Neddylation Pathway Regulates Treg Differentiation and T Cell Function in Chronic Lymphocytic Leukemia (CLL) Ex Vivo and Murine In Vivo Studies

Introduction: Inhibitors of B-cell receptor associated kinases (e.g., Bruton tyrosine kinase and phoshpoinotiside-3 kinase) have led to significant improvement in outcomes of patients with CLL. Furthermore, such therapies have immunomodulatory effects. This is particularly relevant given that T cells from patients with CLL are functionally compromised and demonstrate impaired immune synapse formation, adhesion, migration and cytotoxicity. It is critical to improve understanding of the immunomodulatory properties of the novel agents as this will help understand their effect on the immune system in CLL, mitigate toxicities as well as inform future drug development. Pevonedistat, (MLN4924) forms an irreversible covalent adduct with NEDD8, a ubiquitin-like modifier, thereby inhibiting the NEDD8-activating enzyme (NAE). This leads to decreased neddylation and reduced activity of cullin-RING E3 ubiquitin ligases (CRLs). Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). We have shown that pevonedistat abrogates NFκB activation in CLL cells. Importantly, NFκB activation is indispensable in T-cell activation. However, there is paucity of data regarding the immune effects of targeting neddylation. Here we address this knowledge gap and demonstrate that NAE inhibition may have favorable immunomodulatory effects in CLL. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. Pevonedistat was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with pevonedistat for 1 hour prior to T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were TCR-activated for 7 days under Th1/2/17/Treg-differentiation conditions. BALB/c mice were administered 60 mg/kg pevonedistat SC twice weekly for 3 weeks and T cell populations were analyzed by flow cytometry. Results: In vitro treatment of T cells with pevonedistat led to rapid reduction in neddylated cullins and stabilization of pIκBα. NAE inhibition did not impede proximal TCR signaling following TCR stimulation (pZAP70, pERK). GSEA demonstrated downmodulation of NFκB and IL-2 signaling pathways in pevonedistat-treated cells by 3 h. Despite this, CD4/CD8+ T cells exhibited normal induction of early activation markers (CD40L, CD69). By contrast, we observed reduced expression of CD38, HLA-DR and PD-1 and diminished CD25 following continuous treatment with pevonedistat for 72 h. This was accompanied by dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4/CD8+ T cell subsets (CFSE), but no apoptosis. Sorted naive T cells treated with pevonedistat in Th/Treg polarizing conditions exhibited an increase in IFNγ secretion and a decrease in IL-4, suggesting a shift toward Th1 phenotype. Furthermore, we observed a robust decrease of the inducible Treg (iTreg) FoxP3+ population. Loss of iTregs was accompanied by ablated IL-2/STAT5 signaling. Concurrently, we observed a modest increase in Th17 subpopulation following NAE inhibition. We found increased expression of HIF-1α, a CRL target, in pevonedistat-treated T cells, which may have contributed to this phenomenon. To mimic the clinical pharmacokinetics of pevonedistat, we performed 2 h pulse treatment with pevonedistat prior to TCR stimulation. Under these conditions NFκB activity fully recovered by 24 h. Importantly, allogeneic (OCI-LY19 cells) and autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity, perforin and granzyme B production were not disrupted by NAE inhibition. In vivo administration of pevonedistat in immunocompetent BALB/c mice resulted in a decrease of Treg population, confirming in vitro data. Conclusions: Our data suggest that targeting neddylation may help rebalance T cells towards healthy immune subsets in CLL via the reduction of the Treg/Th2 phenotypes. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. Disclosures Berger: Millennium Pharmaceuticals, Inc., Cambridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Danilov:TG Therapeutics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Seattle Genetics: Consultancy; Gilead Sciences: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Celgene: Consultancy; Curis: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy.

