Multifunctional roles of the autoimmune disease-associated tyrosine phosphatase PTPN22 inregulating T cell homeostasis

The non-receptor tyrosine phosphatase PTPN22 has a vital function in inhibiting antigen-receptor signaling in T cells, while polymorphisms in the PTPN22 gene are important risk alleles in human autoimmune diseases. We recently reported that a key physiological function of PTPN22 was to prevent naïve T cell activation and effector cell responses in response to low affinity antigens. PTPN22 also has a more general role in limiting T cell receptor-induced proliferation. Here we present new data emphasizing this dual function for PTPN22 in T cells. Furthermore, we show that T cell activation modulates the expression of PTPN22 and additional inhibitory phosphatases. We discuss the implication of these findings for our understanding of the roles of PTPN22 in regulating T cell responses and in autoimmunity

Sensory adaptation in naive peripheral CD4 T cells

T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck). Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen. Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness

Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation

Elucidating the mechanisms that controlled T cell activation requires visualization of the spatial organization of multiple proteins on the submicron scale. Here, we use stoichiometrically accurate, multiplexed, singlemolecule super-resolution microscopy (DNA-PAINT) to image the nanoscale spatial architecture of the primary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane association, PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we find that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and are re-recruited at later time points. Unexpectedly, these clusters do not co-localize with PAG at the membrane but instead provide a ready pool of monomers to downregulate signaling. By generating CRISPR-Cas9 knockout T cells, our data also identify that a major risk factor for autoimmune diseases, the protein tyrosine phosphatase non-receptor type 22 (PTPN22) locus, is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population

Deletion of PTPN22 improves effector and memory CD8+ T cell responses to tumors

Adoptive T cell therapy (ACT) has been established as an efficacious methodology for the treatment of cancer. Identifying targets to enhance the antigen recognition, functional capacity and longevity of T cells has the potential to broaden the applicability of these approaches in the clinic. We previously reported that targeting expression of phosphotyrosine phosphatase, non-receptor type (PTPN) 22 in effector CD8+ T cells enhances the efficacy of ACT for tumor clearance in mice. In the current work, we demonstrate that, upon ACT, PTPN22-deficient effector CD8+ T cells afford greater protection against tumors expressing very low affinity antigen, but do not survive long-term in vivo. Persistence of CD8+ T cells following tumor clearance is improved by ACT of memory phenotype cells that have a distinct metabolic phenotype as compared to effector T cells. Importantly, PTPN22-deficient T cells have comparable capacity to form long-lived memory cells in vivo but enhanced anti-tumor activity in vivo and effector responses ex vivo. These findings provide key insight into the regulation of effector and memory T cell responses in vivo, and indicate that PTPN22 is a rationale target to improve ACT for cancer

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

Background Adoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long term protection due to T cell exhaustion induced by chronic TCR stimulation in the tumor microenvironment. One benefit of ACT, however, is that it allows for cellular manipulations, such as deletion of the phosphotyrosine phosphatase non-receptor type 22 (PTPN22), which improves CD8+ T cell anti-tumor efficacy in ACT. We tested whether Ptpn22KO cytolytic T cells (CTL) were also more effective than Ptpn22WT CTL in controlling tumors in scenarios that favor T cell exhaustion. Methods Tumor control by Ptpn22WT and Ptpn22KO CTL was assessed following adoptive transfer of low numbers of CTL to mice with subcutaneously implanted MC38 tumors. Tumor infiltrating lymphocytes were isolated for analysis of effector functions. An in vitro assay was established to compare CTL function in response to acute and chronic re-stimulation with antigen-pulsed tumor cells. The expression of effector and exhaustion-associated proteins by Ptpn22WT and Ptpn22KO T cells was followed over time in vitro and in vivo using the ID8 tumor model. Finally, the effect of PD-1 and TIM-3 blockade on Ptpn22KO CTL tumor control was assessed using monoclonal antibodies and CRISPR/Cas9-mediated knockout. Results Despite having improved effector function at the time of transfer, Ptpn22KO CTL became more exhausted than Ptpn22WT CTL, characterized by more rapid loss of effector functions, and earlier and higher expression of inhibitory receptors (IRs), particularly the terminal exhaustion marker TIM-3. TIM-3 expression, under the control of the transcription factor NFIL3, was induced by IL-2 signaling which was enhanced in Ptpn22KO cells. Anti-tumor responses of Ptpn22KO CTL were improved following PD-1 blockade in vivo, yet knockout or antibody-mediated blockade of TIM-3 did not improve but further impaired tumor control, indicating TIM-3 signaling itself did not drive the diminished function seen in Ptpn22KO CTL. Conclusions This study questions whether TIM-3 plays a role as an IR and highlights that genetic manipulation of T cells for ACT needs to balance short term augmented effector function against the risk of T cell exhaustion in order to achieve longer term protection. What is already known on this topic • T cell exhaustion in the tumor microenvironment is a major factor limiting the potential success of adoptive cell therapy (ACT) in the treatment of solid tumors. • Deletion of the phosphatase PTPN22 in CD8+ T cells improves their response to tumors, but it is not known whether this influences development of exhaustion. What this study adds • Under conditions which promote exhaustion, CTL lacking PTPN22 exhaust more rapidly than WT cells, despite displaying enhanced effector function in their initial response to antigen. • Ptpn22KO CTL express high levels of the inhibitory receptor TIM-3, but TIM-3 signaling does not directly contribute to Ptpn22KO CTL dysfunction. • Ptpn22KO T cells are more responsive to IL-2 through JAK-STAT signaling, which induces TIM-3 expression via the transcription factor NFIL3. How this study might affect research, practice or policy • Strategies aimed at augmenting T cell effector function for ACT should balance improved responses against an increased risk of T cell exhaustion

Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor

Producción CientíficaThe earliest molecular events in T-cell recognition have not yet been fully described, and the initial T-cell receptor (TCR)-triggering mechanism remains a subject of controversy. Here, using total internal reflection/Forster resonance energy transfer microscopy, we observe a two-stage interaction between TCR, CD8 and major histocompatibility complex (MHC)-peptide. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ–CD8 interactions require CD8–MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR-triggering event is induced by free Lck. The early signalling events that trigger initial T-cell receptor signalling are not clearly defined. Here the authors show that this occurs in two stages, the first between the CD8 coreceptor and CD3 requiring Lck association to CD8, while the second interaction requires binding of major histocompatibility molecules

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the α3 domain of MHC I molecules

THE α1 and α2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the α3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CDS. CDS is important in the selection of T cells as anti-CDS antibody injected into perinatal mice interfers with this process. We previously used a hybrid class I molecule with the α1/α2 domains from L^d and the α3 domain from Q7^b and showed that this molecule binds an L^d-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-L^d cytotoxic T lymphocytes. In addition, positive selection of virus-specific L^d-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the α1/α2 domains of class I, the α3 domain plays an important part in both positive and negative selection of antigen-specific cells

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

Background: Adoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long-term protection due to T cell exhaustion induced by chronic TCR stimulation in the tumor microenvironment. One benefit of ACT, however, is that it allows for cellular manipulations, such as deletion of the phosphotyrosine phosphatase non-receptor type 22 (PTPN22), which improves CD8+ T cell antitumor efficacy in ACT. We tested whether Ptpn22KO cytolytic T cells (CTLs) were also more effective than Ptpn22WT CTL in controlling tumors in scenarios that favor T cell exhaustion. Methods: Tumor control by Ptpn22WT and Ptpn22KO CTL was assessed following adoptive transfer of low numbers of CTL to mice with subcutaneously implanted MC38 tumors. Tumor infiltrating lymphocytes were isolated for analysis of effector functions. An in vitro assay was established to compare CTL function in response to acute and chronic restimulation with antigen-pulsed tumor cells. The expression of effector and exhaustion-associated proteins by Ptpn22WT and Ptpn22KO T cells was followed over time in vitro and in vivo using the ID8 tumor model. Finally, the effect of PD-1 and TIM-3 blockade on Ptpn22KO CTL tumor control was assessed using monoclonal antibodies and CRISPR/Cas9-mediated knockout. Results: Despite having improved effector function at the time of transfer, Ptpn22KO CTL became more exhausted than Ptpn22WT CTL, characterized by more rapid loss of effector functions, and earlier and higher expression of inhibitory receptors (IRs), particularly the terminal exhaustion marker TIM-3. TIM-3 expression, under the control of the transcription factor NFIL3, was induced by IL-2 signaling which was enhanced in Ptpn22KO cells. Antitumor responses of Ptpn22KO CTL were improved following PD-1 blockade in vivo, yet knockout or antibody-mediated blockade of TIM-3 did not improve but further impaired tumor control, indicating TIM-3 signaling itself did not drive the diminished function seen in Ptpn22KO CTL. Conclusions: This study questions whether TIM-3 plays a role as an IR and highlights that genetic manipulation of T cells for ACT needs to balance short-term augmented effector function against the risk of T cell exhaustion in order to achieve longer-term protection

Centrosome docking at the immunological synapse is controlled by Lck signaling.

Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor-activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane

Localized Populations of CD8low/− MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium

CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8+. Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer+CD8low/− cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer+CD20− cells inside B cell follicles and that tetramer+ cells did not stain with γδ-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer+CD8low/− cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium