Tr17: A Unique Subset of T Regulatory Cells with Distinct Molecular and Functional Properties

Abstract 1116 A major challenge of the immune system is to fight pathogens and abnormal cell growth while preserving immune tolerance to self-antigen. Active immune suppression by T regulatory (Treg) cells is one of the mechanisms that maintain peripheral T cell tolerance and homeostasis. Recent findings suggest that Treg cells are heterogeneous in functions and phenotypes. Moreover, Treg cells might not be at a terminal stage of differentiation but rather display a significant degree of plasticity particularly under inflammatory conditions. We have previously determined that co-culture of conventional CD4+ T cells and Treg cells in the presence of antigen presenting cells (APC) during T cell receptor mediated stimulation, induced Treg cells producing IL-17 (thereafter named Tr17 cells). IL-1β and IL-2, endogenous products of the co-culture, were essential for the conversion of Treg cells into Tr17 cells. In the present study we sought to identify the phenotypic and functional properties of Tr17 cells. First, we examined the role of IL-2 and IL-1β in the generation of Tr17 cells. Treg cells from Foxp3.GFP knock-in mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-1β-plus-IL-2. A fraction of purified Foxp3+ Treg cells produced significant amounts of IL-17 as determined by ELISA and by intracellular cytokine staining. Very few Tr17 cells were observed during T cell activation in the absence of either IL-1β or IL-2, suggesting that the combination of IL-1β and IL-2 was required for the development of Tr17 cells. Compared to IL-17-Treg, Tr17 cells expressed increased levels of RORγt, the key transcription factor for Th17 cell differentiation. Expression of CCR6 is regulated by RORγt and is one of the features of Th17 cells. To determine whether expression of CCR6+ correlates with the ability of Treg to produce IL-17, we depleted CCR6+ Treg from Foxp3.GFP+ populations and examined the ability of the resulting Treg to produce IL-17. Depletion of CCR6+ Treg resulted in significantly reduced IL-17 production compared with total Foxp3+ Treg cells, suggesting that CCR6+ Treg cells might represent Tr17 cells. Because inducible costimulatory molecule (ICOS) was recently shown to be critical for the differentiation, expansion and function of Th17 cells we examined its role in Tr17 cells. We determined that Tr17 cells expressed strikingly elevated levels of ICOS compared to IL-17− Treg obtained from the same cultures. These results indicate that Tr17 cells acquire phenotypic properties of Th17 cells, which involve RORγt-dependent and independent mechanisms. To determine whether besides obtaining features of Th17 cells, Tr17 cells also retained properties of Treg we first assessed the expression of Foxp3. Compared to IL-17− Treg, Tr17 cells displayed lower levels of Foxp3. However, expression of CTLA-4 and GITR, two other signature makers of Treg cells, was comparable in IL-17− Treg and Tr17 cells. Similarly, both Tr17 and IL-17− Treg cells displayed suppression capacity. Taken together, our results suggest that Tr17 is a unique subset of Treg cells that is differentiated under inflammatory conditions and displays combined phenotypic and functional properties of both Th17 and Treg cells. Because Treg use canonical CD4+ effector cell-associated transcription factors to regulate the immune responses of lineage-specific effectors, our findings suggest that Tr17 cells may have a unique role in regulating Th17-mediated inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

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Overexpression of CTLA-4 in the Bone Marrow of Patients with Multiple Myeloma As a Sign of Local Accumulation of Immunosuppressive Tregs – Perspectives for Novel Treatment Strategies

Blood ◽

10.1182/blood.v118.21.1829.1829 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1829-1829 ◽

Cited By ~ 2

Author(s):

