Citations

Abstract Cerebral malaria (CM) is a severe complication of P. falciparum infection and a leading cause of death, globally. Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice serves as a murine model of CM called experimental cerebral malaria (ECM) due to the neurological and neuropathological features that simulate the infection of human CM. A putative mechanism of disease in human CM requires CD8 T cell to infiltration into the brain and blood-brain barrier disruption. The specific antigen-presenting cell type(s) that prime this CNS infiltrating CD8 T cell response capable of inducing lethal blood-brain barrier disruption has not been defined. To answer this, single class I expressing Kb loxP transgenic mice were generated. Crossing these mice to class I deficient CD11c-cre or LysM-cre mice created the LysM-cre Kb cKO and CD11c-cre Kb cKO mice, respectively. PbA infected Kb loxP and LysM-cre Kb cKO were susceptible to ECM. In contrast, CD11c-cre Kb cKO mice survived acute PbA infection and avoided neurological manifestations. We therefore employed spectral flow cytometry to analyze splenocytes and brain infiltrating immune cells during acute PbA infection in these genotypes. We observed a reduction in activated CD8 T cells in the spleens of PbA infected CD11c-cre Kb cKO mice versus the Kb loxP mice and the LysM-cre Kb cKO. This reduced peripheral CD8 T cell activation in PbA infected CD11c-cre Kb cKO mice also corresponded with reduced brain infiltration of CD8 T cells. We conclude a CD11c+ APC is therefore required for priming and potentially recruitment of CD8 T cells to the brain during lethal ECM pathology. 5R01NS103212-06

Abstract Only a small cohort of patients show durable responses to immune checkpoint inhibitor (ICI) therapy and the mechanisms regulating CD8 T cell responses to ICIs are yet to be elucidated. To that end, we explored T cell intrinsic factors that may be attributed to low ICI therapy response rates. We have identified Dual Specificity Phosphatase 2 (DUSP2) as a potential negative regulator of ICI response within CD8 T cells. We demonstrated that DUSP2 expression in human CD8 T cells is transient, peaking at two-hours post-T cell stimulation with rapid waning thereafter. Downregulation of DUSP2, via siRNA transfection, increased cytotoxicity of human CD8 T cells activated for 48-hours prior to inclusion in a four-hour tumor killing co-culture assay, despite the return of DUSP2 expression to baseline levels at this time-point. Additionally, DUSP2 overexpression, via mRNA transfection, decreased release of effector molecules (Granzyme B and Interferon-gamma) from human CD8 T cells at 24-hours post-activation when compared to control mRNA. When we utilized RNA-sequencing to analyze and compare differentially expressed genes between responders and non-responders of ICI therapy, we found DUSP2 expression in CX3CR1 positive CD8 T cells to be consistently decreased in responders but not in non-responders. Since CX3CR1 positive CD8 T cells identify a highly resilient and cytotoxic subset in response to ICI therapy, we hypothesize that DUSP2 negatively regulates ICI-responsive effector CD8 T cells. Thus, uncovering the role of DUSP2 in CD8 T cells may provide a target to improve the efficacy of ICIs in patients with limited therapeutic alternatives. Mayo Clinic Department of Immunology Pilot Award Mayo Clinic Center for Individualized Medicine

Abstract Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection. R01CA256927

Abstract Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). Recently, mutations in p300 and CBP were identified that abrogate their function as an acetyltransferase but not the ability to mediate crotonylation. These mutants can activate transcription independent of their ability to acetylate histones, demonstrating that crotonylation can also lead to transcriptional activation. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have both decrotonylase and deacetylase activity, but the relative importance of each function in T cells is not known. Genetic knockout of HDAC3 using CD2 icre mice results in a severe block in T cell development and spontaneous development of severe colitis. Examination of HDAC3 cKO mice has shown an aberrant ratio of Treg:Th17 cells within the gut, driving inflammation and loss of mucosal barrier integrity. Our lab has also determined that HDAC3 is required for at least three aspects of T cell development: CD4+ CD8+ (DP) cell survival, positive selection, and CD4+ lineage development. Recently, a mutation was identified in either HDAC1 or HDAC3 that eradicated deacetylase activity but retained decrotonylase activity (“VRPP”). Through this mutation we were able to generate a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP. Strikingly, this mouse does not develop colitis, although T cell development is still impaired. Thus, HDAC3 decrotonylase activity alone is sufficient to impede the IBD phenotype. Support by grants from National Institute of Allergy and Infectious Diseases (R01 AI150100)

