Deep phosphotyrosine characterisation of primary murine T cells using broad spectrum optimisation of selective triggering.

Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimisation of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation. Using 1 mg of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2000 unique pY sites compared to about 300 unique pY sites in non-BOOST control samples. We show that although replicate variation increases when using the BOOST method, BOOST does not jeopardise quantitative precision or the ability to determine statistical significance for peptides measured in triplicate. Many pY previously uncharacterised sites on important T cell signalling proteins are quantified using BOOST, and we identify new TCR responsive pY sites observable only with BOOST. Finally, we determine that the phase-spectrum deconvolution method on Orbitrap instruments can impair pY quantitation in BOOST experiments.

Cerebral venous thrombosis presented with symmetrical crescent-shaped intracranial hemorrhage in alcoholic liver disease: Case reports.

RATIONALE: Cerebral venous thrombosis (CVT) is a relatively uncommon but fatal disease. It can be caused by a variety of hereditary or acquired thrombotic diseases. Initial presentation with intracranial hemorrhage (ICH) in CVT is rare but can further complicate the therapeutic measures and prognosis. Cases of CVT presented with ICH in patients with alcoholic liver disease (ALD) have not been described in the literature, and it might be related with hemostatic abnormalities in ALD patients. PATIENT CONCERNS: We report 2 cases of men admitted to our hospital who were diagnosed with CVT but initially presented with symmetrical crescent-shaped ICH; both of them were ALD patients. DIAGNOSES: Cerebral imaging revealed extended CVT in both cases. The first case was a 64-year-old man with ALD deteriorated with unconsciousness and convulsions; computed tomography showed symmetrical crescent-shaped ICH in the right temporal lobe, and magnetic resonance venography revealed CVT. Another 50-year-old man with ALD complained about dizziness and weakness of his right limbs; computed tomography revealed symmetrical crescent-shaped ICH in bilateral parietal and occipital lobes, and magnetic resonance venography revealed CVT. INTERVENTIONS: The first patient was referred to the endovascular thrombectomy. Both of them were treated with anticoagulation treatment. OUTCOMES: Favorable outcomes were observed in both patients. LESSONS: Symmetrical or multiple crescent-shaped ICH requires a high suspicion in the diagnosis of CVT; even with hemorrhage, it is still important to initiate anticoagulation therapy promptly. The crescent-shaped ICH might be a new sign for CVT, and further studies are needed in the underlying mechanisms of ALD and potential thrombophilia.

Case report: Successful treatment of an anti-D2R and DPPX antibody-associated autoimmune encephalitis patient with high-dose methylprednisolone and intravenous immunoglobulin.

Background: Autoimmune encephalitis is a neurological condition caused by abnormal immune responses, manifesting as cognitive impairments, behavioral abnormalities, and seizures. Its diagnosis depends on the detecting neuronal surface antibodies in serum or cerebrospinal fluid. Despite recent advances in understanding, clinical recognition remains challenging, especially with rare antibodies such as anti-dopamine D2 receptor (D2R) and anti-dipeptidyl-peptidase-like protein 6 (DPPX) antibodies. Delayed diagnosis can lead to severe complications. This case presentation emphasizes the diagnostic intricacies and effective treatment of the anti-D2R and DPPX antibody-associated autoimmune encephalitis. Case description: The patient presented with a 3-day history of fatigue and limb soreness followed by a 3-h episode of confusion and limb convulsions. Upon admission to our facility, the initial diagnosis included status epilepticus, aspiration pneumonia, metabolic acidosis, respiratory alkalosis, and suspected encephalitis. Despite receiving antiepileptic, anti-infection, and antivirus therapy, the patient's condition deteriorated. Both computed tomography (CT) scan and magnetic resonance imaging (MRI) of the brain showed no significant abnormalities. No pathogen was identified in the cerebrospinal fluid (CSF). However, further CSF and serum examination revealed positive results of anti-D2R and anti-DPPX antibodies, confirming a diagnosis of anti-D2R and DPPX antibody-associated autoimmune encephalitis. The patient underwent a comprehensive treatment regimen, including high-dose methylprednisolone pulse therapy combined with intravenous immunoglobulin (IVIG), antiviral and anti-infection treatments, and antiepileptic medications. Significant clinical improvement was observed, and by the 18th day of admission, the patient was stable and coherent. Conclusions: The current patient represents the first reported case of double-positive autoimmune encephalitis for anti-D2R and DPPX antibodies, with epilepsy as a prominent feature. High-dose methylprednisolone pulse therapy combined with IVIG has shown significant safety and efficacy in treating anti-D2R and DPPX antibody-positive autoimmune encephalitis-associated epilepsy.

