the formation of Ag-specific memory T cell immunity capable of activation upon tumor re-encounter. The requirements necessary to overcome the inhibitory signals present in the tumor microenvironment and form such memory T cell responses are unknown. In contrast to previous studies targeting tumors expressing highly immunogenic model Ags, we demonstrate that alleviating tumor-induced suppression along with vaccination against authentic Ags during the perioperative period provides long-lasting protection against a highly suppressive and poorly immunogenic melanoma. In this study, we employed DNA vaccination with an immunologically optimized mouse melanoma-shared Ag, Trp1ee/ng, combined with systemic TGF-ß blockade during the perioperative period of primary tumor resection, to confer protection against B16 melanoma, and against JBRH, an independently derived melanoma unrelated to B16. Importantly, we demonstrate that correlative to memory responses, perioperative immunotherapy increases the formation of tumor-infiltrating and tumor-reactive CD8(+) T cells expressing low levels of the transcription factor T-bet, defined as memory precursor effector cells. We show that conditions for an immunologically fertile environment are met when TGF-ß blockade and vaccination are applied during the perioperative period of primary tumor resection. These findings address limitations of current CD8(+) T cell immunotherapies against cancer by generating effective CD8(+) T cell memory recall responses.

CD8 co-receptor promotes susceptibility of CD8+ T cells to transforming growth factor-ß (TGF-ß)-mediated suppression.

CD8+ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-ß have been studied individually, the influence of CD8 co-receptor on TGF-ß function in CD8+ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-ß-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αßCD8 molecule, we demonstrate that cells expressing full-length αßCD8 were highly susceptible, αßCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-ß. Additionally, we determined that inhibition of Lck rendered mouse CD8+ T cells highly resistant to TGF-ß suppression. Resistance was not associated with TGF-ß receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8+ T cells for response to TGF-ß. Based on the important role which TGF-ß-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8+ T cell-related cancer immunotherapy strategies.