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Ibrutinib Is an Irreversible Molecular Inhibitor of Interleukin-2 Inducible Kinase: Expanding Therapeutic Potential and Modulating a Th1 Selective Pressure in CD4 T-Cells

Blood ◽

10.1182/blood.v120.21.775.775 ◽

2012 ◽

Vol 120 (21) ◽

pp. 775-775 ◽

Cited By ~ 1

Author(s):

Jason A Dubovsky ◽

Kyle A Beckwith ◽

Jennifer A. Woyach ◽

Samantha M. Jaglowski ◽

Joshua Hessler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Adaptive Immunity ◽

Cd4 T Cells ◽

Research Funding ◽

Cell Receptor ◽

Receptor Activation ◽

Genetic Ablation

Abstract Abstract 775 In chronic lymphocytic leukemia (CLL), mounting evidence points to an aberrant tumor associated Th2 bias that drives leukemic cell immune evasion, promotes formation of a supportive niche microenvironment, and functionally cripples innate and adaptive immunity. The end result is a high incidence of infections which is the primary cause of mortality in CLL. This same Th2 bias is induced by many other types of cancer. Th2 CD4 T-cells are singularly dependent upon IL-2-inducible T-cell kinase (ITK) for activation whereas Th1 CD4 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) which conducts T-cell receptor activation even in the absence of ITK. Thus, a clinically viable ITK inhibitor would be ideal for targeting immune suppression associated with CLL and potentially other types of cancer. Unfortunately, no such therapeutic is currently available. Ibrutinib, a confirmed inhibitor of the Bruton's tyrosine kinase (BTK) that irreversibly blocks downstream B-cell receptor activation, has demonstrated outstanding clinical activity in phase I/II clinical trials resulting in durable remissions in CLL. Our studies unveiled a previously uncharacterized Th1 cytokine switch in ibrutinib treated CLL patients which could not be attributed to B-lymphocytes. This ibrutinib-induced Th1 T-cell skewing was confirmed using the EμTCL1 mouse model of leukemia. Such alterations in cytokine patterns were reminiscent of mouse studies in which genetic ablation of ITK subverted Th2 immunity, thereby potentiating Th1-based adaptive immunity. The striking homology between BTK and ITK combined with intriguing in silico docking studies and promising in vitro kinase inhibition profiles with ibrutinib led to the hypothesis that this could be the first clinically viable irreversible ITK inhibitor. Cellular probe assays confirmed that the active site of ITK was covalently blocked by ibrutinib at pharmacologically relevant doses. Our comprehensive molecular analyses of T-cell signaling confirmed this in the Jurkat cell line. We further confirmed both molecular and functional outcomes in primary and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation offering a molecular explanation for the selective outgrowth of cytotoxic Th1 biased immunity. We further confirmed this effect using T-cells directly derived from CLL patients. To demonstrate that ibrutinib-induced ITK inhibition had direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model we clearly demonstrated complete recovery of functional immunity and all ibrutinib treated mice survived a potentially lethal Listeria monocytogenes infection. Our results expose novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib's irreversible ITK inhibitory effects may prove effective in a number of other autoimmune, inflammatory, and viral diseases, including influenza A and HIV/AIDS. Disclosures: Jaglowski: Pharmacyclics: Research Funding. Chang:Pharmacyclics, Inc.: Employment. Maddocks:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

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Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models

Blood ◽

10.1182/blood-2021-146347 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3719-3719

Author(s):

Vi Lam ◽

Xiaoguang Wang ◽

Scott R Best ◽

Nur Bruss ◽

Tingting Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

Foxp3 Expression ◽

Tcr Signaling ◽

Advisory Committees

Abstract Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs. Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.

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The Absence of NLRP6 in Donor T Cells Exacerbates Gvhd

Blood ◽

10.1182/blood-2021-152089 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2766-2766

Author(s):

Masahiro Suto ◽

Eri Matsuki ◽

Masahiro Miyata ◽

Erika Sekiguchi ◽

Hiroya Tamaki ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Cell Responses ◽