CARD19, a Novel Regulator of the TAK1/NF-κB Pathway in Self-Reactive B Cells.

The caspase recruitment domain family member (CARD)11-Bcl10-Malt1 signalosome controls TGF-ß-activated kinase 1 (TAK1) activation and regulates BCR-induced NF-κB activation. In this study, we discovered that CARD19 interacted with TAK1 and inhibited TAB2-mediated TAK1 ubiquitination and activation. Although CARD19 deficiency in mice did not affect B cell development, it enhanced clonal deletion, receptor editing, and anergy of self-reactive B cells, and it reduced autoantibody production. Mechanistically, CARD19 deficiency increased BCR/TAK1-mediated NF-κB activation, leading to increased expression of transcription factors Egr2/3, as well as the E3 ubiquitin ligases c-Cbl/Cbl-b, which are known inducers of B cell tolerance in self-reactive B cells. RNA sequencing analysis revealed that although CARD19 deficiency did not affect the overall Ag-induced gene expression in naive B cells, it suppressed BCR signaling and increased hyporesponsiveness of self-reactive B cells. As a result, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus. In summary, CARD19 negatively regulates BCR/TAK1-induced NF-κB activation and its deficiency increases Egr2/3 and c-Cbl/Cbl-b expression in self-reactive B cells, thereby enhancing B cell tolerance.

Phenylacetyl glutamine: a novel biomarker for stroke recurrence warning.

BACKGROUND: Stroke is the second leading cause of disease-related death and the third leading cause of disability worldwide. However, how to accurately warn of stroke onset remains extremely challenging. Recently, phenylacetyl glutamine (PAGln) has been implicated in the onset of stroke, but evidences from cohort studies of onset are lacking, especially in patients with first-onset or recurrent. It is necessary to deeply demonstrate the effectiveness of PAGln level on warning stroke onset. METHODS: One hundred fifteen first onset stroke patients, 33 recurrent stroke patients, and 135 non-stroke controls were included in the analysis. Risk factors associated with stroke attacking were evaluated, and plasma PAGln levels were detected via HPLC-MS based method. LASSO regression, Pearson correlation analysis, and univariate analysis were carried out to demonstrate the associations between PAGln levels and risk factors of stroke. Random forest machine learning algorithm was used to build classification models to achieve the distinction of first-onset stroke patients, recurrent stroke patients, and non-stroke controls, and further demonstrate the contribution of PAGln levels in the distinction of stroke onset. RESULTS: The median level of PAGln in the first-onset stroke group, recurrent stroke group, and non-stroke group was 933 ng/mL, 1014 ng/mL, and 556 ng/mL, respectively. No statistical correlation was found between PAGln level and subject's living habits, eating preferences, and concomitant diseases (hypertension, hyperlipidemia, and diabetes). Stroke severity indicators, mainly age and NIHSS score, were found associate with the PAGln levels. Machine learning classification models confirmed that PAGln levels, as the main contributing variable, could be used to distinguish recurrent stroke patients (but not first-onset stroke patients) from non-stroke controls. CONCLUSION: PAGln may be an effective indicator to monitor the recurrence in stroke patients.

Cloned antibodies from patients with HIT provide new clues to HIT pathogenesis.

Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

Exosomes from human urine-derived stem cells carry NRF1 to alleviate bladder fibrosis via regulating miR-301b-3p/TGFßR1 pathway.

