TIGIT expression is upregulated in T cells and causes T cell dysfunction independent of PD-1 and Tim-3 in adult B lineage acute lymphoblastic leukemia

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti- CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.

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CD19/CD3 Bispecific Antibody Blinatumomab (MT-103) Is Highly Effective In Treatment of Patients with Minimal Residual Disease From Chemotherapy-Resistant B-Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.174.174 ◽

2010 ◽

Vol 116 (21) ◽

pp. 174-174 ◽

Cited By ~ 4

Author(s):

Max S Topp ◽

Gerhard Zugmaier ◽

Nicola Goekbuget ◽

Svenja Neumann ◽

Heinz-August Horst ◽

...

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Minimal Residual Disease ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Clinical Relapse ◽

B Lineage ◽

Minimal Residual

Abstract Abstract 174 Blinatumomab, a bispecific, T cell-engaging (BiTE®) antibody, can effectively redirect T cells for highly selective lysis of CD19+ target cells. The B-cell differentiation antigen CD19 is a marker for B-ALL cells. In B-lineage acute lymphoblastic leukemia (ALL), persistence or relapse of minimal residual disease (MRD) is an independent poor prognostic factor, and new treatments are urgently needed. MRD relapse during or after maintenance treatment in adult standard risk patients generally heralds a hematological relapse in 90% of patients. A phase 2 study was conducted to determine the efficacy of blinatumomab in ALL patients with MRD persistence or relapse (MRD level <310-4) after induction and consolidation therapy. MRD was assessed by qRT-PCR for rearrangements of immunoglobulin (Ig) or T-cell receptor (TCR) genes, or for specific fusion genes. Blinatumomab was administered as a 4-week continuous i.v. infusion at a dose of 15 μ g/m2/d followed by a 2-week treatment free period (1 cycle). Primary endpoint was the proportion of patients with MRD response defined by individual rearrarrangement of Ig or TCR below 10-4. Patients who showed neither MRD progression nor response were permitted to receive up to 7 cycles of treatment. Patients who had achieved MRD response received 3 additional consolidation cycles. Between May 2008 and November 2009, 21 patients (16 Ph-negative; 2 patients with MLL-AF4; 5 patients with Ph+ ALL) were enrolled. The cut-off date for data analysis was May 15, 2010. Patients received between 1 and 7 cycles of blinatumomab (total of 66 cycles). Transient pyrexia (100%) and chills (43%) were the most common clinical AEs. There were no blinatumomab related deaths. Sixteen patients became MRD-negative. One patient was not evaluable due to a grade 3 adverse event (AE) leading to treatment discontinuation. Of the responding patients, 13 had never before achieved a negative MRD status on chemotherapy. Regardless of their MRD level prior to study treatment, all 16 (13/15 patients with Ph− and 3/5 patients with Ph+ ALL) became MRD-negative after the first cycle of blinatumomab. Nine patients were enrolled with a MRD load >10-2 prior to study treatment and all reached complete MRD response. Thirteen out of 16 patients with persisting MRD prior to study treatment and 3 out of 4 patients with MRD relapse showed complete MRD response. Overall relapse-free survival (RFS) currently is 78% at a median follow up of 405 days. RFS is 100% for the 8 patients who received subsequent allogeneic stem cell transplantation (median follow up 434 days). Blinatumomab is a highly active treatment for patients with MRD-positive B-lineage ALL after intensive chemotherapy and has an acceptable safety profile. T cells engaged by blinatumomab seem capable of eradicating chemotherapy-resistant tumor cells in bone marrow that otherwise might cause clinical relapse. A long RFS suggests that blinatumomab may improve outcome in patients with B-precursor ALL. A multicenter international study of blinatumomab in patients with MRD-positive B-lineage ALL has been initiated. Disclosures: Zugmaier: Micromet Inc.: Employment. Degenhard:Micromet Inc.: Employment. Schmidt:Micromet Inc.: Employment. Scheele:Micromet Inc.: Employment. Kufer:Micromet Inc.: Employment. Klinger:Micromet Inc.: Employment. Nagorsen:Micromet Inc.: Employment. Bargou:Micromet Inc.: Consultancy.

