Kristi Marquardt

Uncoupling of Proliferative Potential and Gain of Effector Function by CD8+ T Cells Responding to Self-Antigens

Professional antigen-presenting cells (APCs) are capable of transporting self-antigens from peripheral tissues to secondary lymphoid organs where they are presented to potentially autoreactive CD8+ T cells. In the absence of an inflammatory response, this results in immune tolerance. The presence of activated, antigen-specific CD4+ T cells converts this tolerogenic encounter into an immunogenic one by promoting extensive proliferation of CD8+ T cells and their development into effectors. Surprisingly, activation of APCs with an agonistic antibody specific for CD40 could not substitute for CD4+ help in this task. Anti-CD40 induced recruitment of dendritic cells expressing high levels of B7 costimulatory molecules into the lymph nodes, which in turn, greatly enhanced activation and expansion of CD8+ T cells. However, these activated CD8+ cells did not demonstrate effector function. We conclude that proliferative potential and gain of effector function are separable events in the differentiation program of CD8+ T cells.

The Fate of Low Affinity Tumor-Specific CD8+ T Cells in Tumor-Bearing Mice

A major challenge in tumor immunology is how best to activate the relatively low avidity self-specific and tumor-specific T cells that are available in the self-tolerant repertoire. To address this issue, we produced a TCR transgenic mouse expressing a class I-restricted hemagglutinin (HA)-specific TCR (clone 1 TCR) derived from a mouse that expressed HA as a self-Ag in the insulin-producing β cells of the pancreatic islets (InsHA) mice. Upon transfer of clone 1 TCR CD8+ T cells into InsHA mice, very few cells were activated by cross-presented HA, indicating that the cells were retained in InsHA mice because they ignored the presence of Ag, and not because they were functionally inactivated by anergy or tuning. Upon transfer into recipient mice in which HA is expressed at high concentrations as a tumor-associated Ag in spontaneously arising insulinomas (RIP-Tag2-HA mice), a high proportion of clone 1 cells were activated when they encountered cross-presented tumor Ag in the pancreatic lymph nodes. However, the activated cells exhibited very weak effector function and were soon tolerized. The few activated cells that did migrate to the tumor were unable to delay tumor progression. However, when HA-specific CD4 helper cells were cotransferred with clone 1 cells into RIP-Tag2-HA recipients and the mice were vaccinated with influenza, clone 1 cells were found to exert a significant level of effector function and could delay tumor growth. This tumor model should prove of great value in identifying protocols that can optimize the function of low avidity tumor-specific T cells.

Adjuvants targeting innate and adaptive immunity synergize to enhance tumor immunotherapy

Because of mechanisms of self-tolerance, many tumor-specific CD8 T cells exhibit low avidity for tumor antigens and would benefit from strategies that enhance their numbers and effector function. Here we demonstrate that the combined use of two different types of immune adjuvants, one that directly targets the CD8 cell, IL-2/anti-IL-2 mAb complexes, and one that targets the innate immune system, poly(I:C), can achieve this goal. Provision of IL-2/mAb complexes was found to enhance the activation and effector function of low-avidity tumor-specific T cells, yet this was insufficient to achieve tumor eradication. The addition of poly(I:C) further increased the accumulation of granzyme B-expressing effectors within the tumor and resulted in tumor eradication. This strategy presents many of the benefits of whole-body irradiation, including the provision of high levels of homeostatic cytokines, enhanced expansion of effector cells relative to regulatory T cells, and provision of inflammatory cytokines, and is therefore likely to serve as a strategy for both tumor vaccines and adoptive immunotherapy of cancer.

PTPN22 Controls the Germinal Center by Influencing the Numbers and Activity of T Follicular Helper Cells

A single nucleotide polymorphism in PTPN22 (R620W), which encodes the Lyp tyrosine phosphatase, has been linked to a number of autoimmune diseases including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Studies in PTPN22 knockout (KO) mice and in mice expressing the mouse homolog of the pro-autoimmune allele, PEPR619W, have reported increased germinal center activity and enhanced Ab production. In this article, we present findings that explain the basis for increased germinal center activity in PTPN22 mutant mice. As compared with their wild type equivalents, T follicular helper cells from PTPN22 KO mice proliferate and accumulate to a greater extent, and exhibit enhanced production of IL-21. The follicular regulatory T cells in PTPN22 KO mice do not expand to effectively regulate these T follicular helper cells, resulting in an increase in B cell numbers and Ab production. This is evident in the KBxN mouse model of arthritis in which PTPN22 deficiency results in increased severity of disease. Our findings demonstrate the importance of cell type–specific PTPN22 activity on regulation of Ab production.

Functional differences between low- and high-affinity CD8+ T cells in the tumor environment

Weak T-cell antigen receptor (TCR)-ligand interactions are sufficient to activate naïve CD8+ T cells, but generally do not result in tumor eradication. How differences in TCR affinity affect the regulation of T-cell function in an immunosuppressive tumor environment has not been investigated. We have examined the functional differences of high- vs. low-affinity CD8+ T cells and we observed that infiltration, accumulation, survival and cytotoxicity within the tumor are severely impacted by the strength of TCR-ligand interactions. In addition, high-affinity CD8+ T cells were found to exhibit lower expression of inhibitory molecules including PD-1, LAG-3 and NKG2A, thus being less susceptible to suppressive mechanisms. Interferon γ and autocrine interleukin-2 were both found to influence the level of expression of these molecules. Interestingly, although high-affinity CD8+ T cells were superior to low-affinity CD8+ T cells in their ability to effect tumor eradication, they could be further improved by the presence of tumor specific CD4+ T cells. These findings illustrate the importance of both TCR affinity and tumor-specific CD4 help in tumor immunotherapy.