Walter Moises Tobias Braga ◽

Andre Luiz Vettore ◽

Ana Carolina Carvalho ◽

Djordje Atanackovic ◽

Gisele W.B. Colleoni

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

T Cell Activation ◽

Th17 Cells ◽

Conflicts Of Interest ◽

Treg Cells ◽

Genetic Alterations ◽

The Other ◽

Other Hand

Abstract Abstract 1829 Introduction: Multiple myeloma (MM) development involves a series of genetic alterations and changes in the bone marrow (BM) microenvironment, favoring the growth of the tumor and failure of local immune control. T regulatory (Treg) cells play an important role in the maintenance of self-tolerance and modulation of overall immune responses against infections and tumor cells. T-helper-17 (Th17) cells, on the other hand, have a critical function in eradicating extracellular pathogens and tumor cells. The balance between Treg and Th17 cells may be essential for maintaining homeostasis of anti-tumor immunity. The aim of our study was to characterize the expression of Treg- (FOXP3 and CTLA-4) and Th17-related (RORγt) genes in total MM bone marrow samples to assess the local immune milieu as a potential therapeutic target in this still incurable disease. Material and Methods: Expression of FOXP3, CTLA-4 and RORγt was determined by quantitative real-time PCR (qPCR) in BM aspirates of 55 MM patients, 4 patients with monoclonal gammopathy of undetermined significance (MGUS), and 5 healthy BM donors. Genes were considered overexpressed when their expression was at least two times higher in myeloma BM than in normal samples. Results:RORγt showed a non-significant overexpression in MM patients compared to controls. FOXP3, on the other hand, showed a 3.6-fold higher expression in MM patients compared to controls but the difference was not significant and was overexpressed in 63% of MM cases. CTLA-4 expression was 14.6-fold higher in MM patients compared to controls (p=0.03, Mann-Whitney test) and was overexpressed in 70% of MM cases. Importantly the CTLA-4/RORyt ratio was significantly higher in MM samples compared to controls (p = 0.009) while this difference was not significant when controls were compared to MGUS patients, suggesting that the immunological imbalance worsens with the progression of disease. Conclusions: CTLA-4 (cytotoxic T-lymphocyte antigen-4), an intracellular and membrane inhibitory protein expressed on Treg cells, modulates T-cell activation and is critical in maintaining immune tolerance to self-antigens. Monoclonal antibodies targeting CTLA-4 have improved the survival of patients with metastatic melanoma in clinical trials. The myeloma-related overexperssion of CTLA-4 and the increased CTLA-4/RORyt ratio suggest an accumulation of immunosuppressive Tregs in the tumor microenvironment and/or an immediate involvement of this gene in the development and progression of myeloma. If protein expression confirms gene expression imbalance between Treg and Th17 subpopulations, therapeutic approaches that specifically target CTLA-4-expressing Treg myeloma cases may provide a new treatment strategy for this disease. (Supported by CNPq, Brazil). Disclosures: No relevant conflicts of interest to declare.

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CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity

Frontiers in Immunology ◽

10.3389/fimmu.2021.697840 ◽

2021 ◽

Vol 12 ◽

Author(s):

Alsya J. Affandi ◽

Katarzyna Olesek ◽

Joanna Grabowska ◽

Maarten K. Nijen Twilhaar ◽

Ernesto Rodríguez ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Inflammatory Responses ◽

Lavage Fluid ◽

Type I ◽

Inflammatory Conditions ◽

Activated Monocytes ◽

And Function

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

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Combination Treatment With Metformin and Tacrolimus Improves Systemic Immune Cellular Homeostasis by Modulating Treg and Th17 Imbalance

Frontiers in Immunology ◽

10.3389/fimmu.2020.581728 ◽

2021 ◽

Vol 11 ◽

Author(s):

Soon Kyu Lee ◽

Min-Jung Park ◽

Joo Yeon Jhun ◽

Jin-Ah Beak ◽

Jeong Won Choi ◽

...

Keyword(s):

T Cell ◽

Combination Therapy ◽

T Cell Activation ◽

Th17 Cells ◽

Cell Activation ◽

Treg Cells ◽

T Cell Proliferation ◽

Allogeneic Stimulation

We examined the effect of combination therapy with metformin and tacrolimus on immune parameters including T regulatory (Treg) and type 17 helper T (Th17) cells in vitro and in vivo in mice and in liver transplantation (LT) patients. T cell proliferation and subtypes after in vitro T cell activation or allogeneic stimulation were evaluated. RNA sequencing and microarray analysis were used to evaluate differences in gene expression. Metformin and tacrolimus were administered to mice with graft-versus-host disease (GVHD) and the effects in vivo were assessed. Five LT patients were treated with metformin and the changes in Treg and Th17 cells examined. Combination therapy decreased Type 1 helper T (Th1) and Th17 cells present after in vitro T cell activation, whereas genes associated with Treg were overexpressed. During in vitro allogeneic stimulation, combination therapy increased Treg cells and decreased T cell proliferation and pro-inflammatory markers. In mice with GVHD, combination treatment decreased the clinical and pathological severity of GVHD. In LT patients, addition of metformin increased the peripheral percentage of CD4+Treg and CD8+Treg cells and decreased CD4+Th17. Our study suggests that the addition of metformin to tacrolimus may improve immunological balance by increasing Treg cells and decreasing Th17 cells.

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Potent Alloreactive Effector T Cells Cause Limited Damage to Non-Hematopoietic Tissues Under Non-Inflammatory Conditions, Despite Proper Expression of the Relevant Target Antigens.