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Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell–engaging CD19/CD3-bispecific BiTE antibody blinatumomab

Blood ◽

10.1182/blood-2012-01-400515 ◽

2012 ◽

Vol 119 (26) ◽

pp. 6226-6233 ◽

Cited By ~ 255

Author(s):

Matthias Klinger ◽

Christian Brandl ◽

Gerhard Zugmaier ◽

Youssef Hijazi ◽

Ralf C. Bargou ◽

...

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

B Cell ◽

Continuous Infusion ◽

Lymphoblastic Leukemia ◽

Molecular Response ◽

Entire Treatment Period ◽

Cell Counts ◽

B Lineage

T cell–engaging CD19/CD3-bispecific BiTE Ab blinatumomab has shown an 80% complete molecular response rate and prolonged leukemia-free survival in patients with minimal residual B-lineage acute lymphoblastic leukemia (MRD+ B-ALL). Here, we report that lymphocytes in all patients of a phase 2 study responded to continuous infusion of blinatumomab in a strikingly similar fashion. After start of infusion, B-cell counts dropped to < 1 B cell/μL within an average of 2 days and remained essentially undetectable for the entire treatment period. By contrast, T-cell counts in all patients declined to a nadir within < 1 day and recovered to baseline within a few days. T cells then expanded and on average more than doubled over baseline within 2-3 weeks under continued infusion of blinatumomab. A significant percentage of reappearing CD8+ and CD4+ T cells newly expressed activation marker CD69. Shortly after start of infusion, a transient release of cytokines dominated by IL-10, IL-6, and IFN-γ was observed, which no longer occurred on start of a second treatment cycle. The response of lymphocytes in leukemic patients to continuous infusion of blinatumomab helps to better understand the mode of action of this and other globally T cell–engaging Abs. The trial is registered with www.clinicaltrials.gov identifier NCT00560794.

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Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion

Science Advances ◽

10.1126/sciadv.abd2710 ◽

2021 ◽

Vol 7 (18) ◽

pp. eabd2710

Author(s):

Chen Zhu ◽

Karen O. Dixon ◽

Kathleen Newcomer ◽

Guangxiang Gu ◽

Sheng Xiao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adaptor Protein ◽

Transcriptional Analysis ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Cell Dysfunction ◽

Autoreactive T Cell ◽

Autoimmune Conditions ◽

T Cell Dysfunction

T cell exhaustion has been associated with poor prognosis in persistent viral infection and cancer. Conversely, in the context of autoimmunity, T cell exhaustion has been favorably correlated with long-term clinical outcome. Understanding the development of exhaustion in autoimmune settings may provide underlying principles that can be exploited to quell autoreactive T cells. Here, we demonstrate that the adaptor molecule Bat3 acts as a molecular checkpoint of T cell exhaustion, with deficiency of Bat3 promoting a profound exhaustion phenotype, suppressing autoreactive T cell–mediated neuroinflammation. Mechanistically, Bat3 acts as a critical mTORC2 inhibitor to suppress Akt function. As a result, Bat3 deficiency leads to increased Akt activity and FoxO1 phosphorylation, indirectly promoting Prdm1 expression. Transcriptional analysis of Bat3−/− T cells revealed up-regulation of dysfunction-associated genes, concomitant with down-regulation of genes associated with T cell effector function, suggesting that absence of Bat3 can trigger T cell dysfunction even under highly proinflammatory autoimmune conditions.

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TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

10.21203/rs.3.rs-641407/v1 ◽

2021 ◽

Author(s):

qi shao ◽

Lei Wang ◽

maoling yuan ◽

Xiaohong Jin ◽

changping wu

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

T Cell ◽

Glucose Metabolism ◽

Tumor Growth ◽

Tumor Cells ◽

Peripheral Blood ◽

Cancer Tissue ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

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Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+T cell dysfunction and maintain memory phenotype

Science Immunology ◽

10.1126/sciimmunol.aat7061 ◽

2018 ◽

Vol 3 (29) ◽

pp. eaat7061 ◽

Cited By ~ 44

Author(s):