Abstract Aging dramatically impacts T cells, contributing to diminished and delayed responses to infection. While the development and maintenance of lymphocytes certainly undergo intrinsic changes with age, the contribution of cell-extrinsic forces, such as the aging tissue microenvironment, has been difficult to uncouple. Macroscopic changes to the lymph nodes have been well described, but the impact of aging on lymph node stromal subsets is less understood. Here, we visualized naïve T cell migration in the aged murine lymph node using live-cell 2-photon microscopy and determined that T cell motility was impaired in regions of increased fibrotic deposition. Using fluorescent reporters for Ccl19 and Cxcl12, homeostatic chemokines important for stromal:lymphocyte crosstalk, we also found that the distribution of stromal cells expressing these chemokines was altered with age, and that migrating naïve T cells were excluded from regions of chemokine expression. Flow cytometric analyses of fibroblastic reticular cells (FRCs) revealed decreased proportions of FRCs within the mesenteric lymph nodes and decreased putative FRC progenitors in the skin-draining lymph nodes. FRC progenitors at both sites had reduced surface expression of lymphotoxin-beta receptor, an important receptor for FRC differentiation and maintenance. We hypothesize that age-associated changes to FRC maturation contribute to increased lymph node fibrosis and altered localization of homeostatic factors. Current studies have indicated that in vitro cultured lymph node fibroblasts are sensitive to TNF-alpha and respond by increasing expression of alpha-smooth muscle actin, suggesting a mechanism by which aging remodels the lymph node microenvironment.

Abstract Chimeric antigen receptor (CAR) T-cells are T-cells engineered with synthetic receptors that directs their specificity and function toward an antigen of choice. This application has been highly successful in treating a variety of blood-borne cancers such as B-cell lymphomas. CAR T-cells against solid tumors have been less efficacious, potentially due to the resistance of tumor cells to T cell-mediated cytotoxicity. Current paradigm suggests that tumor cells are killed by perforin/granzyme-secreting CD8 cytotoxic T cells (CTLs). However, activated CD8 T cells also produce TNF and IFNg, two cytokines that have been reported to have anti-tumor function, but it is not clear whether these cytokines can directly kill tumor targets. It has been reported that defects in kinases in the TNFR1 signaling pathway can sensitize cells to TNF-induced death. Therefore, we hypothesize that by inactivating these kinases, we can sensitize solid tumors to killing by CD8 CAR T-cells producing TNF and IFNg. We intend to conduct proof-of-concept studies to show that sensitizing tumor cells to cytokine-mediated cytotoxicity will enhance the effectiveness of CAR T-cell therapy against solid tumors. Institutional Funding

Abstract Peripherally-derived RORγt+ regulatory T cells (Tregs) induced in early life are crucial for maintaining oral tolerance later in life, and the loss of these long-lived cells is associated with increased allergic and inflammatory phenotypes in several animal models. We have identified a predominant pathway of luminal antigen delivery that is associated with Treg development. Around weaning, microbial and dietary proteins are delivered to the colonic lamina propria via goblet cell-associated antigen passages (GAPs) and taken up by antigen presenting cells. GAP formation is inhibited by breastmilk-derived epidermal growth factor (EGF) which is highest in maternal milk at parturition and is diminished by weaning. We have previously shown that disruption of this process – via EGF receptor inhibition (EGFRi) day of life (DOL)4–8 or by conditional deletion of EGFR from goblet cells – results in premature antigen delivery. Consequently, this results in an early initial increase in FoxP3 expression in the colon that is unstable and, ultimately, lost by Tregs primed pre-weaning; this suggests that earlier education of T cells is incompatible with regulatory T cell differentiation in early life. EGFRi-treated FoxP3eGFP-Cre-ERT2ROSAlsl-YFP mice, whose FoxP3+ cells were YFP-labeled at DOL21, had a larger number of YFP+ T cells that lost FoxP3 expression than that of control mice, by DOL28. Further, compared to controls, wild-type mice treated with EGFRi DOL4–8 had increased IFNγ, TNFα, and IL-17a-expressing CD4 T cells when challenged with DSS colitis later in life. This suggests that early antigen delivery-associated FoxP3 instability may contribute to an increased propensity toward an inflammatory phenotype later in life.