Contribution of TCR Signaling Strength to CD8+ T Cell Peripheral Tolerance Mechanisms

Peripheral tolerance mechanisms are in place to prevent T cells from mediating aberrant immune responses directed against self and environmental Ags. Mechanisms involved in the induction of peripheral tolerance include T cell–intrinsic pathways, such as anergy or deletion, or exogenous tolerance mediated by regulatory T cells. We have previously shown that the density of peptide-MHC class I recognized by the TCR determines whether CD8+ T cells undergo anergy or deletion. Specifically, using a TCR-transgenic CD8+ T cell model, we demonstrated that persistent peripheral exposure to low- or high-dose peptides in the absence of inflammatory signals resulted in clonal deletion or anergy of the T cell, respectively. In this study, by altering the affinity of the peptide-MHC tolerogen for TCR, we have confirmed that this mechanism is dependent on the level of TCR signaling that the CD8+ T cell receives. Using altered peptide ligands (APLs) displaying high TCR affinities, we show that increasing the TCR signaling favors anergy induction. Conversely, using APLs displaying a decreased TCR affinity tilted our system in the direction of deletional tolerance. We demonstrate how differential peripheral CD8+ T cell tolerance mechanisms are controlled by both the potency and density of MHC class I–peptide tolerogen.

Tissue-Resident Memory CD8+ T Cells Can Be Deleted by Soluble, but Not Cross-Presented Antigen

Under noninflammatory conditions, both naive and central memory CD8 T cells can be eliminated in the periphery with either soluble peptide or cross-presented Ag. Here, we assess the tolerance susceptibility of tissue-resident memory CD8 T cells in mice to these two forms of tolerogen. Soluble peptide specifically eliminated the majority of memory CD8 cells present in both lymphoid and extralymphoid tissues including lung and liver, but was unable to reduce the number present in the CNS. In contrast, systemic cross-presentation of Ag by dendritic cells resulted in successful elimination of memory cells only from the spleen, with no significant reduction in the numbers of tissue-resident memory cells in the lung. The fact that tissue-resident memory cells were unable to access cross-presented Ag suggests that either the memory cells in the lung do not freely circulate out of the tissue, or that they circulate through a region in the spleen devoid of cross-presented Ag. Thus, although tissue-resident memory cells are highly susceptible to tolerance induction, both the form of tolerogen and location of the T cells can determine their accessibility to tolerogen and the degree to which they are successfully deleted from specific tissues.

PTPN22 contributes to exhaustion of T lymphocytes during chronic viral infection

Significance Some viruses, including lymphocytic choriomeningitis virus clone 13, shut down the ability of CD4 T lymphocytes to produce IL-2, a cytokine required for the survival and function of T lymphocytes. This shutdown contributes to exhaustion of CD4 and CD8 T lymphocytes and chronic viral infection of the host. The underlying mechanism responsible for the loss of cytokine production by CD4 T cells remains poorly understood. We demonstrate that the expression of a protein tyrosine phosphatase, PTPN22, contributes to chronic viral infection. PTPN22 increases the production of IFN-β following infection, resulting in increased expression of the cAMP response element modulator (CREM) in CD4 T lymphocytes. CREM prevents production of IL-2, thereby contributing to T-cell exhaustion and chronic viral infection. The protein encoded by the autoimmune-associated protein tyrosine phosphatase nonreceptor type 22 gene, PTPN22, has wide-ranging effects in immune cells including suppression of T-cell receptor signaling and promoting efficient production of type I interferons (IFN-I) by myeloid cells. Here we show that mice deficient in PTPN22 resist chronic viral infection with lymphocytic choriomeningitis virus clone 13 (LCMV cl13). The numbers and function of viral-specific CD4 T lymphocytes is greatly enhanced, whereas expression of the IFNβ-induced IL-2 repressor, cAMP-responsive element modulator (CREM) is reduced. Reduction of CREM expression in wild-type CD4 T lymphocytes prevents the loss of IL-2 production by CD4 T lymphocytes during infection with LCMV cl13. These findings implicate the IFNβ/CREM/IL-2 axis in regulating T-lymphocyte function during chronic viral infection.

CRISPR-Cas9 mediated modification of the NOD mouse genome with Ptpn22R619W mutation increases autoimmune diabetes

An allelic variant of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), PTPN22R620W, is strongly associated with type 1 diabetes (T1D) in humans and increases the risk of T1D by two- to fourfold. The NOD mouse is a spontaneous T1D model that shares with humans many genetic pathways contributing to T1D. We hypothesized that the introduction of the murine orthologous Ptpn22R619W mutation to the NOD genome would enhance the spontaneous development of T1D. We microinjected CRISPR-Cas9 and a homology-directed repair template into NOD single-cell zygotes to introduce the Ptpn22R619W mutation to its endogenous locus. The resulting Ptpn22R619W mice showed increased insulin autoantibodies and earlier onset and higher penetrance of T1D. This is the first report demonstrating enhanced T1D in a mouse modeling human PTPN22R620W and the utility of CRISPR-Cas9 for direct genetic alternation of NOD mice.

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non‐obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well‐characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus‐like particles produced by co‐expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes‐resistant mice developed anti‐Env autoantibodies that increase in titer as disease progresses. A lentiviral‐based RNA interference knockdown of Gag revealed that Gag contributes to the MV‐induced T‐cell response, whose diabetogenic function can be demonstrated via cell‐transfer into immune‐deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet‐derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes‐resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti‐retroviral responses to trigger autoimmunity.