Bei Wang ◽

Wen Zhang ◽

Vladimir Jankovic ◽

Jacquelynn Golubov ◽

Patrick Poon ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Programmed Cell Death ◽

Cancer Immunotherapy ◽

Effector Memory ◽

Cell Phenotype ◽

Checkpoint Inhibition ◽

Cell Dysfunction ◽

T Cell Dysfunction

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor–related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1–Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

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G19.4(alpha CD3) x B43(alpha CD19) monoclonal antibody heteroconjugate triggers CD19 antigen-specific lysis of t(4;11) acute lymphoblastic leukemia cells by activated CD3 antigen-positive cytotoxic T cells

Blood ◽

10.1182/blood.v80.11.2826.bloodjournal80112826 ◽

1992 ◽

Vol 80 (11) ◽

pp. 2826-2834

Author(s):

PM Anderson ◽

W Crist ◽

D Hasz ◽

AJ Carroll ◽

DE Myers ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cytotoxic T Cells ◽

Lymphoblastic Leukemia ◽

Interleukin 2 ◽

Cytolytic Activity ◽

Multiparameter Flow Cytometry ◽

Specific Lysis

A highly purified, 300-Kd bispecific monoclonal antibody (MoAb) heteroconjugate was prepared by covalently linking the anti-CD3 MoAb, G19.4, to the anti-CD19 MoAb, B43. Dual-color staining techniques and multiparameter flow cytometry confirmed that this alpha CD3 x alpha CD19 heteroconjugate was able to bind to both CD3+ T cells and CD19+ t(4;11) acute lymphoblastic leukemia (ALL) cells. T-cell-mediated lysis of freshly isolated primary bone marrow blasts from nine newly diagnosed ALL patients with a t(4;11)(q21;q23) chromosomal translocation were studied with 51Cr-release assays. Picomolar concentrations of alpha CD3 x alpha CD19 MoAb heteroconjugate effectively triggered lysis of CD19+ t(4;11) ALL cells by interleukin-2- activated CD3+ peripheral blood T-cell (PBTC) effectors but did not augment the cytolytic activity of the same effectors against CD19- T- ALL cells. In contrast to the alpha CD3 x alpha CD19 heteroconjugate, neither the alpha CD3 x alpha CD3 homoconjugate control nor the alpha CD19 x alpha CD72 heteroconjugate control facilitated the cytolysis of t(4;11) ALL blasts. Occupation of the target CD19 binding sites on t(4;11) ALL blasts by preincubation with excess unconjugated alpha CD19 MoAb abrogated the potentiating effects of the alpha CD3 x alpha CD19 heteroconjugate on PBTC-mediated cytolysis. Thus, the cell type- specific cytolysis of t(4;11) ALL blasts by PBTC effectors is dependent on both the alpha CD19 and alpha CD3 moieties of the alpha CD3 x alpha CD19 heteroconjugate. To our knowledge, this is the first description of an effective bispecific antibody that facilitates the T-cell- mediated lysis of t(4;11) ALL blasts.

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663 Media based on the metabolic composition of tumor interstitial fluid reveals persistent T cell dysfunction induced through arginine deprivation and exposure to the oncometabolite phosphoethanolamine

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.663 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A691-A691

Author(s):