Abstract Dendritic cells (DCs) function as principal sensors of PAMPs and DAMPs, connecting the innate and adaptive arms of the immune system. DC subset deficiencies have been observed in untreated CLL patients compared to age-matched healthy controls. Studies investigating the specific DC subsets that differ in function and localization; conventional DCs (cDCs), plasmacytoid DCs (pDCs), and Axl+ Siglec-6+ (AS) DCs are lacking in CLL. In mice, DC development and maintenance is highly dependent on the Flt3/Flt3L signaling axis. Activated T cells are believed to be the major source of Flt3L to DCs. Previous studies have reported elevated Flt3L in the serum of untreated CLL patients. Interestingly, increased serum levels of Flt3L have been associated with DC deficiencies, as DCs are believed to be the principle immune cell requiring this cytokine. This study will utilize extensive flow cytometry panels to investigate the prevalence and functional status of DCs in untreated CLL patient blood, including cDC1, cDC2, pDCs, and AS DCs. In addition, we will interrogate the ability of CLL-derived exhausted T cells to provide Flt3L upon activation.

Abstract In this study, we leveraged immunomodulatory Siglec-sialic acid interactions to protect beta-cells against autoimmune rejection in Type 1 Diabetes (T1D). Our lab previously demonstrated that the sialyltransferase ST8Sia6 generates ligands that bind the Siglec-E receptor expressed on murine islet-resident macrophages. Here, mice were engineered to express ST8Sia6 in beta cells (βST mice), backcrossed to the non-obese diabetic (NOD) mouse model of T1D, and followed for disease. 60% of female littermates spontaneously developed disease by 300 days, compared to only 6% in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration in early life, but drastically reduced insulitis only in βST mice at later timepoints. This was not due to an absence of autoreactive T cells, as an anti-PD-L1 challenge resulted in similarly rapid induction of disease in both βST and littermate mice. Our genetic model allows for temporal control of ST8Sia6 expression in βST mice using the tet-off system. Treating our mice with doxycycline at 20 weeks of age resulted in persistent protection compared to age matched littermates, while the same treatment at 8 weeks of age led to similar incidence of diabetes between βST mice and littermate controls. Flow cytometric analysis of islet infiltrating immune cells from euglycemic mice at 14 weeks of age demonstrated reductions in short-lived effector CD8 T cells and increased thymically derived Tregs in βST mice compared to littermate controls. These results highlight the therapeutic potential of sialyltransferase mediated immune regulation and present a potential path toward improved curative approaches for T1D. Supported by grants from NIH (5R21AI135858-02, 1F30DK127564)

Abstract Invariant natural killer T (iNKT) cells are unique innate immune cell which secrete cytokines and immune mediators. These cells recognize self or microbial glycolipids presented in the context of CD1d. iNKT cells can contribute to host protection or pathogenesis during intestinal inflammation. Colonic iNKT cells are established under the influence of the microbiota in early life and the current understanding is that colonic iNKT cells are fixed and not manipulable in later life. Goblet cells (GCs) or/and goblet cell associated passages (GAPs) deliver luminal antigens and maintain peripheral T regulatory cells in the steady state. However, GAPs in the colon are absent in adult mice due to GCs microbial sensing. We found that the glycolipids can be delivered through GAPs and that inducing GAPs in the colon in adult mice resulted in significant iNKT cell expansion. Deletion of GCs or deletion of CD1d on GCs prevented iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of colonic-iNKT cells showed significantly increased iNKT2 and iNKT1 subsets after colonic GAP induction. Proteomic analysis of these iNKT cells further confirmed the characteristics of expanded iNKT subsets. Moreover, iNKT cells expanding after colonic GAP induction persisted and were protective in DSS-induced colitis. Adoptive transfer of the expanded colonic iNKT cells protected in the DSS-induced colitis model, indicating that expanded colonic iNKT cells were sufficient for protection in this model. These findings indicate that colonic iNKT cells are not fixed during early life but can be expanded in a GAP dependent and GC CD1d dependent manner and can be protective in some models of colitis. Crohn’s and Colitis Foundation