158 Inhibition of PI3Kδ improves tumor specific T cell immunity and metabolic fitness

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A172-A172
Author(s):  
Guillermo Rangel Rivera ◽  
Guillermo Rangel RIvera ◽  
Connor Dwyer ◽  
Dimitrios Arhontoulis ◽  
Hannah Knochelmann ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Therapy ◽  
Tumor Immunity ◽  
Mitochondrial Function ◽  
T Cell Differentiation ◽  
Tumor Control ◽  
T Cell Therapy

BackgroundDurable responses have been observed with adoptive T cell therapy (ACT) in some patients. However, current protocols used to expand T cells often exhibit suboptimal tumor control. Failure in these therapies has been attributed to premature differentiation and impaired metabolism of the infused T cells. Previous work done in our lab showed that reduced PI3Kδ signaling improved ACT. Because PI3Kγ and PI3Kδ have critical regulatory roles in T cell differentiation and function, we tested whether inhibiting PI3Kγ could recapitulate or synergize PI3Kδ blockade.MethodsTo test this, we primed melanoma specific CD8+ pmel-1 T cells, which are specific to the glycoprotein 100 epitope, in the presence of PI3Kγ (IPI-459), PI3Kδ (CAL101 or TGR-1202) or PI3Kγ/δ (IPI-145) inhibitors following antigen stimulation with hgp100, and then infused them into 5Gy total body irradiated B16F10 tumor bearing mice. We characterized the phenotype of the transferred product by flow cytometry and then assessed their tumor control by measuring the tumor area every other day with clippers. For metabolic assays we utilized the 2-NBDG glucose uptake dye and the real time energy flux analysis by seahorse.ResultsSole inhibition of PI3Kδ or PI3Kγ in vitro promoted greater tumor immunity and survival compared to dual inhibition. To understand how PI3Kδ or PI3Kγ blockade improved T cell therapy, we assessed their phenotype. CAL101 treatment produced more CD62LhiCD44lo T cells compared to IPI-459, while TGR-1202 enriched mostly CD62LhiCD44hi T cells. Because decreased T cell differentiation is associated with mitochondrial metabolism, we focused on CAL101 treated T cells to study their metabolism. We found that CAL101 decreased glucose uptake and increased mitochondrial respiration in vitro, indicating augmented mitochondrial function.ConclusionsThese findings indicate that blocking PI3Kδ is sufficient to mediate lasting tumor immunity of adoptively transferred T cells by preventing premature differentiation and improving mitochondrial fitness. Our data suggest that addition of CAL101 to ACT expansion protocols could greatly improve T cell therapies for solid tumors by preventing T cell differentiation and improving mitochondrial function.

scholarly journals 668 Lipid-instructed metabolic rewiring unleash the anti-tumor potential of CD8+ T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A696-A696
Author(s):  
Teresa Manzo ◽  
Carina Nava Lauveson ◽  
Teresa Maria Frasconi ◽  
Silvia Tiberti ◽  
Ignazio Caruana ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd8 T Cells ◽  
Mitochondrial Function ◽  
Cd8 T Cell ◽  
T Cell Differentiation ◽  
Metabolic Reprogramming ◽  
Tumor Control ◽  
Metabolic Fitness