Blood ◽

10.1182/blood.v114.22.2454.2454 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2454-2454

Author(s):

Boris van der Zouwen ◽

Alwine B. Kruisselbrink ◽

Peter A. Von Dem Borne ◽

Roelof Willemze ◽

J.H. Frederik Falkenburg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Conflicts Of Interest ◽

Human Fibroblasts ◽

Hematopoietic Cells ◽

Effector T Cells ◽

Inflammatory Conditions

Abstract Abstract 2454 Poster Board II-431 Donor T cells specific for minor histocompatibility antigens (mHags) contribute to graft-versus-leukemia (GvL) reactivity and graft versus host disease (GvHD) following HLA-matched stem cell transplantation. Hematopoiesis-restricted mHags may give rise to a more specific GvL effect without GvHD, whereas ubiquitously expressed mHags can be targets for both GvHD and GvL. Interestingly, high frequencies of circulating T cells specific for ubiquitously expressed mHags (e.g. male-specific epitopes, LB-ADIR-1F) have been detected in patients showing strong GvL with no or limited GvHD. Although both hematopoietic and non-hematopoietic cells express the proteins encoding these mHags, the immune effector response in these patients appears to be skewed towards hematopoietic cells. Therefore, we hypothesized that non-hematopoietic tissues are relatively unsusceptible to the cytotoxic effect of these mHag-specific T cells. To test this hypothesis, we developed an in vitro system in which HLA-A2+, ADIR+ primary human fibroblasts were exposed to mHag LB-ADIR-1F or allo-A2 specific CD8+ T cells. ADIR+ EBV-LCL were used as a control for hematopoietic cells. CTL-mediated cytotoxicity was measured using 4, 8 and 20 hours chromium release assays. Whereas all EBV-LCL were killed within 4 hours, 10% lysis of fibroblasts was found after 4 hours, only gradually increasing to 70% lysis after 20 hours in the continuous presence of the CTLs at 10/1 effector/target (E/T) ratios. To analyze whether this inefficient killing was due to low antigen expression by the human primary fibroblasts, we examined cytotoxicity against targets loaded with increasing concentrations of ADIR-peptide. Exogenous loading of fibroblasts with saturating concentrations (10-6M) of ADIR-peptide did not increase the sensitivity to T cell mediated cell death. To exclude that the observed inferior cell killing was due to the number of T cells available for engaging the fibroblasts, E/T ratios were increased to >100/1, but this did not change the poor susceptibility of the fibroblasts to CTL-mediated killing. Since apparently inefficient elimination of non-hematopoietic targets was found despite proper expression of the relevant antigen and the presence of functional T cells, we investigated whether insufficient engagement of T cells during contact with the fibroblasts was the cause of the impaired killing. To analyze this, kinetics of T cell degranulation upon target encounter was measured by CD107a, perforin and granzyme B expression. Although the onset of T cell activation was already seen after 2 hours, stimulation with fibroblasts resulted in degranulation in only a proportion of the T cells (50-60%) after 4hrs, resulting in full activation only after prolonged co-culture. In contrast, stimulation with EBV-LCL resulted in rapid, complete, and profound degranulation (100% of T cells within 2hrs). In concordance with this, T cell activation, as measured by the production of interferon gamma (IFNg) after stimulation with the fibroblasts, was suboptimal as compared to stimulation with EBV-LCL. These data suggest formation of an initial low avidity interaction with fibroblasts under steady state conditions, possibly only leading to efficient T cell engagement and fibroblast killing after a prolonged period of co-culture, resulting in the formation of a local pro-inflammatory environment. To confirm whether susceptibility to mHag-specific T cells was increased under pro-inflammatory conditions, we pre-treated fibroblasts with IFNg. Analysis of the expression of a panel of known adhesion and co-stimulatory molecules revealed that IFNg treatment resulted in a 2-5 fold upregulation of expression of HLA class I, HLA class II and ICAM-1, leading to a more uniform and profound T cell activation. This increased T cell activation resulted in more efficient direct fibroblast killing. In conclusion, despite proper expression of the relevant target antigens, low-avidity interaction between effector T cells and non-hematopoietic tissues results in minimal damage under non-inflammatory conditions. Disclosures: No relevant conflicts of interest to declare.