Bladder outlet obstruction (BOO) is a common disease that always make the bladder develops from inflammation to fibrosis. This study was to investigate the effect of exosomes from human urine-derived stem cells (hUSCs) on bladder fibrosis after BOO and the underlying mechanism. The BOO mouse model was established by inserting a transurethral catheter, ligation of periurethral wire, and removal of the catheter. Mouse primary bladder smooth muscle cells (BSMCs) were isolated and treated with TGFß1 to mimic the bladder fibrosis model in vitro. Exosomes from hUSCs (hUSC-Exos) were injected into the bladder of BOO mice and added into the culture of TGFß1-induced BSMCs. The associated factors in mouse bladder tissues and BSMCs were detected. It was confirmed that the treatment of hUSC-Exos alleviated mouse bladder fibrosis and down-regulated fibrotic markers (a-SMA and collagen III) in bladder tissues and TGFß1-induced BSMCs. Overexpression of NRF1 in hUSC-Exos further improved the effects of hUSC-Exos on bladder fibrosis both in vivo and in vitro. TGFßR1 was a target of NRF1 and miR-301b-3p, and miR-301b-3p was a target of NRF1. It was next characterized that hUSC-Exos carried NRF1 to up-regulate miR-301B-3p, thereby reducing TGFßR1level. Our results illustrated that hUSC-Exos carried NRF1 to alleviate bladder fibrosis through regulating miR-301b-3p/TGFßR1 pathway.

A lineage-specific requirement for YY1 Polycomb Group protein function in early T cell development.

Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group protein (PcG) with important functions for regulating lymphocyte development and stem cell self-renewal. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that result in histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in the hematopoietic system results in an early T cell developmental blockage at the double negative (DN) 1 stage with reduced Notch1 signaling. There is a lineage-specific requirement for YY1 PcG function. YY1 PcG domain is required for T and B cell development but not necessary for myeloid cells. YY1 functions in early T cell development are multicomponent and involve both PcG-dependent and -independent regulations. Although YY1 promotes early T cell survival through its PcG function, its function to promote the DN1-to-DN2 transition and Notch1 expression and signaling is independent of its PcG function. Our results reveal how a ubiquitously expressed PcG protein mediates lineage-specific and context-specific functions to control early T cell development.

Kras-Deficient T Cells Attenuate Graft-versus-Host Disease but Retain Graft-versus-Leukemia Activity.

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.

Generation of PECAM-1 (CD31) conditional knockout mice.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM-1 functions, we generated mice in which PECAM1, the gene encoding PECAM-1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3' loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT-flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 flox/flox offspring, which expressed PECAM-1 at normal levels and had no overt phenotype. PECAM1 flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY-box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 del/WT ), which were crossbred to generate Sox2Cre; PECAM1 del/del offspring. Sox2Cre; PECAM1 del/del mice recapitulated the phenotype of conventional global PECAM-1 knockout mice. PECAM1 flox/flox mice will be useful for studying distinct roles of PECAM-1 in tissue specific contexts and to gain insights into the roles that PECAM-1 plays in blood and vascular cell function.

CXCR5+PD-1+ follicular helper CD8 T cells control B cell tolerance.

Many autoimmune diseases are characterized by the production of autoantibodies. The current view is that CD4+ T follicular helper (Tfh) cells are the main subset regulating autoreactive B cells. Here we report a CXCR5+PD1+ Tfh subset of CD8+ T cells whose development and function are negatively modulated by Stat5. These CD8+ Tfh cells regulate the germinal center B cell response and control autoantibody production, as deficiency of Stat5 in CD8 T cells leads to an increase of CD8+ Tfh cells, resulting in the breakdown of B cell tolerance and concomitant autoantibody production. CD8+ Tfh cells share similar gene signatures with CD4+ Tfh, and require CD40L/CD40 and TCR/MHCI interactions to deliver help to B cells. Our study thus highlights the diversity of follicular T cell subsets that contribute to the breakdown of B-cell tolerance.