Yupeng Wang ◽

Chufan Cai ◽

Dayana Rivadeneira ◽

Alexander Muir ◽

Greg Delgoffe

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Interstitial Fluid ◽

Cytokine Production ◽

Cell Dysfunction ◽

Kennedy Pathway ◽

T Cell Dysfunction ◽

Tumor Interstitial Fluid

BackgroundWhile CD8 T cells are crucial for anti-tumor immunity, tumor infiltrating CD8 T cells encounter stressors which deviate their differentiation to a dysfunctional, exhausted phenotype. T cell functions are closely regulated by T cell metabolism, and the dysfunctional vasculature in tumor tissues and the deregulated metabolism of tumor cells lead to depletion of nutrients and accumulation of metabolic wastes in the tumor microenvironment (TME). Thus, the unbalanced levels of the nutrients and the metabolic wastes might skew the metabolism of T cells thus contributing to T cell dysfunction.MethodsOvalbumin-specific OT-I cells were activated with SIINFEKL/IL2 and cultured with IL2. The tumor interstitial fluid media (TIFM) was formulated based on the concentrations of the metabolites measured in the tumor interstitial fluid of pancreatic ductal adenocarcinoma.1 Purified arginine and phosphoethanolamine (PEtn) were used to change their levels in TIFM/RPMI1640 culture. Expression level of cytokines and PD-1 was measured by flow cytometry.ResultsWe sought to determine how T cells would differentiate, in vitro, if they were exposed only to the metabolites present in the TME. Using media formulated to model the metabolic composition of tumor interstitial fluid (TIFM),1 we show that CD8 T cells develop features of exhausted T cells in the TIFM culture: reduced proliferation, increased expression of PD-1 and decreased cytokine production. Using 'dropout' and 'add-back' approaches, we found arginine levels as a major contributor to the proliferation defect observed in TIFM-cultured T cells. Arginine was sufficient to restore proliferative capacity to T cells cultured in TIFM, but had no effect on the inhibited cytokine production. We then asked which metabolites were enriched in the TIFM, finding that PEtn, an intermediate in the ethanolamine branch of the Kennedy pathway and an oncometabolite enriched in the interstitial of many solid tumors, up-regulates PD-1 expression and compromises the cytokine production of the cells in culture. Depletion of Pcyt2, the metabolizing enzyme of PEtn and the rate limiting enzyme in the Kennedy pathway, makes CD8 T cells resistant to the effects of PEtn.ConclusionsOur data shows that the metabolic environment in the TME can be recapitulated in vitro and is sufficient to drive T cell dysfunction. Arginine depletion acts as a major inhibitor of T cell proliferation in the TME, but the oncometabolite PEtn drives a hypofunctional effector fate of T cells. Targeting PEtn metabolism via Pcyt2 depletion or inhibition is a potential target to reinvigorate T cells and enhance anti-tumor immunity.ReferenceSullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. Elife 2019;;8:e44235. doi: 10.7554/eLife.44235. PMID: 30990168; PMCID: PMC6510537.

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583 Exploring the potential of T cell acute lymphoblastic leukemia (T-ALL) for generating leukemia-specific T cell responses that can be therapeutically harnessed with immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.583 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A613-A613

Author(s):

Todd Triplett ◽

Joshua Rios ◽

Alexander Somma ◽

Sarah Church ◽

Khrystyna North ◽

...

