Erica J. Huelsmann

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.

Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

Vitiligo can be reversed through immune targeting with mutant heat shock protein 70. New Treatment Makes Vitiligo Beat It Whether your grant application is due, you have a paper that needs to be submitted, or your patient load is too high, medical science is not a relaxing profession. High stress is known to negatively affect your health at both the whole body and cellular level. One way the body responds to cellular stressors is through the induction of heat shock proteins (HSPs). Now, Mosenson et al. suggest that mutant HSP70 could be a potential treatment for autoimmune vitiligo. The authors noticed that mutant inducible HSP70 (HSP70i) could prevent T cell–mediated depigmentation in a mouse model of vitiligo, perhaps by shifting dendritic cells from an inflammatory to a regulatory phenotype. Moreover, a DNA vaccine of the mutant HSP70i could be used therapeutically to partially restore pigmentation in a second model of depigmentation. The authors then took these studies into ex vivo human skin, showing that their mutant HSP70i could prevent the disease-related shift from quiescent to effector T cell phenotype. Although these observations still need to be translated into the clinic, they form the basis for a new potential treatment for autoimmune 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.

HIV Infection Leads to Redistribution of Leaky Claudin-2 in the Intestine of Humanized SCID IL-2R(-/-) Hu-PBMC Mice.

FIG. 1. HIV-induced Claudin-2 in humanized mice in vivo. Immunostaining of the mouse colon was performed 2 weeks after HIV infection. Tissues were fixed, stained with Claudin-2 antibody, and further stained with a secondary antibody. HIV infection in these humanized mice not only increased Claudin-2 (green) staining at the base of the crypts but also induced Claudin-2 expression in the middle and top of the crypts.

Combination immunotherapy with anti-CTLA-4 and interleukin-2 redirects regulatory T cells into tumor-draining lymph nodes and expands anti-tumor CD8+ T cells in the tumor microenvironment

Meeting abstracts Monotherapy with Ipilimumab (anti-CTLA-4 antibody) and monotherapy with IL-2 (T cell stimulating cytokine) are approved for the treatment of metastatic melanoma. Combination immunotherapy has been suggested as a more potent regimen but has not been sufficiently investigated. We

Tu1692 HIV Infection Accelerates Gastrointestinal Tumor Outgrowth in Humanized NSG-HuPBL Mice

Objective(s): HIV infection is a risk factor for the tumorigenesis including non-AIDS defining cancers like those of the gastrointestinal tract. However, the mechanisms underlying such cancer outgrowth are still unknown. Further, combined HIV/cancer studies are difficult to evaluate using primatemodels or in the clinical patient setting. To understand themechanisms of tumor outgrowth in the context of HIV infection, we adopted a humanized mouse model permissive to infection and cancer. Design: In vivo humanized mouse challenge with colon cancer in the context of HIV infection. Methods: Immunodeficient NOD SCID IL-2R-/mice were immunologically reconstituted by adoptive transfer of HIV-negative donor peripheral blood leukocytes and challenged with 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. Results: 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 β-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. Conclusions: 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.

Role of dose selection in successful interleukin-2 immunotherapy: solving the Goldilock’s Complex

Background The results of multiple IL-2 clinical trials (using a range of doses) have shown similar trends, in which IL-2 immunotherapy leads to an objective response in 15-20% of patients and complete response in 5-10%. Importantly, the majority (> 80%) of patients with a complete response maintain long-term responses. However, why only a small proportion of patients attain a complete response at any dose is unknown. We hypothesized that the dose of IL-2 utilized, differentially targets CD8+ effector versus CD4+ regulatory T cells based on patient-specific characteristics, and that an optimal IL-2 dose may be needed to achieve therapeutic benefit.

PD-1 blockade reverses viral infection-induced loss of anti-tumor CD8+ T cell responses

Background Emerging epidemiologic studies describe an increased prevalence of tumors in patients with non-oncogenic chronic viral disease (e.g., non-AIDS-defining cancers in HIV and non-hepatic cancers in HCV). However, there is a lack of basic understanding by what mechanism infections result in increased unrelated cancer formation (i.e., of cancers in tissues not associated with the infection), and what role immunotherapy may have in the rescue of immune responses under this condition. We hypothesized that viral infections establish an immunological environment that leads to the preferential loss of anti-tumor (i.e., anti-self antigen) CD8+ T cells, resulting in the inability of the host to avert tumor growth, and further that immunotherapy can reverse this detrimental loss. Methods

Combination immunotherapy with Interleukin-2 and CTLA-4 blockade decreases tumor growth and improves overall survival

Background Combination immunotherapy is quickly gaining attention in the field of cancer treatment. IL-2, a cytokine which activates T cells, and ipilimumab, a monoclonal antibody that blocks CTLA-4, are both approved as monotherapy for metastatic melanoma. To date, combination immu- notherapy with IL-2 and anti-CTLA-4 has not been adequately investigated. We hypothesized that that this combination may work synergistically owing to its dis- tinct but complementary mechanisms of T cell activation. mean tumor area with combination IL-2 and anti-CTLA- 4 was 2mm2, while in the anti-CTLA-4 only, IL-2 only, and placebo groups it was 14, 29, and 68 mm2, respec- tively (P<0.01 for all comparisons, except IL-2 only ver- sus anti-CTLA-4 only (not significant)). At day 30, the overall survival with combination IL-2 and anti-CTLA-4 was 50%, while with IL-2 only and anti-CTLA-4 only it was 10% and 20%, respectively (P<0.01 for all compari- sons). All animals treated in the placebo group suc- cumbed to their tumors by day 19. These findings were confirmed in a second experiment with similar results. Conclusions Combination immunotherapy with IL-2 activation and CTLA-4 blockade significantly decreases tumor growth and increases overall survival when compared to IL-2 or anti-CTLA-4 monotherapy or placebo. The role of CD8+ effector versus CD4+ regulatory T cells in the success of this immunotherapy is ongoing and will be included in our formal presentation. Ultimately, we aim to translate this work into a combination immunotherapy clinical trial.