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Allergic asthma is a chronic intermittent inflammatory lung disease where patients experience relatively few symptoms between exacerbations precipitated by allergen-challenge of allergic memory responses. Allergen-specific CD4+ T cells are believed to be a major mediator of allergic memory responses through the production of type 2 cytokines including IL-4, IL-5, IL-13 and IL-9. Recently, tissue-resident memory (Trm) cells have been identified in peripheral tissues that mediate mucosal barrier immunity. However, the precise role of Trm CD4+ T cells in allergic exacerbations is not clearly defined. In studies to examine the role of IL-9 in allergen-specific Trm responses to Aspergillus fumigatus we observed that all IL-9-secreting T cells had a resident memory phenotype. Inhibition of circulating T cells by administering FTY720 in the last month of rest, diminished the total CD4 T cells population in the lung, while the Th9 cells remained stable. Blockade of IL-9 prior to recall challenge phase reduced overall allergic lung inflammation as assessed both by flow cytometry and single cell RNA-seq. Within eight hours after challenge, IL-9 is one of the top genes identified by scRNA-seq that distinguishes ST2+ and ST2− CD4+ T cell populations in the lung. Using IL-9-reporter mice we also compared gene expression in isolated IL-9+ T cells and other type 2 T cells. These findings demonstrate that IL-9-producing Trms play an important role in mediating allergic memory responses. IL-9 could be a promising therapeutic target specifically in patients showing symptoms of intermittent allergic response.

Chimeric antigen receptor (CAR) T cell therapy involves genetically manipulating T cells to express an antigen receptor specific for tumor protein, resulting in a powerful anti-cancer therapeutic. However, trafficking to tumor sites and the suppressive nature of the tumor microenvironment have prevented effective treatment of solid tumors. Previous work has shown that antigen specific T helper (Th) subsets have variable tumor killing capabilities, however, it is not completely defined how different transcriptional differentiation programs effect the fitness and function of CAR T cells. It was hypothesized that differentiation of T cells transduced with CAR improves cancer cell killing in an antigen specific manner. To test this hypothesis, splenic CD4+ and CD8+ T cells were cultured under T cell activating (T0), Th2 (T2), or Th9 (T9) cell differentiation conditions and transduced with a 2nd generation CD19-specific CAR. Control cells were transduced with CD19-specific CAR lacking the intracellular domain. CAR T cells were co-cultured with CD19+ B cells and target cell killing was measured by flow cytometry. Preliminary results show that CAR T9 cells, but not T2 cells, kill target cells similar to the clinic relevant T0 cells. FACS analysis of T cells after co-culture with target cells revealed antigen-experience promoted production of lineage specific cytokines as well as granzymes A and B. T9 conditions maintained a larger proportion of CD8+ T cells than T2 conditions, suggesting that T9 conditions promote expansion of effector CD8+ T cells. These results suggest that the Th9 transcriptional program promotes target cell killing, transduction efficiency, and generates a better ratio of CD4:CD8 T cells for adoptive cell therapy.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic β cells are destroyed by auto-aggressive T cells. Tregs in T1D have been shown to lose their suppressive function and/or become inflammatory, complicating Treg immunotherapy. Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae, protects against autoimmunity. Although CFA/I fimbriae act in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific Tregs. Our initial findings showed that oral dosing with a Lactococcus lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45%. We hypothesized that dosing NOD mice more frequently would induce Foxp3+ Tregs in the pancreas and PaLNs to provide more robust protection. To test this, 4 wk-old NOD mice were orally dosed with LL-CFA/I varying the dosage and frequency of treatment. When dosed every 2 wks, the frequency of insulitis was reduced by more than half at 11 wks of age, and tetramer staining revealed a 2-fold reduction of insulin-specific CD4+ and CD8+ T cells within the PaLNs. Additional groups were followed until 16 wks of age, and examined for Treg and inflammatory T cell phenotypes. Treatment with LL-CFA/I reduced 2-fold IFN-γ-producing CD8+ T cells in the spleen and PaLNs. PaLN Foxp3+ T cells from the PBS and vector control groups, but not the LL-CFA/I-treated mice, co-expressed Tbet and IFN-γ, suggesting that Tregs from LL-CFA/I treated mice maintained their regulatory function at 16 wks. These data show that orally dosing with LL-CFA/I diminishes or halts T1D progression via activation of stably functional Foxp3+ Tregs. Work supported by AI121745.

Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae protect in murine models of multiple sclerosis and rheumatoid arthritis. Although CFA/I fimbriae initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection was found to be ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). Our recent findings show that oral dosing with a L. lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45% with a concomitant 8-fold increase in their splenic Foxp3+CD25+ Tregs. However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. We hypothesized that LL-CFA/I stimulates dendritic cells (DCs) to establish a regulatory microenvironment since DCs play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic. In this study, 4 wk-old NOD mice were orally dosed with LL-CFA/I and treated every 2 wks; control groups were given L. lactis vector or PBS. At 11 wks of age, frequency of insulitis was reduced by more than half, and insulin-specific T cells were reduced in the pancreatic lymph nodes (PaLNs; P < 0.05). To discern how this was mediated, groups of mice were examined at 3, 7, 14, and 21 days post-treatment for changes in DC and T cell phenotypes. As early as 3 days post-treatment, DCs exhibited significantly less expression of costimulatory molecules, CD40 and CD86, and stably maintained this phenotype for at least 7 wks. Splenic DCs showed reduced IL-6 production. These data show that orally dosing with LL-CFA/I ameliorates T1D in NOD mice by establishing a regulatory microenvironment via DCs. Work is supported by AI121745.