Research

ANTIGEN PRESENTATION IN TOLERANCE AND AUTOIMMUNITY

We are interested in autoimmunity and self-tolerance and how antigen presenting cells acquire, process, and present antigens. Several models are used to examine antigen presentation in vivo in which autoreactive T cells can be monitored at each step of the immune response. Understanding how antigen presenting cells influence CD4 and CD8 T cells responses is necessary for developing potential therapies for subduing or preventing autoimmune diseases. 

Tolerance induction by dendritic cells and lymph node stroma.


T cell tolerance to self-antigens is crucial for preventing autoimmune diseases, and several mechanisms have evolved to enable lymphocytes to discriminate between self and non-self. Peripheral induction of tolerance in adulthood occurs primarily in lymph nodes where self-reactive T cells are either deleted or silenced. Deletion of such cells, which is arguably the best safeguard against T cell-mediated autoimmunity, relies on the presentation of peptide-MHC complexes to circulating T cells. In the prevailing model of this process, antigen capturing cells acquire proteins from parenchymal tissues and then transport them to nearby lymph nodes, where CD8 dendritic cells (DCs) present self-peptide-MHC complexes to circulating T cells. Under non-inflammatory conditions, these events can lead to the elimination or inactivation of self-reactive T cells. However, exposure of DCs to selected inflammatory stimuli can change the outcome of the immune response from tolerance to immunity.

We recently discovered a novel mechanism of self-tolerance induction that is mediated by a unique lymph node-resident population of nonhematopoietic stromal cells that we coined lymph node stromal cells (LNSCs).  We, and now others, have shown that LNSCs express peripheral tissue antigens and the autoimmune regulator (Aire) gene that partially controls promiscuous gene expression in the thymic stroma. Moreover, we demonstrated that presentation of endogenously-expressed self-antigens by LNSCs results in deletion of antigen-specific CD8 T cells. More recently we have shown that LNSCs can present antigen on MHC class II molecules and promote the conversion of naïve CD4 T cells into FoxP3 T regulatory cells. Antigen presentation by LNSCs appears to represent an important mechanism for controlling the peripheral repertoire of self-reactive T cells and may resolve the long-standing dilemma of how tolerance to self is preserved under inflammatory conditions. The long-term objective of our work is to elucidate how LNSCs and DCs present intestinal and pancreatic antigens to T cells in lymph nodes, and thereby regulate immunity and tolerance; this information is important for understanding how the T cell repertoire is amended in the periphery to prevent untoward responses to vital tissues.

  


Immature dendritic cells and lymph node stromal cells sample exogenous antigens. Localization of endo-cytosed FITC-OVA inside immature dendritic cells (imDCs; left) and lymph node stromal cells (LNSCs; right). Cells were exposed to FITC-OVA (2.5mg/ml) for 1hr at 37oC, washed, and processed for immuno-fluorescence microscopy. FITC-OVA is depicted in green and DAPI in blue.
 
 

Loss of mucosal tolerance upon PD-L1 blockade. Representative histology of the small intestine from a control (left) and anti-PD-L1-treated iFABP-tOVA mice (right). OT-I T cell infused iFABP-tOVA mice develop T cell-mediated enteritis following PD-L1 blockade. Tissues were fixed and processed for H&E staining (20X).
 

Dendritic cells and environmental triggers in autoimmunity.

Type-1 diabetes is an autoimmune disease characterized by the annihilation  destruction of the insulin-secreting beta cells of the endocrine pancreas.  In the first stage of pancreatic autoimmunity, or insulitis, leukocytes invade the pancreatic islets and form insulitic lesions.  The prelude to insulitis unfolds in pancreatic lymph nodes when naïve T lymphocytes reactive to islet beta cells encounter stimulatory DCs presenting beta cell peptides in association with MHC molecules.  Our studies aim to understand how T cell responses against pancreatic beta cells are provoked. It is generally thought that environmental factors impinge on an individual’s genetic susceptibility to control the penetrance and course of pancreatic autoimmunity.  Several lines of evidence point to the influence of extraneous factors associated with the gastrointestinal tract. Associations between type-1 diabetes and diverse viral infections, particularly with enteroviruses such as coxsackievirus B4, have been invoked, although a clear-cut causal agent has yet to be identified. Several other lines of evidence suggest that celiac disease and Crohn’s disease may be linked to autoimmunity in the pancreas, and possibly precipitate type-1 diabetes. The common route of entry of potential environmental triggers such as enteroviruses and dietary antigens raises the questionof how the gastrointestinal tract relates to the pancreatic axis and autoantigens presentation, however, the precise mechanisms by which such environmental factors influence the autoimmune response against pancreatic beta cells remain elusive. We are investigating the influence of the intestinal microenvironment on dendritic cell function and self-antigen presentation in gut-associated lymphoid tissues.