Donor T Cells ◽

Induced Apoptosis

Abstract The Nlrp6 (NOD-like receptor family pyrin domain containing 6) inflammasome is important for intestinal epithelial cell innate immune responses and for maintaining gut homeostasis by preventing microbial dysbiosis. Contrary to its role in epithelial cell inflammasome-mediated responses, we recently showed that Nlrp6 in gut epithelial cells exacerbates GVHD in a manner independent of the inflammasome or gut microbiota. However, donor allogeneic T cells are also critical for GVHD development, yet, the function of Nlrp6 in allogeneic T cells is unknown. We hypothesized that Nlrp6 deficient donor T cells would ameliorate experimental GVHD. To test our hypothesis, WT-BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x10 6 bone marrow and 1.0x10 6 splenic CD90 +T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Nlrp6 -/- donors. Contrary to our hypothesis, the survival of allogeneic recipients of Nlrp6 -/- donor T cells was significantly worse than those receiving WT-B6 T cells (p<0.05). Nlrp6 -/- donor T cells also caused greater GVHD mortality and morbidity in an MHC mismatched haploidentical B6 into B6D2F1 model (p<0.05) and an MHC mismatched B10.BR into B6 model. Similar results were obtained using B6 into BALB/c and B6 into B6D2F1 models performed at the University of Michigan, suggesting our results were not unique to local environmental factors. By contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. Because the B6 into C3H.sw model is largely driven by CD8+ T cells whereas the previous models are mediated by both CD4+ and CD8+ T cells, we examined whether Nlrp6 separately regulates CD4+ and CD8+ T cell-mediated GVHD. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x10 6 CD4+ or CD8+ T cells from B6-WT or Nlrp6 -/- animals. GVHD severity and mortality (P<0.05) were enhanced only when Nlrp6 -/- CD4+ T cells were transplanted. These data suggested that Nlrp6 regulates allogeneic T cell responses in a subset-specific manner. To explore how Nlrp6 regulates intrinsic responses in donor T cell subsets, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation. Consistent with the lack of increased GVHD induced by CD8+ Nlrp6 -/- donor T cells in the B6 into C3H.sw model, Nlrp6 -/- CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, when stimulated with either anti-CD3/CD28 antibodies or lethally irradiated allogeneic antigen presenting cells in a mixed lymphocyte reaction. In addition, activation-induced apoptosis was decreased in Nlrp6 -/- CD4+ T cells compared to WT T cells. Importantly, Treg suppressive function was not altered in Nlrp6 -/- T cells. Therefore, increased proliferative responses and resistance to activation-induced apoptosis may have contributed to the enhanced GVHD caused by Nlrp6 -/- donor T cells. Increased Th1 and Th17 polarization is associated with worse GVHD. Because only CD4+ Nlrp6 -/- T cells enhanced GVHD, we tested whether Nlrp6 influenced T helper cell differentiation into Th1, Th17, and Th2 subsets. Consistent with our in vivo data, Th1 in vitro differentiation was enhanced in Nlrp6 -/- CD4+ T cells. To determine the molecular signaling events altered by Nlrp6 deficiency, we tested various T cell activation signaling pathways and found that phosphorylation of ZAP-70 was increased in Nlrp6 -/- T cells. These data suggested that Nlrp6 in donor T cells may regulate allo-immune responses via ZAP-70 pathway. GVH and graft-versus-tumor (GVT) responses are intricately linked. Because CD8+ responses were not affected by Nlrp6 deficiency, we hypothesized that GVT responses would be unaltered in Nlrp6 -/- donor T cells. Indeed, Nlrp6 -/- T cells showed equivalent in vivo GVL responses to MLL-AF4 leukemia cells as WT-T cells. Hence Nlrp6in donor T cells is not required for GVT responses. Altogether our data suggested that Nlrp6 negatively-regulates allogeneic donor CD4+ T cell responses, possibly via negative regulation of ZAP-70 signaling, resulting in mitigation of GVHD and maintenance of robust GVT responses. Disclosures Ishizawa: AbbVie: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; Bristol Myers Squibb: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Consultancy; SymBio: Honoraria, Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; IQVIA: Research Funding.

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Cd40-Independent Pathways of T Cell Help for Priming of Cd8+ Cytotoxic T Lymphocytes

Journal of Experimental Medicine ◽

10.1084/jem.191.3.541 ◽

2000 ◽

Vol 191 (3) ◽

pp. 541-550 ◽

Cited By ~ 144

Author(s):

Zhengbin Lu ◽

Lingxian Yuan ◽

Xianzheng Zhou ◽

Eduardo Sotomayor ◽

Hyam I. Levitsky ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cytotoxic T Lymphocyte ◽

Cell Communication ◽

Cd8 T Cell ◽

Cd4 Help ◽

T Cell Help

In many cases, induction of CD8+ CTL responses requires CD4+ T cell help. Recently, it has been shown that a dominant pathway of CD4+ help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4+ T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide–specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4+ T helper cells, respectively. We found that CD4+ T cells can provide potent help for DCs to activate CD8+ T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4+ help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4+–CD8+ T cell communication via lymphokines. Therefore, we conclude that CD4+ help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4+–CD8+ T cell communication.