Gab2 and Gab3 Redundantly Suppress Colitis by Modulating Macrophage and CD8+ T-Cell Activation.

Inflammatory Bowel Disease (IBD) is a multi-factorial chronic inflammation of the gastrointestinal tract prognostically linked to CD8+ T-cells, but little is known about their mechanism of activation during initiation of colitis. Here, Grb2-associated binding 2/3 adaptor protein double knockout mice (Gab2/3-/-) were generated. Gab2/3-/- mice, but not single knockout mice, developed spontaneous colitis. To analyze the cellular mechanism, reciprocal bone marrow (BM) transplantation demonstrated a Gab2/3-/- hematopoietic disease-initiating process. Adoptive transfer showed individual roles for macrophages and T-cells in promoting colitis development in vivo. In spontaneous disease, intestinal intraepithelial CD8+ but much fewer CD4+, T-cells from Gab2/3-/- mice with rectal prolapse were more proliferative. To analyze the molecular mechanism, reduced PI3-kinase/Akt/mTORC1 was observed in macrophages and T-cells, with interleukin (IL)-2 stimulated T-cells showing increased pSTAT5. These results illustrate the importance of Gab2/3 collectively in signaling responses required to control macrophage and CD8+ T-cell activation and suppress chronic colitis.

Frontline Science: PECAM-1 (CD31) expression in naïve and memory, but not acutely activated, CD8+ T cells.

Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-ß-mediated suppression of T cell activity. It is well established in CD4+ T cells that PECAM-1 is expressed in naïve recent thymic emigrants, but is down-regulated after acute T cell activation and absent from memory cells. The extent to which PECAM-1 expression is similarly regulated in CD8+ T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8+ T cells, PECAM-1 expression was strongly down-regulated during acute infection but re-expressed to intermediate levels in memory cells. Down-regulation of PECAM-1 expression in CD8+ T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM-1 was also detected on the surface of human activated/memory CD8+ , but not CD4+ T cells. These data demonstrate that PECAM-1 expression is dynamically regulated, albeit differently, in both CD4+ and CD8+ T cells. Furthermore, unlike memory CD4+ T cells, memory CD8+ T cells retain PECAM-1 expression and have the potential to be modulated by this inhibitory receptor.

PLCγ-dependent mTOR signalling controls IL-7-mediated early B cell development.

The precise molecular mechanism underlying the regulation of early B cell lymphopoiesis is unclear. The PLCγ signaling pathway is critical for antigen receptor-mediated lymphocyte activation, but its function in cytokine signaling is unknown. Here we show that PLCγ1/PLCγ2 double deficiency in mice blocks early B cell development at the pre-pro-B cell stage and renders B cell progenitors unresponsive to IL-7. PLCγ pathway inhibition blocks IL-7-induced activation of mTOR, but not Stat5. The PLCγ pathway activates mTOR through the DAG/PKC signaling branch, independent of the conventional Akt/TSC/Rheb signaling axis. Inhibition of PLCγ/PKC-induced mTOR activation impairs IL-7-mediated B cell development. PLCγ1/PLCγ2 double-deficient B cell progenitors have reduced expression of genes related to B cell lineage, IL-7 signaling, and cell cycle. Thus, IL-7 receptor controls early B lymphopoiesis through activation of mTOR via PLCγ/DAG/PKC signaling, not via Akt/Rheb signaling.

Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development.

Pre-T cell receptor (TCR) signaling is required for pre-T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre-TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, that are important to mediate PKC activation and intracellular Ca2+ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR-mediated signaling, development and T-cell activation, but the role of PLCγ1 in pre-TCR signal transduction and pre-T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre-T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre-TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre-T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre-TCR mediated Ca2+ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre-TCR mediated signal transduction and pre-T cell development.

Evaluation of nestin or osterix promoter-driven cre/loxp system in studying the biological functions of murine osteoblastic cells.