TUMOR CELL-IMMUNE CELL INTERACTIONS

Pancreatic cancer is an incurable malignancy and the fourth leading cause of cancer fatality. Innovative therapies are urgently needed to cure this disease since radiation, chemotherapy, and surgical interventions have failed. The immune system can recognize and destroy tumors by a process termed immunosurveillance. Thus, a promising approach to defeating pancreatic cancer is the augmentation of anti-tumor immunity.

Cancer is the result of a complex chain of events from initiation of transformation due to loss- or gain-of-function of cell cycle control mechanisms to invasion of multiple organs by metastasis. Although tumor cells arise from self, due to genetic instabilities, they may express novel proteins or proteins with unique post-translational modifications that can be recognized by T cells as foreign or altered self. These tumor-associated antigens appear as a result of overexpression of certain proteins, mutations, expression of developmental phase- or tissue-specific antigens, or from dying tumor cells and could potentially become targets of tumoricidal lymphocytes. Leukocytes such as T cells, DCs, NK cells and macrophages have been observed in human and rodent tumors suggesting an ongoing immune response however their prognostic value depends on the type and the frequency of the infiltrating immune cells. For example, an accumulation of mature DCs and CD8 T cell is a sign of better prognosis, whereas immature DCs and Tregs is a sign of a poor prognosis.

Unfortunately, this straightforward picture hides the complexities of the tumor microenvironment. High genetic drift in tumor cell populations allows them to be selected against pressure from the immune system by immunoediting. This selective outgrowth results in the generation of non-immunogenic tumor variants, which employ various mechanisms to escape from the immune system. For instance, certain defects in tumor cells such as loss of antigen or antigen processing machinery, downregulation of MHC or MHC-related molecules, and lack of costimulatory molecules can cause T cells and NK cells to ignore tumor cells. Expression of cell surface molecules such as PD-L1 and B7-H4, and immunosuppressive soluble factors such as TGF-beta, IL-10 and VEGF can directly suppress the effector cell functions. In addition, tumor cells may counterattack the immune system by killing leukocytes via apoptosis-inducing molecules while protecting themselves from programmed cell death by upregulating anti-apoptotic molecules.

Another major mechanism of immune evasion is the accumulation of suppressive immune cells within tumors. There are dynamic and complex interactions between tumors and suppressive subsets of these accumulating leukocytes including Tregs, immature DCs and myeloid-derived suppressor cells (MDSCs). Tregs can inhibit effector CD4 and CD8 T cells as well as NK cells by direct cell contact or secretion of immunosuppressive cytokines such as TGF-beta and IL-10. DCs and MDSCs can suppress the proliferation and function of T cells by various mechanisms. Products of tryptophan metabolism by indoleamine 2,3-dioxygenase, and L-arginine metabolism by arginase I  and inducible nitric oxide synthase are some of the molecules involved in suppression of T cell proliferation and function by these cells. Exposure to such factors within the tumor microenvironment can paralyze antigen-specific effector functions of CD8 T cells by impairing cytokine signaling pathways, downregulating TCR signaling molecules such as CD3-zeta chain, and altering TCR recognition of peptide-MHC complexes. We are studying the dysfunction(s) of dendritic cells, macrophages, and T cells in models of pancreatic cancer and melanoma. Our work aims to develop new approaches to unleash robust anti-tumor immunity by restoring the stimulatory potential of tumor-resident dendritic cells and the effector function of tumor-infiltrating lymphocytes.
 


(Left) Pancreatic tumor cells metastasize to pancreatic lymph nodes. Immunofluorescence microscopy of metastatic lesion (top) in pancreatic lymph node. Cryosections from 12-week old (CD11c-YFP x RIP-Tag2) mice were stained with DAPI (blue) and anti-MHCII (red). Dendritic cells (CD11c+)
are YFP+ and depicted in green.
(Right) CD8 T cells reside for long periods of time in solid tumors of the endocrine pancreas. Immunofluorescence microscopy of tumor-resident CD8 T cells in a large islet tumor in the RIP1-Tag2 pancreas. Cryosection from an 11-week old RIP1-Tag2 mice was stained with DAPI (blue), anti-CD31 (green), and anti-CD8 (red).