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Systemic Disease ◽

Flow Cytometric Analysis ◽

T Cell Responses ◽

Lymphoid Tissues ◽

Cell Responses ◽

Cell Acute Lymphoblastic Leukemia

BackgroundT cell Acute Lymphoblastic Leukemia (T-ALL) is a devastating malignancy found primarily in pediatric populations. Unfortunately, standard of care for T-ALL has not progressed from highly toxic, intensive regimens of chemotherapy, which fails to cure all patients. Immunotherapies designed to activate patients‘ leukemia-specific T cells may provide a new therapeutic avenue to increase complete response rates, reduce toxicity without the need to engineer (e.g. CAR) cells. However, it is unknown whether T-ALL is capable of being recognized by T cells due given its relatively low mutation-rate. These studies therefore sought to investigate whether signs of leukemia-specific T cell responses are generated by T-ALL. Because T-ALL results in systemic disease and infiltrates multiple lymphoid and non-lymphoid tissues, these studies also determined how the divergent immune contextures of these TMEs impacts T cell responses to T-ALL. From this, we aim to identify immunotherapeutic targets capable of activating T cells across tissues to eradicate leukemia systemically.MethodsPrimary leukemia cells isolated from a spontaneous murine model (LN3 mice) into immune-competent, congenic (CD45.1) recipient mice. Tissues were harvested at distinct stages of disease for analysis by flow cytometry or utilizing NanoString Technologies’ GeoMX Digital Spatial Profiling (DSP) platform.ResultsFlow cytometric analysis of T cells revealed extensive changes in response to T-ALL that included multiple features of exhaustion typically associated with anti-tumor responses as determined by upregulation of co-inhibitory receptors and TOX. This included a surprisingly high-frequency of PD1+ T cells, which was accompanied by PDL1- and PDL2-expressing myeloid cells that likely are restraining these subsets. Importantly, combination immunotherapy with OX40 agonists while inhibiting PD1 resulted in drastically reduced tumor burden and concomitant expansion of proliferating granzyme-expressing CD8 T cells. To gain better insight into T cell responses within distinct organs, we analyzed tissue sections using DSP. This technique enabled us to evaluate T cells in direct contact with leukemia infiltrates compared to T cells in regions without T-ALL, which further revealed an enrichment of activated subsets. Importantly, these studies have provided critical insight needed to better understand how T cells responding to T-ALL diverge between distinct types of tissues.ConclusionsThe results from these studies collectively suggest that T cells are activated by T-ALL and that they can be therapeutically harnessed despite relatively low mutation-rates. Future studies will continue analysis of individual organs and use these results to rationally design combinations of immunotherapies by tailoring to activate T cells in all tissue types.AcknowledgementsSpecial thanks to all the support and analysis from everyone at NanoString, along with financial support provided by a SITC-NanoString DSP Fellowship awarded to Dr. Todd Triplett used for DSP analysis of all frozen tissues in these studies. Salary support for Dr. Triplett and pilot funding was provided by departmental funds via a Cancer Prevention and Research Institute of Texas (CPRIT) Scholar Award (Grant #RR160093; awarded to Dr. Gail Eckhardt).

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GB Virus C E2 Inhibits PD-1-Mediated T Cell Signaling Dysfunction during Chronic Viral Infection

Proceedings ◽

10.3390/proceedings2020050062 ◽

2020 ◽

Vol 50 (1) ◽

pp. 62

Author(s):

Nirjal Bhattarai ◽

Jennifer L. Welch ◽

Jinhua Xiang ◽

Muthu Saravanan Manoharan ◽

Jeffrey A. Martinson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Viral Infections ◽

Activated T Cells ◽

E2 Protein ◽

Gb Virus C ◽

Cell Dysfunction ◽

Chronic Viral Infections ◽

T Cell Dysfunction ◽

Virus C

Background: Program death receptor 1 (PD-1) is a co-inhibitory receptor that is upregulated and contributes to T cell dysfunction (exhaustion) during chronic viral infections, including HIV and HCV. GB virus C (GBV-C) is a persistent human virus, and co-infection is associated with reduced immune activation and improved clinical outcomes in HIV- and Ebola-infected individuals. Methods: PD-1 levels were measured by flow cytometry on CD38+ T cells from 45 HIV-infected individuals, 20 of whom were co-infected with GBV-C. Jurkat cell lines that stably express GBV-C E2 protein and vector control were used to purify total cellular RNA before, and 24 h following, activation using anti-CD3/CD28 treatment. Gene expression was analyzed by RNA-seq and qRT-PCR. Results: HIV-infected individuals with GBV-C viremia had reduced PD-1 expression on activated CD4+ and CD8+ T cells compared to HIV-infected GBV-C negative individuals. GBV-C particles and GBV-C E2 protein each inhibited PD-1 expression on T cells in vitro. Consistent with this, GBV-C E2 reduced gene expression of PD-1, and its ligand PD-L1, in both resting and activated T cells. GBV-C E2 regulated transcription of the PD-1 signaling pathway and T cell activation associated genes, without downregulation of the interferon-stimulated and innate immunity-related genes needed to resolve viral infections. Conclusions: Our current understanding of chronic RNA virus infections is that upregulation of PD-1 with T cell exhaustion is critical for viral persistence. However, these data demonstrate that GBV-C infection reduced PD-1 expression on activated T cells during HIV infection, and that the GBV-C E2 protein inhibits PD-1 signaling in T cells. This may preserve T cell function and contribute to the lack of immune deficiency in people with chronic GBV-C infection. Understanding the mechanisms by which GBV-C E2 alters PD-1 signaling may aid in the development of novel immunomodulatory therapeutics to prevent T cell dysfunction (exhaustion) during chronic viral infections.

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