BackgroundAdoptive cell therapy (ACT) harnesses the immune system to recognise tumor cells and carry out an anti-tumor function. However, metabolic constraints imposed by the tumour microenvironment (TME) suppress anti-tumor responses of CTL by reshaping their metabolism and epigenetic landscape. We have recently demonstrated that progressive accumulation of specific long-chain fatty acids (LCFAs) impair mitochondrial function and drives CD8+ T cell dysfunction. In this scenario, maintaining T cells in a less-differentiated state and with high metabolic plasticity during ex vivo T cell production and after infusion may have a strong therapeutic impact. Here, we propose a novel strategy to boost ACT efficacy by implementing T cell long-term functionality, metabolic fitness and preventing exhaustion through lipid-induced mitochondrial rewiring.MethodsWe screen different LCFAs and assess their ability to shape CD8+ T cell differentiation using multi-parametric flow cytometry, proliferation and cytotoxic assays, together with a complete transcriptomic and epigenomic profiling. Metabolic reprogramming of lipid-treated CD8+ T cell was examined by bioenergetic flux measurements paired with metabolomic and lipidomic analysis. Finally, the anti-tumor responses of lipid-instructed CD8 T cells was evaluated in a melanoma mouse model, known to poorly respond to immunotherapy.ResultsLCFAs-treated CD8+ T cells are endowed with highly effector and cytotoxic features but still retaining a memory-like phenotype with decreased PD1 protein levels. Consistently, analysis of the bioenergetic profile and mitochondrial activity has shown that LCFA-instructed CD8+ T cells display a greater mitochondrial fitness. Thus, in vitro LCFA-instructed CD8+ T cells are characterized by higher mitochondrial fitness, potent functionality, memory-like phenotype and PD-1 down-regulation, overall evoking the ideal T cell population associated with a productive anti-tumor response. The therapeutic potential of CD8 T cells lipid-induced metabolic rewiring was further confirmed in vivo. ACT performed with LCFA-reprogrammed CD8 T cells induces higher frequency of memory T cells, which show high polyfunctionality and mitochondrial function, decreased PD1 expression, ultimately resulting in improved tumor control. In addition, LCFA-induced metabolic rewiring during manufacturing of human CAR-redirected T cells, generated a CD8+ T cell memory-like population with higher mitochondrial fitness coupled with a much potent cytotoxic activity.ConclusionsThese results suggest that LCFAs dictate the fate of CD8+ T cell differentiation and could be considered as a molecular switch to fine-tune memory T cell formation and metabolic fitness maintenance, linking lipid metabolism to anti-tumor surveillance. This will be of fundamental importance for a new generation of adoptive T cell-based therapies.Ethics ApprovalThe experiments described were performed in accordance with the European Union Guideline on Animal Experiments and mouse protocols were approved by Italian Ministry of Health and the IEO Committee.

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

Allogeneic Vδ1 gamma delta T cells engineered with glypican-3 (GPC3)-specific CAR expressing soluble IL-15 have enhanced antitumor efficacy against hepatocellular carcinoma in preclinical models.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14511-e14511
Author(s):  
Amani Makkouk ◽  
Xue (Cher) Yang ◽  
Taylor Barca ◽  
Anthony Lucas ◽  
Mustafa Turkoz ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Healthy Donor ◽  
Cytokine Production ◽  
Tumor Control ◽  
Gamma Delta ◽  
T Cell Therapy ◽  
Donor Pbmcs

e14511 Background: Autologous αβ chimeric antigen receptor (CAR) T cell therapy has shown promising clinical results in hematologic malignancies but limited success in solid tumors. Allogeneic αβ T cell therapy may overcome several challenges faced by autologous therapy but carries the risk of graft-versus-host disease (GvHD) and does not readily recognize multiple tumor-associated antigens. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells in an MHC-unrestricted manner without causing GvHD. The Vδ1 subset is preferentially localized in peripheral tissue and is critical for tumor immunosurveillance. Engineering Vδ1 T cells with CARs can further enhance antitumor activity and represents an attractive and safe approach to treating solid tumors. However, their clinical use has been hindered by the limited number of circulating Vδ1 T cells. Here, we describe the development of the first allogeneic Vδ1 T cells that have been expanded from healthy donor PBMCs and genetically modified to secrete IL-15 (sIL15) and express a CAR targeting glypican-3 (GPC3), a rational target for hepatocellular carcinoma (HCC). Methods: Vδ1 T cells in healthy donor PBMCs were activated by a Vδ1-specific monoclonal antibody and transduced with 41BBζ or 41BBζ-sIL15 GPC3-CARs prior to cell expansion, αβ T cell depletion and cryopreservation. In vitro characterization included: 1) co-culture assays with GPC3-expressing HCC targets HepG2 and PLC/PRF/5, 2) phenotypic analysis by flow cytometry, and 3) cytokine production by multiplexed immunoassay. For in vivo assessment of tumor control, immunodeficient NSG mice were subcutaneously injected with HepG2 cells and treated with a single dose of 41BBζ or 41BBζ-sIL15 GPC3-CAR Vδ1 T cells. Additionally, tissues were harvested 7 days post transfer and analyzed by flow cytometry for Vδ1 T cell tissue homing and proliferation, or at end of study and analyzed for GvHD by immunohistochemistry. Results: Vδ1 T cells expanded over 10,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low and high GPC3 levels in vitro. In a HepG2 mouse model, GPC3-CAR Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose was able to efficiently control tumor burden without causing GvHD. Importantly, 41BBζ-sIL15 GPC3-CAR Vδ1 cells displayed enhanced tumor-specific proliferation that resulted in better tumor control without any toxicity. Conclusions: Our results show that expanded Vδ1 T cells engineered with GPC3-CAR and sIL-15 represent a promising platform for safe and effective off-the-shelf treatment of HCC.

scholarly journals CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lena Andersch ◽  
Josefine Radke ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
Annika Winkler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Lines ◽  
T Cell Therapy ◽  
Retinoblastoma Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

scholarly journals Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

2020 ◽  
Vol 8 (2) ◽  
pp. e000896
Author(s):  
Talia Velasco-Hernandez ◽  
Samanta Romina Zanetti ◽  
Heleia Roca-Ho ◽  
Francisco Gutierrez-Aguera ◽  
Paolo Petazzi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
High Affinity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundThere are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19– either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19– B-ALL relapses and CD19– preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.MethodsHere, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.ResultsConformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.ConclusionsWe report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22–CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

scholarly journals In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

scholarly journals Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 139 ◽  
Author(s):  
Jing Cui ◽  
Herui Wang ◽  
Rogelio Medina ◽  
Qi Zhang ◽  
Chen Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Therapeutic Potential ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

scholarly journals Targeted in-vitro-stimulation reveals highly proliferative multi-virus-specific human central memory T cells as candidates for prophylactic T cell therapy

PLoS ONE ◽  
2019 ◽  
Vol 14 (9) ◽  
pp. e0223258 ◽  
Author(s):  
Benjamin Faist ◽  
Fabian Schlott ◽  
Christian Stemberger ◽  
Kevin M. Dennehy ◽  
Angela Krackhardt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Memory T Cells ◽  
Central Memory ◽  
T Cell Therapy ◽  
Central Memory T Cells

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1431-1439 ◽  
Author(s):  
Ross N. La Motte-Mohs ◽  
Elaine Herer ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Development ◽  
T Cell Differentiation ◽  
Human Cord Blood ◽  
Hematopoietic Stem

AbstractThe Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood–derived HSCs cultured on Delta-like 1–expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 αβ–T-cell receptor (TCR)–bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro–differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

scholarly journals CD69 Association with Jak3/Stat5 Proteins Regulates Th17 Cell Differentiation

2010 ◽  
Vol 30 (20) ◽  
pp. 4877-4889 ◽  
Author(s):  
Pilar Martín ◽  
Manuel Gómez ◽  
Amalia Lamana ◽  
Arantxa Cruz-Adalia ◽  
Marta Ramírez-Huesca ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Th17 Cells ◽  
T Cell Differentiation ◽  
Orphan Receptor ◽  
Interleukin 17 ◽  
Deficient Mice

ABSTRACT T-cell differentiation involves the early decision to commit to a particular pattern of response to an antigen. Here, we show that the leukocyte activation antigen CD69 limits differentiation into proinflammatory helper T cells (Th17 cells). Upon antigen stimulation in vitro, CD4+ T cells from CD69-deficient mice generate an expansion of Th17 cells and the induction of greater mRNA expression of interleukin 17 (IL-17), IL 23 receptor (IL-23R), and the nuclear receptor retinoic acid-related orphan receptor γt (RORγt). In vivo studies with CD69-deficient mice bearing OTII T-cell receptors (TCRs) specific for OVA peptide showed a high proportion of antigen-specific Th17 subpopulation in the draining lymph nodes, as well as in CD69-deficient mice immunized with type II collagen. Biochemical analysis demonstrated that the CD69 cytoplasmic tail associates with the Jak3/Stat5 signaling pathway, which regulates the transcription of RORγt and, consequently, differentiation toward the Th17 lineage. Functional experiments in Th17 cultures demonstrated that the selective inhibition of Jak3 activation enhanced the transcription of RORγt. Moreover, the addition of exogenous IL-2 restored Stat5 phosphorylation and inhibited the enhanced Th17 differentiation in CD69-deficient cells. These results support the early activation receptor CD69 as an intrinsic modulator of the T-cell differentiation program that conditions immune inflammatory processes.

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