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.
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, PEP(R619W), 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.
A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.
Nonobese diabetic (NOD) mice congenic for C57BL/10 (B10)-derived genes in the Idd9 region of chromosome 4 are highly protected from type 1 diabetes (T1D). Idd9 has been divided into three protective subregions (Idd9.1, 9.2, and 9.3), each of which partially prevents disease. In this study we have fine-mapped the Idd9.1 and Idd9.2 regions, revealing further genetic complexity with at least two additional subregions contributing to protection from T1D. Using the NOD sequence from bacterial artificial chromosome clones of the Idd9.1 and Idd9.2 regions as well as whole-genome sequence data recently made available, sequence polymorphisms within the regions highlight a high degree of polymorphism between the NOD and B10 strains in the Idd9 regions. Among numerous candidate genes are several with immunological importance. The Idd9.1 region has been separated into Idd9.1 and Idd9.4, with Lck remaining a candidate gene within Idd9.1. One of the Idd9.2 regions contains the candidate genes Masp2 (encoding mannan-binding lectin serine peptidase 2) and Mtor (encoding mammalian target of rapamycin). From mRNA expression analyses, we have also identified several other differentially expressed candidate genes within the Idd9.1 and Idd9.2 regions. These findings highlight that multiple, relatively small genetic effects combine and interact to produce significant changes in immune tolerance and diabetes onset.
In the NOD mouse model of type 1 diabetes, insulin-dependent diabetes (Idd) loci control the development of insulitis and diabetes. Independently, protective alleles of Idd3/Il2 or Idd5 are able to partially protect congenic NOD mice from insulitis and diabetes, and to partially tolerize islet-specific CD8(+) T cells. However, when the two regions are combined, mice are almost completely protected, strongly suggesting the existence of genetic interactions between the two loci. Idd5 contains at least three protective subregions/causative gene candidates, Idd5.1/Ctla4, Idd5.2/Slc11a1, and Idd5.3/Acadl, yet it is unknown which of them interacts with Idd3/Il2. Through the use of a series of novel congenic strains containing the Idd3/Il2 region and different combinations of Idd5 subregion(s), we defined these genetic interactions. The combination of Idd3/Il2 and Idd5.3/Acadl was able to provide nearly complete protection from type 1 diabetes, but all three Idd5 subregions were required to protect from insulitis and fully restore self-tolerance. By backcrossing a Slc11a1 knockout allele onto the NOD genetic background, we have demonstrated that Slc11a1 is responsible for the diabetes protection resulting from Idd5.2. We also used Slc11a1 knockout-SCID and Idd5.2-SCID mice to show that both loss-of-function alleles provide protection from insulitis when expressed on the SCID host alone. These results lend further support to the hypothesis that Slc11a1 is Idd5.2.
Type 1 diabetes genes within the interleukin (IL)-2, cytotoxic T-lymphocyte--associated protein 4 (CTLA-4), and natural resistance-associated macrophage protein (NRAMP1) pathways influence development of autoimmune diabetes in humans and NOD mice. In NOD mice, when present together, protective alleles encoding IL-2, Idd3 candidate gene, CTLA-4, NRAMP1, and acetyl-coenzyme A dehydrogenase, long-chain (ACADL) (candidate genes for the Idd5.1, Idd5.2, and Idd5.3 subregions) provide nearly complete diabetes protection. To define where the protective alleles of Idd3 and the Idd5 subregions must be present to protect from diabetes and tolerize islet-specific CD8(+) T cells, SCID mice were reconstituted so that the host and lymphocytes expressed various combinations of protective and susceptibility alleles at Idd3 and Idd5. Although protective Idd3 alleles in the lymphocytes and protective Idd5 alleles in the SCID host contributed most significantly to CD8 tolerance, both were required together in both lymphocyte and nonlymphocyte cells to recapitulate the potent diabetes protection observed in intact Idd3/5 mice. We conclude that genetic regions involved in autoimmune disease are not restricted in their influence to individual cell types. Even a single protective gene product, such as IL-2, must be expressed in both the lymphocytes and dendritic cells to exert its full extent of disease protection. These studies highlight the pleiotropic effects of genes that determine autoimmune disease susceptibility.
CD4 help for CD8(+) T lymphocytes prevents tolerance and promotes the survival of effector and memory CD8(+) T cells. Here, we describe additional helper functions that require CD4(+) T cells within the tumor environment. CD8(+) T-cell recruitment, proliferation, and effector function within the tumor were greatly enhanced by tumor-specific CD4(+) T cells. Recruitment of CD8(+) T cells was accelerated by IFN-?-dependent production of chemokines. Production of interleukin-2 by tumor resident CD4(+) T cells enhanced CD8(+) T-cell proliferation and upregulated expression of granzyme B. These results highlight a novel role for tumor-specific CD4(+) T cells in promoting CD8(+) T-cell recruitment and cytolytic function, two previously unappreciated aspects of tumor-specific CD4 help.
Multiple type 1 diabetes susceptibility genes have now been identified in both humans and mice, yet mechanistic understanding of how they impact disease pathogenesis is still minimal. We have sought to dissect the cellular basis for how the highly protective mouse Idd9 region limits the expansion of autoreactive CD8(+) T-cells, a key cell type in destruction of the islets.
In humans and NOD mice, defects in immune tolerance result in the spontaneous development of type-1-diabetes. Recent studies have ascribed a breakdown in tolerance to dysfunction in regulatory T cells that is secondary to reduced IL-2 production by T cells having the NOD diabetes susceptibility region insulin-dependent diabetes 3 (Idd3). In this study, we demonstrate a peripheral tolerance defect in the dendritic cells of NOD mice that is independent of regulatory T cells. NOD CD8 T cells specific for islet Ags fail to undergo deletion in the pancreatic lymph nodes. Deletion was promoted by expression of the protective alleles of both Idd3 (Il2) and Idd5 in dendritic cells. We further identify a second tolerance defect that involves endogenous CD4 T cell expression of the disease-promoting NOD alleles of these genetic regions. Pervasive insulitis can be reduced by expression of the Idd3 and Idd5 protective alleles by either the Ag-presenting cell or lymphocytes.
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 ? 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.
Vemurafenib is an orally available small molecule that targets constitutively activated BRAFV600E, an integral part of the MAPK pathway involved in melanomagenesis. We examined the effects of vemurafenib on cytokine production and antitumor response in a BRAF wild-type (WT) non-tumor-bearing murine model and a BRAF WT murine insulinoma system to determine its effect on immune function during immunotherapy. We demonstrate no significant effect from vemurafenib on CD4+ and CD8+ T-cell cytokine production or on a T-cell-mediated antitumor response. Our data demonstrate that vemurafenib does not significantly affect BRAF WT targets, suggesting that there may be a role for combining vemurafenib treatment with T-cell-directed immunotherapy.
PTPN22 encodes a tyrosine phosphatase that inhibits Src-family kinases responsible for Ag receptor signaling in lymphocytes and is strongly linked with susceptibility to a number of autoimmune diseases. As strength of TCR signal is critical to the thymic selection of regulatory T cells (Tregs), we examined the effect of murine PTPN22 deficiency on Treg development and function. In the thymus, numbers of pre-Tregs and Tregs increased inversely with the level of PTPN22. This increase in Tregs persisted in the periphery and could play a key part in the reduced severity observed in the PTPN22-deficient mice of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This could explain the lack of association of certain autoimmune conditions with PTPN22 risk alleles.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.