Hoelzinger, Dominique B.

ABSTRACT Biofilm‐associated staphylococcal infections remain exceptionally difficult to treat due to the presence of resilient staphylococcal biofilms and the limited effectiveness of currently available antibiotics. To combat persistent infections, providers are often forced to use prolonged courses of combination antimicrobial therapies that have significant toxicities with limited effectiveness, leading to increased hospital stays, substantial health care costs and a rise in patient morbidity and mortality. Staphylococcus aureus is a dominant cause of recalcitrant biofilms, for which novel non‐antibiotic therapeutics are critically needed. Lysins, a class of protein‐based antimicrobials, rapidly kill staphylococci, exhibit potent anti‐biofilm activity, have a low propensity of resistance development and synergy with antibiotics. To address this unmet need, we evaluate an extracellular vesicle (EV)‐based delivery platform for the engineered anti‐staphylococcal lysin LYSG101, designed to improve stability and localization at sites of infection. Initial proof‐of‐concept work is provided here, demonstrating that human serum‐derived EVs can be loaded with LYSG101 to exert a potent in vitro antimicrobial effect against both planktonic and biofilm forms of S. aureus . EV‐mediated delivery achieved activity equivalent to free lysin, with additional translational advantages including stability and the potential for sustained intra‐articular retention. This work supports further development of the EV‐mediated lysin delivery as a broadly applicable antimicrobial platform, with potential future applications in biofilm‐associated infections, including prosthetic joint infection (PJI).

Comparative analysis of fibrin sealant and recombinant thrombin hydrogels reveals distinct microarchitectures and cellular responses, emphasizing recombinant thrombin’s potential to enhance cellular activity and enable vesicle delivery.

Abstract Age related changes compromise T cell mediated immunity. Previous findings suggest that the aged immune T cell repertoire can be exploited to mount effective anti-tumor responses to immunogenic tumors. However, antitumor immune responses against tumor self-antigens are significantly dysregulated in the old; hence, the salient differences in the biology and function of T cells between young and old, should be elucidated. These differences fall into three categories: (i) there are lower surface levels of T cell receptor (TCR) on old CD4 and CD8 T cells, (ii) the TCR complex is cycled differently in aged T cells and (iii) the TCR complex may be disassociated in older animals. Therefore these preliminary findings support the hypothesis that aging causes a decline in T cell function. Data analyses from a T cell activation time-course microarray study show that there are large differences in the gene expression profiles of old and young naive cells in CD4 (2022 genes) and CD8 (3666 genes) subsets. These differing genetic signatures point to potentially dysregulated proteins in the old immune system. Further investigation of selected gene products using appropriate mouse models, may provide valuable preclinical information with significant therapeutic potential which could have the opportunity of being translated into clinical trials. This work was sponsored in part by a grant from AFAR.

Abstract IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.

Abstract Macrophages with suppressive characteristics such as Tumor associated macrophages or Myeloid derived suppressor cells have been defined showing regulatory function under inflammatory or polarizing conditions. Until now, there are no reports indicating that under normal, non-inflammatory or non-polarizing conditions, macrophages could have immunoregulatory functions. Utilizing knock-in Foxp3-GFP mice, we identified a new population of Foxp3 positive cells that were CD11b+F4/80+ cells. These cells were observed in spleen, lymph nodes, bone marrow and thymus. Similar to Tregs, the CD11b+F4/80+Foxp3+ macrophages inhibited T cell proliferation, whereas Foxp3neg macrophages did not. The Foxp3neg macrophages could be converted to express Foxp3. These induced-CD11b+F4/80+Foxp3+ cells displayed inhibitory properties similar to natural Foxp3pos macrophages. Based on the immunoregulatory properties of these cells, we termed them Macrophage regulatory cells (Mac-regs). These Mac-regs express CD68hiF4/80hiCTLA4hiGITRhiIL-4Rhi. Interestingly, GR-1 is not a differentiation marker. The cytokine, chemokine, growth factor and genomic profiles of Mac-regs are different compared to CD11b+F4/80+Foxp3-cells. For the first time, we demonstrated the occurrence of a natural population of macrophages displaying regulatory function which may contribute to the homeostatic balance of the immune system. This research was supported by Grants from the NIH and AFAR.

