Low-dose interleukin-2 impairs host anti-tumor immunity and inhibits therapeutic responses in a mouse model of melanoma.

Recombinant interleukin-2 (rIL-2) is associated with objective responses in 15-20 % of patients with metastatic melanoma and renal cell carcinoma. More recently, rIL-2 has also demonstrated improved clinical activity in patients with melanoma. Given the toxicity of high-dose rIL-2 and the availability of many new immunotherapy agents, it has been suggested that lower doses of rIL-2 may be preferred for combination clinical studies. In order to determine the impact of low doses of rIL-2 on anti-tumor immunity and therapeutic effectiveness, we challenged C57BL/6 mice with poorly immunogenic B16-F10 melanoma and treated them with varying doses of rIL-2 (range 103-105 IU). Tumor growth at day 14 was significantly reduced when rIL-2 was administered at 10,000 (P < 0.02) and 100,000 (P < 0.02) IU doses, but tumor growth was significantly increased when mice were treated at 1000 IU rIL-2 (P < 0.02), as compared to placebo treatment. While the proportions of CD8+ and CD4+ T cells in the tumor were similar at all doses tested, the proportion of NK cells was decreased and the proportion of Tregs was increased in tumors exposed to low-dose rIL-2. The ratio of gp100-specific CD8+ to CD4+ regulatory T cells was increased in tumors treated at 10,000 and 100,000 IU of rIL-2 but was decreased at the 1000 IU dose compared to placebo-treated mice. These findings suggest that low-dose rIL-2 may impair host anti-tumor immunity and promote tumor growth. Early-phase adjuvant and combination clinical studies should include patient cohorts with higher doses of rIL-2.

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.

NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade.

BACKGROUND: Melanoma is one of the few types of cancer with an increasing annual incidence. While a number of immunotherapies for melanoma have been associated with significant clinical benefit, including high-dose IL-2 and cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade, clinical response to either of these single agents has been limited to 11-20% of treated patients. Therefore, in this study, we sought to test the hypothesis that the combination of IL-2 and CTLA-4 blockade could mediate a more profound therapeutic response. METHODS: Here, B6 mice were challenged with poorly immunogenic B16 melanoma on day 0, and treated with CTLA-4 blocking antibody (100 µg/mouse) on days 3, 6, and 9, and IL-2 (100,000 units) twice daily on days 4-8, or both. RESULTS: A highly significant synergistic effect that delayed tumor growth and prolonged survival was demonstrated with the combination immunotherapy compared to either monotherapy alone. The therapeutic effect of combination immunotherapy was dependent on both CD8+ T and NK cells and co-depletion of these subsets (but not either one alone) abrogated the therapeutic effect. CTLA-4 blockade increased immune cell infiltration (including CD8+ T cells and NK cells) in the tumor and IL-2 reduced the proportion of highly differentiated/exhausted tumor-infiltrating NK cells. CONCLUSIONS: These results have implications for the design of clinical trials in patients with metastatic melanoma and provide new insights into how the immune system may be mediating anti-tumor activity with combination IL-2 and CTLA-4 blockade in melanoma.

Pro-inflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response.

Prior studies suggest that the impaired healing seen in diabetic wounds derives from a state of persistent hyper-inflammation characterized by harmful increases in inflammatory leukocytes including macrophages. However, such studies have focused on wounds at later time points (day 10 or older), and very little attention has been given to the dynamics of macrophage responses in diabetic wounds early after injury. Given the importance of macrophages for the process of healing, we studied the dynamics of macrophage response during early and late phases of healing in diabetic wounds. Here, we report that early after injury, the diabetic wound exhibits a significant delay in macrophage infiltration. The delay in the macrophage response in diabetic wounds results from reduced Chemokine (C-C motif) ligand 2 (CCL2) expression. Importantly, one-time treatment with chemoattractant CCL2 significantly stimulated healing in diabetic wounds by restoring the macrophage response. Our data demonstrate that, rather than a hyper-inflammatory state; the early diabetic wound exhibits a paradoxical and damaging decrease in essential macrophage response. Our studies suggest that the restoration of the proper kinetics of macrophage response may be able to jumpstart subsequent healing stages. CCL2 chemokine-based therapy may be an attractive strategy to promote healing in diabetic wounds.

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.

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.

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.