Mirza, Noweeda

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 Chronic inflammation in the aged closely resembles tumor-induced immune suppression, manifested as a progressive T1→T2 shift and an increased presence of MDSCs. We hypothesize that reversing these age-related phenomena should reduce the elderly’s heightened susceptibility to malignancy. We have now optimized real-time analyses of phosphorylated STATs in murine T cells and MDSCs in order to correlate STAT activation with immune suppression. Both young and old T cells responded to IL-4 stimulation with STAT6 activation and to IFN-γ stimulation with STAT1 activation. Uniquely, however, young T cells also phosphorylated STAT1, STAT3 and STAT5 in response to IL-4, and responded to IL-6 with STAT3 activation, all consistent with a greater susceptibility to T1/T17 differentiation. Regarding MDSCs, both old and young displayed constitutive STAT3 activation which could be enhanced by G-CSF or IL-6 treatment. Both young and old MDSCs activated STAT1 briskly in response to IFN-γ, STAT5 in response to GM-CSF and STAT6 in response to IL-4. However, younger MDSCs, like younger T cells, displayed more diverse responsiveness to IL-4, activating not only STAT6 but also STAT1 and STAT3. While chronic in vivo exposure to T2 cytokines may explain the decreased responsiveness of older MDSCs and T cells to IL-4, paradoxically IL-4 still robustly activated STAT6 in these cells. Such selective responsiveness may be required to preserve the immune suppressive potential of older MDSCs.

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.

Regulatory T cells (T reg cells) are characterized by the expression of the forkhead lineage-specific transcription factor Foxp3, and their main function is to suppress T cells. While evaluating T reg cells, we identified a population of Foxp3-positive cells that were CD11b+F4/80+CD68+, indicating macrophage origin. These cells were observed in spleen, lymph nodes, bone marrow, thymus, liver, and other tissues of naive animals. To characterize this subpopulation of macrophages, we devised a strategy to purify CD11b+F4/80+Foxp3+ macrophages using Foxp3-GFP mice. Analysis of CD11b+F4/80+Foxp3+ macrophage function indicated that these cells inhibited the proliferation of T cells, whereas Foxp3− macrophages did not. Suppression of T cell proliferation was mediated through soluble factors. Foxp3− macrophages acquired Foxp3 expression after activation, which conferred inhibitory properties that were indistinguishable from natural Foxp3+ macrophages. The cytokine and transcriptional profiles of Foxp3+ macrophages were distinct from those of Foxp3− macrophages, indicating that these cells have different biological functions. Functional in vivo analyses indicated that CD11b+F4/80+Foxp3+ macrophages are important in tumor promotion and the induction of T reg cell conversion. For the first time, these studies demonstrate the existence of a distinct subpopulation of naturally occurring macrophage regulatory cells in which expression of Foxp3 correlates with suppressive function.

Abstract Intratumoral accumulation of T regulatory cells (Tregs) creates an immunosuppressive environment that reduces the efficacy of antitumor immunotherapy. The immunosuppressive milieu within tumors is largely brought about by the presence of Tregs, which maintain self-tolerance by directly inhibiting T cells, NK cells, and dendritic cells. Depletion of Tregs enhances antitumor immune responses; however, current depletion therapies also affect the function of CD4 and CD8 T effector cells. Previous studies from our laboratory indicate that intratumoral delivery of CpG-ODN strongly reduces the levels of Tregs within the tumor, which is mainly mediated by IL-6. Because IL-6 promotes growth of some human cancers, alternate pathways to inactivate Tregs were sought through microarray analysis, resulting in gene candidates that can be exploited to modulate the function of Tregs. Analysis of these candidates indicates that neutralization of chemokine (C-C motif) ligand 1 (CCL1) prevented de novo conversion and suppressive function of Tregs without affecting the function of T effector cells. The combination of CpG-ODN and anti-CCL1 treatments induced complete rejection of tumors in BALB-neuT tolerant mice, and result in the generation of long-term protective memory responses. Tumor rejection correlated with changes in the lymphocyte composition within the tumor; we observed decreased Treg numbers and a concomitant accumulation of tumoricidal cells such as CD8+NKG2D+ and NK cells. These studies demonstrate that neutralization of CCL1 can be used as an adjuvant to antitumor immunotherapy, as a means of reversing the immunosuppressive function of Tregs without compromising T cell effector function.

Abstract T cell responses are compromised in the elderly. The B7-CD28 family receptors are critical in the regulation of immune responses. We evaluated whether the B7-family and CD28-family receptors were differentially expressed in dendritic cells, macrophages, and CD4+ and CD8+ T cells from young and old mice, which could contribute to the immune dysfunction in the old. Although most of the receptors were equally expressed in all cells, >85% of the old naive CD8+ T cells expressed B7-H1 compared with 25% in the young. Considering that B7-H1 negatively regulates immune responses, we hypothesized that expression of B7-H1 would downregulate the function of old CD8+ T cells. Old CD8+ T cells showed reduced ability to proliferate, but blockade of B7-H1 restored the proliferative capacity of old CD8+ T cells to a level similar to young CD8+ T cells. In vivo blockade of B7-H1 restored antitumor responses against the B7-H1− BM-185–enhanced GFP tumor, such that old animals responded with the same efficiency as young mice. Our data also indicate that old CD8+ T cells express lower levels of TCR compared with young CD8+ T cells. However, following antigenic stimulation in the presence of B7-H1 blockade, the levels of TCR expression were restored in old CD8+ T cells, which correlated with stronger T cell activation. These studies demonstrated that expression of B7-H1 in old CD8+ T cells impairs the proper activation of these cells and that blockade of B7-H1 could be critical to optimally stimulate a CD8 T cell response in the old.