Abstract Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (>65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.

Abstract T-regulatory cells (Tregs) create an immunosuppressive environment that reduces the efficacy of anti-tumor immunotherapy. Depletion of Tregs enhances anti-tumor immune responses, but also affects T-effector cells. Previously we showed that blockade of chemokine ligand 1 (CCL1) inhibits Treg conversion, their suppressive function, and in combination with intra-tumoral CpG-ODN results in complete rejection of tumors in BALB-NeuT tolerant mice. Most importantly, this therapeutic combination does not affect T-effector function and increases tumor-lytic CD8+NKG2D+ cells within the tumor. Here we expand on the role of blocking CCL1 in enhancing anti-tumor immunotherapy. Using CCL1 knockout (CCL1ko) mice, we found no impairment in TGFβ dependent de novo Treg conversion, however CCL1ko Tregs are less suppressive in vitro, than their wild type counterparts. CCL1ko mice treated with intratumoral CpG showed a marked reduction in tumor growth and a significant increase in tumor rejection as compared to similarly treated C57b control mice. Examination of lymphocyte populations within B16 and MucI tumors showed elevated levels of CD8+ cells. Finally the evaluation of levels of intra-tumoral cytokines and chemokines revealed elevated levels of IFNγ, CXCL9, CCL3 and CXCL10 in tumors grown in CCL1ko mice, suggesting a pro-inflammatory environment. Taken together these data support the role of CCL1as a critical factor for the maintenance of the suppressive function of Tregs.

Abstract Effective anti-tumor immunotherapy must overcome the immunosuppressive networks created by the tumor. Previous studies from our group indicate that intratumoral injection of CpG-ODN reduces Treg levels within the tumor by 80%. This decrease in Tregs is mainly mediated by the activation of antigen presenting cells secreting IL-6. To explore the effect of IL6 on Tregs, an expression microarray analysis of genes involved in TGFÎ2 dependent Treg conversion, which are also sensitive to IL6, was done. It yielded gene candidates which can be exploited to modulate the function of Tregs. In vitro analysis of two such candidates: chemokine ligand 1 (CCL1) and integrinαE (ITGαE), shows that blocking these proteins reduces de novo conversion of Tregs (CCL1 and ITGαE) and inhibits the suppressive function of Tregs (CCL1 only). In vivo assays using the neu tolerant breast cancer mouse model demonstrates that injection of αCCL1 or αITGαE blocking antibodies significantly reduced the level of Tregs within the tumor and the tumor draining lymph nodes. Furthermore, the combination of intratumoral injections of CpG-ODN and αCCL1 induce complete tumor rejection in neu mice. These results demonstrate that blockage of CCL1 signaling modulated the function of Tregs and subsequently enhances immunotherapeutic approaches.

Abstract Previous studies have established that 4-1BB and OX40, members of the TNFR family, enhance antitumor immune responses. Recent studies from our laboratory indicate that intratumoral injections of CpG-ODN induce antitumor responses in Her-2/neu tolerant mice. However, this treatment alone is not sufficient to completely eradicate tumors. Here we investigate whether the combination of CpG+anti-OX40 or anti-4-1BB enhance the antitumor responses in neu mice. Our results indicate that CpG+anti-4-1BB but not CpG+anti-OX40 eradicate tumor in neu mice. CpG+anti-4-1BB treatment significantly increased the number of CD8 cells within lymphoid organs as compared to control or anti-OX40 treated animals. More importantly, we observed that anti-4-1BB but not anti-OX40 inhibits the suppressive function of Tregs. Microarray analysis of Tregs revealed specific transcriptional differences brought about by treatment with anti-OX40 or anti-4-1BB. 4-1BB modulates differential gene expression in Tregs of over 300 genes that remain unaffected by OX40. Potential gene candidates include interleukin-9 and protein-tyrosine phosphatase receptor-type zeta-1, which are transcriptionally down and up regulated in the presence of anti-4-1BB. These proteins may play a role in Treg function, and could explain the enhanced anti-tumor effect observed in the presence of anti-4-1BB.

AbstractIdentifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.