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HLA-G and CD86 Expression on Malignant Plasma Cells Convey a Poor Prognosis and Immunoregulate T Cells In Patients with Myeloma

Blood ◽

10.1182/blood.v116.21.983.983 ◽

2010 ◽

Vol 116 (21) ◽

pp. 983-983

Author(s):

Ross D. Brown ◽

Karieshma Kabani ◽

Shihong Yang ◽

Esther Aklilu ◽

Phoebe Joy Ho ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Poor Prognosis ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Costimulatory Molecule ◽

Prognostic Significance ◽

Tumor Escape

Abstract Abstract 983 HLA-G is a “non-classical” HLA class I molecule which is considered to be responsible for the immune tolerance of the fetus, allografts or tumors by inhibiting the antigen-specific cytotoxic T lymphocyte response and decreasing NK cell function. CD86 is a costimulatory molecule which binds to a T cell counter receptor (eg CD28) during antigen stimulation and T cell receptor engagement. T cells do not express CD86mRNA but can acquire CD86 and HLA-G by trogocytosis. We have studied the incidence and prognostic significance of HLA-G and CD86 on malignant plasma cells, the incidence of HLA-G and CD86 on T cells and the immunosuppressive function of HLA-G on malignant plasma cells and T cells. HLA-G and CD86 expression were determined on T cells (CD3+) and plasma cells (CD38++) of patients with myeloma (MM). Flow-sorted CD3+ HLA-G- cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti-CD3/CD2/CD28 beads in the presence and absence of either HLA-G+ T cells or HLA-G+ plasma cells. Suppression of the proliferation of CFSE-tracked HLA-G- T cells by HLA-G+ T cells or plasma cells was determined by flow cytometry. CD86+ plasma cells are present in 54% of patients and are associated with a poor prognosis (χ2=4.6;p<0.03) (Blood 96:1274). We now report that the incidence of HLA-G expression on plasma cells was heterogeneous (0.2 to 96%; mean = 21.3%) and that the clinical relevance of HLA-G+ plasma cells was demonstrated by a significant reduction in overall survival (13 months vs median not achieved) for the 11/46 patients with HLA-G+ plasma cells (>5% CD38++ cells and expressing >12% HLA-G; χ2=12.4; p<0.0004) who had no difference in β2M, age or M protein isotype. Biotinylated cell membrane protein transfer from plasma cells to T cells (trogocytosis) was demonstrated using flow cytometry and confocal microscopy. Trogocytosis was unidirectional, common in MM but not from CLL or WM cells and there was with a significantly greater acquisition of biotinylated proteins by CD3+ cells (mean=13.6%) than B cells (mean=2.4%; t=2.80; p<0.05) or NK cells (mean=3.1%; t=2.57; p<0.05). Low HLA-G expression was found on both CD4 and CD8 normal T cells (n=15; mean=0.19%). Twenty six % of MM patients had HLA-G-expressing CD3+ T cells above the normal range (χ2=4.9;p<0.03). There was a significant acquired expression of CD86 by T cells (t=6.06; p<0.004) after T cell activation (CD3/2/28 beads) and exposure to the CD86+ cell line RPMI8226. Trogocytosis of HLA-G has been reported by others (Blood 109:2040). Increased CD86 surface (but not mRNA) expression was found on the CD3+ cells of 32% of patients (n=98; F=38.6; p<0.0001). Flow-sorted HLA-G+ T cells significantly reduced the proliferation of CFSE-labeled T cells in 4 day cultures. Inhibition by HLA-G T cells was greater than the inhibition due to HLA-G+ plasma cells (t=3.03; p=0.039) and was neutralised by anti HLA-G. HLA-G+ T cells were both CD4 and CD8 but were CD25- and thus were not Treg cells. In conclusion, HLA-G and CD86 expression on plasma cells and T cells are clinically relevant in patients with myeloma. Tumor escape in patients with myeloma may be due to the inhibition of normal T cell proliferation by distinct T cell cohorts which develop novel immunoregulatory functions following the acquisition and ectopic expression of antigens which include HLA-G and CD86. Disclosures: No relevant conflicts of interest to declare.

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CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

Infection and Immunity ◽

10.1128/iai.00297-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4922-4935 ◽

Cited By ~ 9

Author(s):

Maíra Felonato ◽

Adriana Pina ◽

Simone Bernardino ◽

Flávio V. Loures ◽

Eliseu Frank de Araujo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Inflammatory Cytokines ◽

T Cell Activation ◽

Paracoccidioides Brasiliensis ◽

Costimulatory Molecule ◽

Fungal Growth ◽

Treg Cells ◽

Cell Immunity ◽

Anti Inflammatory

ABSTRACT T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28− / −) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28− / − mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.

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Superagonistic CD28 Protects against Renal Ischemic Injury by Expansion of Regulatory T-Cell

American Journal of Nephrology ◽

10.1159/000470918 ◽

2017 ◽

Vol 45 (5) ◽

pp. 389-399 ◽

Cited By ~ 4

Author(s):

Yiran Liang ◽

Yan Li ◽

Qing Kuang ◽

Xiaoqiang Ding ◽

Zheng Wei ◽

...

