Immunotherapeutic implications of negative regulation by protein tyrosine phosphatases in T cells: the emerging cases of PTP1B and TCPTP.

In the context of inflammation, T cell activation occurs by the concerted signals of the T cell receptor (TCR), co-stimulatory receptors ligation, and a pro-inflammatory cytokine microenvironment. Fine-tuning these signals is crucial to maintain T cell homeostasis and prevent self-reactivity while offering protection against infectious diseases and cancer. Recent developments in understanding the complex crosstalk between the molecular events controlling T cell activation and the balancing regulatory cues offer novel approaches for the development of T cell-based immunotherapies. Among the complex regulatory processes, the balance between protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTPs) controls the transcriptional and metabolic programs that determine T cell function, fate decision, and activation. In those, PTPs are de facto regulators of signaling in T cells acting for the most part as negative regulators of the canonical TCR pathway, costimulatory molecules such as CD28, and cytokine signaling. In this review, we examine the function of two close PTP homologs, PTP1B (PTPN1) and T-cell PTP (TCPTP; PTPN2), which have been recently identified as promising candidates for novel T-cell immunotherapeutic approaches. Herein, we focus on recent studies that examine the known contributions of these PTPs to T-cell development, homeostasis, and T-cell-mediated immunity. Additionally, we describe the signaling networks that underscored the ability of TCPTP and PTP1B, either individually and notably in combination, to attenuate TCR and JAK/STAT signals affecting T cell responses. Thus, we anticipate that uncovering the role of these two PTPs in T-cell biology may lead to new treatment strategies in the field of cancer immunotherapy. This review concludes by exploring the impacts and risks that pharmacological inhibition of these PTP enzymes offers as a therapeutic approach in T-cell-based immunotherapies.

The induction of SHP-1 degradation by TAOK3 ensures the responsiveness of T cells to TCR stimulation.

Thousand-and-one-amino acid kinase 3 (TAOK3) is a serine and threonine kinase that belongs to the STE-20 family of kinases. Its absence reduces T cell receptor (TCR) signaling and increases the interaction of the tyrosine phosphatase SHP-1, a major negative regulator of proximal TCR signaling, with the kinase LCK, a component of the core TCR signaling complex. Here, we used mouse models and human cell lines to investigate the mechanism by which TAOK3 limits the interaction of SHP-1 with LCK. The loss of TAOK3 decreased the survival of naïve CD4+ T cells by dampening the transmission of tonic and ligand-dependent TCR signaling. In mouse T cells, Taok3 promoted the secretion of interleukin-2 (IL-2) in response to TCR activation in a manner that depended on Taok3 gene dosage and on Taok3 kinase activity. TCR desensitization in Taok3-/- T cells was caused by an increased abundance of Shp-1, and pharmacological inhibition of Shp-1 rescued the activation potential of these T cells. TAOK3 phosphorylated threonine-394 in the phosphatase domain of SHP-1, which promoted its ubiquitylation and proteasomal degradation. The loss of TAOK3 had no effect on the abundance of SHP-2, which lacks a residue corresponding to SHP-1 threonine-394. Modulation of SHP-1 abundance by TAOK3 thus serves as a rheostat for TCR signaling and determines the activation threshold of T lymphocytes.

Loss of T-cell protein tyrosine phosphatase in the intestinal epithelium promotes local inflammation by increasing colonic stem cell proliferation.

T-cell protein tyrosine phosphatase (TC-PTP) has a critical role in the development of the immune system and has been identified as a negative regulator of inflammation. Single-nucleotide polymorphisms in the TC-PTP locus have been associated with increased susceptibility to inflammatory bowel diseases (IBDs) in patients. To further understand how TC-PTP is related to IBDs, we investigated the role of TC-PTP in maintaining the intestinal epithelial barrier using an in vivo genetic approach. Intestinal epithelial cell (IEC)-specific deletion of TC-PTP was achieved in a mouse model at steady state and in the context of dextran sulphate sodium (DSS)-induced colitis. Knockout (KO) of TC-PTP in IECs did not result in an altered intestinal barrier. However, upon DSS treatment, IEC-specific TC-PTP KO mice displayed a more severe colitis phenotype with a corresponding increase in the immune response and inflammatory cytokine profile. The absence of TC-PTP caused an altered turnover of IECs, which is further explained by the role of the tyrosine phosphatase in colonic stem cell (CoSC) proliferation. Our results suggest a novel role for TC-PTP in regulating the homeostasis of CoSC proliferation. This supports the protective function of TC-PTP against IBDs, independently of its previously demonstrated role in intestinal immunity.

Mining the Complex Family of Protein Tyrosine Phosphatases for Checkpoint Regulators in Immunity.