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CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer

Journal of Experimental Medicine ◽

10.1084/jem.20011845 ◽

2002 ◽

Vol 196 (4) ◽

pp. 481-492 ◽

Cited By ~ 62

Author(s):

Kristin V. Tarbell ◽

Mark Lee ◽

Erik Ranheim ◽

Cheng Chi Chao ◽

Maija Sanna ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Receptor ◽

Glutamic Acid Decarboxylase ◽

Cd4 T Cells ◽

Cell Receptor ◽

Acid Decarboxylase ◽

Gad 65

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

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CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽

10.1182/blood-2009-01-200923 ◽

2009 ◽

Vol 114 (3) ◽

pp. 580-588 ◽

Cited By ~ 45

Author(s):

Kathrin Gollmer ◽

François Asperti-Boursin ◽

Yoshihiko Tanaka ◽

Klaus Okkenhaug ◽

Bart Vanhaesebroeck ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Early Time ◽

Cell Receptor ◽

Tcr Signaling ◽

Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

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173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.173 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A185-A185

Author(s):

Michelle Fleury ◽

Derrick McCarthy ◽

Holly Horton ◽

Courtney Anderson ◽

Amy Watt ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

T Cell Receptor ◽

Cytokine Production ◽

Cell Receptor ◽

Great Promise ◽

And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

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BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to In Vitro Differentiation into Functional Foxp3+ T Regulatory Cells

PLoS ONE ◽

10.1371/journal.pone.0112242 ◽

2014 ◽

Vol 9 (11) ◽

pp. e112242 ◽

Cited By ~ 2

Author(s):

Ghanashyam Sarikonda ◽

Georgia Fousteri ◽

Sowbarnika Sachithanantham ◽

Jacqueline F. Miller ◽

Amy Dave ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

T Regulatory Cells ◽

Cell Receptor ◽

Regulatory Cells ◽

In Vitro Differentiation

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Selective anergy of V beta 8+,CD4+ T cells in Staphylococcus enterotoxin B-primed mice.

Journal of Experimental Medicine ◽

10.1084/jem.172.4.1065 ◽

1990 ◽

Vol 172 (4) ◽

pp. 1065-1070 ◽

Cited By ~ 216

Author(s):

Y Kawabe ◽

A Ochi

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

T Cell Anergy ◽

Specific Cytotoxicity ◽

Enterotoxin B ◽

Staphylococcus Enterotoxin B

The cellular basis of the in vitro and in vivo T cell responses to Staphylococcus enterotoxin B (SEB) has been investigated. The proliferation and cytotoxicity of V beta 8.1,2+,CD4+ and CD8+ T cells were observed in in vitro response to SEB. In primary cytotoxicity assays, CD4+ T cells from control spleens were more active than their CD8+ counterparts, however, in cells derived from SEB-primed mice, CD8+ T cells were dominant in SEB-specific cytotoxicity. In vivo priming with SEB abrogated the response of V beta 8.1,2+,CD4+ T cells despite the fact that these cells exist in significant number. This SEB-specific anergy occurred only in V beta 8.1,2+,CD4+ T cells but not in CD8+ T cells. These findings indicate that the requirement for the induction of antigen-specific anergy is different between CD4+ and CD8+ T cells in post-thymic tolerance, and the existence of coanergic signals for the induction of T cell anergy is suggested.

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Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Blood ◽

10.1182/blood.v99.6.2084 ◽

2002 ◽

Vol 99 (6) ◽

pp. 2084-2093 ◽

Cited By ~ 101

Author(s):

Alexander D. McLellan ◽

Michaela Kapp ◽

Andreas Eggert ◽

Christian Linden ◽

Ursula Bommhardt ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Antigen Expression ◽

Cell Receptor ◽

In Vitro Assays ◽

Peptide Complexes ◽

Marginal Zones

Abstract Mouse spleen contains CD4+, CD8α+, and CD4−/CD8α− dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule–associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8α+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8α+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)–transgenic T cells. CD8α− or CD8α+DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I–peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8α DCs was responsible for the low allostimulatory capacity of CD8α+ DCs in vitro. Surprisingly, both CD8α+ DCs and CD4−/CD8− DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8α+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8α+ DCs and CD8+ T cells.

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