Keyword(s):

T Cell ◽

Peripheral Blood ◽

Inflammatory Responses ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Ischemic Precondition ◽

Cell Receptor ◽

Treg Cells ◽

Tubular Damage ◽

Renal Protection

Background: Regulatory T (Treg) cells are a highly suppressive subset of CD4+ lymphocytes and have recently been proved to be crucial to suppress the inflammatory responses of ischemic kidney injury. CD28 superagonists (CD28sa) are monoclonal antibodies that preferentially expand Treg cells without a T-cell receptor and a costimulatory signal. This study aims to test the protection and discover the mechanisms of CD28sa treatment against renal ischemia-reperfusion (IR) injury (IRI). Methods: Male C57BL/6N mice were treated with CD28sa via peritoneal injection (0.1 mg) 6 days before the induction of IRI, or with 18-min ischemic precondition (IPC). IRI was induced by bilateral clamping of renal pedicles for 35 min followed by reperfusion. The role of Treg expansion in renal protection conferred by CD28sa treatment was examined using anti-CD25 antibody. Results: CD28sa treatment alone significantly increased the percentage of Treg cells in the spleen (18.10 ± 2.00 vs. 6.64 ± 0.86%, p < 0.01), peripheral blood (16.43 ± 5.94 vs. 2.57 ± 1.09%, p < 0.01), and kidney (2.69 ± 0.90 vs. 0.53 ± 0.14%, p < 0.01) of C57BL/6N mice 6 days after the administration. Mice pretreated with CD28sa or IPC had less renal injury at 24 h after IRI with attenuation of renal tubular damage and lower serum creatinine compared with the mice that underwent renal IRI alone. The number of infiltrating macrophages in the kidney and IFN-γ secreting CD4+ T cells in peripheral blood were diminished in the CD28sa-IR group and the IPC-IR group. The renal protection bestowed by CD28sa or IPC was abolished by anti-CD25 antibody administration. Conclusions: Treg expansion induced by CD28sa ameliorated renal IRI.

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The Role of Protein Tyrosine Phosphatase PTP4A3 in T-Cell Acute Lymphoblastic Leukemia Progression

Blood ◽

10.1182/blood-2019-126256 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2539-2539

Author(s):

Min Wei ◽

Jessica Blackburn

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

T Cell Activation ◽

Protein Tyrosine Phosphatase ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Tyrosine Phosphatase ◽

Causative Role ◽

Protein Tyrosine ◽

Cell Acute Lymphoblastic Leukemia

The tyrosine protein tyrosine phosphatase PTP4A3 has been extensively reported to play a causative role in numerous cancers, including several types of acute leukemia. We found PTP4A3 to be highly expressed in T-cell Acute Lymphoblastic Leukemia samples, and show that PTP4A3 accelerates T-ALL onset and increases the invasive ability of T-ALL cells in a zebrafish model, and is required for T-ALL engraftment and progression in mouse xenograft. Our in vitro studies showed that PTP43A3 enhances T-ALL migration, in part via modulation of SRC signaling. However, whether SRC is a direct substrate of PTP4A3, and whether the phosphatase activity of PTP4A3 actually plays a role in T-ALL or other types of leukemia progression is unknown and remains a major question in the field. We used a BioID-based proximity labeling approach combined with PTP4A3 substrate trapping mutant pull down assay to capture the PTP4A3 substrates candidates. BioID, a biotin ligase, was fused to PTP4A3 to generate a Biotin-PTP4A3 (BP) fusion protein. The overexpression of BP in T-ALL cell lines led to biotin modification of 288 PTP4A3 proximal proteins, including the potential direct PTP4A3 substrates. PANTHER pathway analysis showed that PTP4A3 interacting proteins are largely clustered in the T-cell activation, PDGF signaling, and angiogenesis. We are in process of validating potential substrates using immunoprecipitation and phosphoenrichement assays. Finally, we are using a novel zebrafish Myc+PTP4A3 induced T-ALL model to assess the function of PTP4A3 in leukemia progression. We have created several PTP4A3 protein mutants, including a phosphatase-dead mutant, a mutant unable to bind magnesium transporter, and a prenylation deficient mutant, and are in process of assessing the effects of these mutants in T-ALL onset and progression in our in vivo model. In total, these studies will allow us to better understand function of PTP4A3 in T-ALL progression, and may provide a strong rationale for the development of PTP4A3 inhibitors for use in leukemia. Disclosures No relevant conflicts of interest to declare.

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