The family of protein tyrosine phosphatases (PTPs) includes 107 genes in humans that are diverse in their structures and expression profiles. The majority are present in immune cells and play various roles in either inhibiting or promoting the duration and amplitude of signaling cascades. Several PTPs, including TC-PTP (PTPN2) and SHP-1 (PTPN6), have been recognized as being crucial for maintaining proper immune response and self-tolerance, and have gained recognition as true immune system checkpoint modulators. This chapter details the most recent literature on PTPs and immunity by examining their known functions in regulating signaling from either established checkpoint inhibitors or by their intrinsic properties, as modulators of the immune response. Notably, we review PTP regulatory properties in macrophages, antigen-presenting dendritic cells, and T cells. Overall, we present the PTP gene family as a remarkable source of novel checkpoint inhibitors wherein lies a great number of new targets for immunotherapies.

SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin.

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.

A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri.

The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

X-linked lymphoproliferative syndromes and related autosomal recessive disorders.

PURPOSE OF REVIEW: X-linked lymphoproliferative (XLP) syndromes and related autosomal disorders are severe primary immune deficiencies triggered by infection with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis. Recent findings reviewed herein provided key new insights into the genetic and immunological basis of these diseases. They also improved our comprehension of the immunological mechanisms controlling EBV infection. RECENT FINDINGS: Mutations of an X-linked gene, SH2D1A, which encodes the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), are responsible for most cases of XLP disorders. More recently, other genetic causes for XLP syndromes and autosomal recessive variants of this disease were elucidated. Mutations in genes such as XIAP, ITK, and CD27 were identified. The clinical manifestations and immunological defects seen in these patients were characterized. SUMMARY: The similarities and differences in immunological defects and clinical manifestations between XLP syndromes and related autosomal recessive disorders enabled important new insights into the pathogenesis of these diseases. They also helped our comprehension of the mechanisms implicated in the control of EBV infection. They suggested that CD8+ T cells, natural killer (NK) cells, and NKT cells are critically involved.

The adaptor SAP controls NK cell activation by regulating the enzymes Vav-1 and SHIP-1 and by enhancing conjugates with target cells.

The adaptor SAP, mutated in X-linked lymphoproliferative disease, has critical roles in multiple immune cell types. Among these, SAP is essential for the ability of natural killer (NK) cells to eliminate abnormal hematopoietic cells. Herein, we elucidated the molecular and cellular bases of this activity. SAP enhanced NK cell responsiveness by a dual molecular mechanism. It coupled SLAM family receptors to the kinase Fyn, which triggered the exchange factor Vav-1 and augmented NK cell activation. SAP also prevented the inhibitory function of SLAM family receptors. This effect was Fyn independent and correlated with uncoupling of SLAM family receptors from the lipid phosphatase SHIP-1. Both mechanisms cooperated to enable conjugate formation with target cells and to stimulate cytotoxicity and cytokine secretion by NK cells. These data showed that SAP secures NK cell activation by a dichotomous molecular mechanism, which is required for conjugate formation. These findings may have implications for the role of SAP in other immune cell types.

Importance and mechanism of 'switch' function of SAP family adapters.

The signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) family of adapters includes SAP, Ewing's sarcoma-associated transcript-2 (EAT-2), and EAT-2-related transducer (ERT). These Src homology-2 (SH2) domain-only molecules play critical roles in immune regulation. The prototype of the SAP family, SAP, is mutated in X-linked lymphoproliferative disease in humans. Moreover, genetically engineered mice lacking one or more SAP family members have defects in multiple immune cell types including T cells, natural killer (NK) cells, NKT cells, and B cells. Accumulating data show that SAP family adapters regulate immunity by influencing the functions of SLAM family receptors, through two distinct but cooperative mechanisms. First, SAP family adapters couple SLAM family receptors to active biochemical signals, which promote immune cell functions. Second, SAP family adapters interfere with the intrinsic ability of SLAM family receptors to trigger inhibitory signals, which could be mediated via molecules such as SH2 domain-containing 5'-inositol phosphatase-1. The latter effect of SAP family adapters does not seem to be because of direct blocking of inhibitory effector binding to SLAM family receptors. Rather, it appears to implicate alternative mechanisms such as functional competition, trans-regulation, or steric hindrance. In the absence of SAP family adapters, the inhibitory signals mediated by SLAM family receptors suppress critical activating receptors, explaining in part the pronounced phenotypes seen in SAP family adapter-deficient humans and mice. Thus, SAP family adapters are molecular switches that regulate immunity as a result of their capacity to control the type of signals and functions emanating from SLAM family receptors.