OBJECTIVE: To compare Osterix and Nestin-Cre/Loxp system in studying the biological functions of murine osteoblastic cells including primary osteoblasts (OBs) and osteolineage mesenchymal progenitor cells (MPCs). METHODS: We isolated primary osteoblasts (OBs) from neonatal Nestin-cre-R26-loxP-YFP (Nes-OBs) and Osterix-cre-R26-loxP-YFP (Osx-OBs) mice and bone marrow mesenchymal stromal cells (BMMSCs) from the adults (termed as Nes-BMMSCs and Osx-BMMSCs). Then we detected the percentage of YFP(+) subpopulation in Nes/Osx-OBs and the percentage of CD45(-)YFP(+) progenitor population in Nes/Osx-BMMSCs and sorted them out (termed as Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs) by using the sorting machine. We also analyzed the expression of surface antigens on Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs by Flow cytometry. PDGF-BB induced proliferation of Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs was measured by H3-Thymidine incorporation assay. We then did OB maturation and mineralization assays of Nes/Osx-YFP(+) OBs and CFU and multi-lineage differentiation assays of Nes/Osx-CD45(-)YFP(+) MPCs. RESULTS: YFP(+)% in Nes-OBs and Osx-OBs and CD45(-)YFP(+)% in Nes-BMMSCs and Osx-BMMSCs was respectively 5.56%±3.56% (n=5), 10.12%±2.7% (n=4), 1.29%±0.98% (n=13) and 16.38%±6.98% (n=17). Both Nes-YFP(+) OBs and Osx-YFP(+) OBs were positive for CD51. Nes/Osx-CD45(-)YFP(+) MPCs were positive for CD51, CD105 and Sca1, and negative for CD31 and CD45. PDGFR expression in Osx-YFP(+) OBs was a bit higher than that in Nes-YFP(+) OBs, and slightly higher in Osx-CD45(-)YFP(+) MPCs than in Nes-CD45(-)YFP(+) MPCs. Proliferation ability of Nes/Osx-YFP(+) OBs increased dramatically after stimulated with PDGF-BB for 48 h, while it was not statistically significant that PDGF-BB induced the increase of proliferation ability in either Nes-CD45(-)YFP(+) MPCs or Osx-CD45(-)YFP(+) MPCs. We observed that no significant difference of OB maturation and mineralization ability existed between Nes-YFP(+) OBs and Osx-YFP(+) OBs, and there was little difference of self-renewal and multi-lineage differentiation potential between Nes-CD45(-)YFP(+) MPCs and Osx-CD45(-)YFP(+) MPCs, either. CONCLUSION: Both Nestin and Osterix could be selected as useful markers for the osteoblastic cells, while Osterix was a prior choice due to larger number of Osterix-expressing cells than Nestin-expressing cells in distinct subpopulations of bone-forming cells.

The adhesion molecule PECAM-1 enhances the TGF-ß-mediated inhibition of T cell function.

Transforming growth factor-ß (TGF-ß) is an immunosuppressive cytokine that inhibits the proinflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical TGF-ß signaling results in the activation of Smad proteins, which are transcription factors that regulate target gene expression. We found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitated noncanonical (Smad-independent) TGF-ß signaling in T cells. Subcutaneously injected tumor cells that are dependent on TGF-ß-mediated suppression of immunity for growth grew more slowly in PECAM-1(-/-) mice than in their wild-type counterparts. T cells isolated from PECAM-1(-/-) mice demonstrated relative insensitivity to the TGF-ß-dependent inhibition of interferon-γ (IFN-γ) production, granzyme B synthesis, and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-ß in a manner that was partially restored by reexpression of PECAM-1. Co-incubation of T cells with TGF-ß and a T cell-activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 (SH2) domain-containing tyrosine phosphatase-2 (SHP-2). Such conditions also induced the colocalization of PECAM-1 with the TGF-ß receptor complex as identified by coimmunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-ß in T cells and suggest that therapeutic targeting of the PECAM-1-TGF-ß inhibitory axis represents a means to overcome TGF-ß-dependent immunosuppression within the tumor microenvironment.