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Pancreatic ductal adenocarcinoma (PDA) is a lethal and metastatic malignancy resistant to therapy. Elucidating how pancreatic tumor-specific T cells differentiate and are maintained in vivo could inform novel therapeutic avenues to promote T cell antitumor activity. Here, we show that the spleen is a critical site harboring tumor-specific CD8 T cells that functionally segregate based on differential Cxcr3 and Klrg1 expression. Cxcr3+ Klrg1- T cells express the memory stem cell marker Tcf1, whereas Cxcr3-Klrg1 + T cells express GzmB consistent with terminal differentiation. We identify a Cxcr3+ Klrg1+ intermediate T cell subpopulation in the spleen that is highly enriched for tumor specificity. However, tumor-specific T cells infiltrating primary tumors progressively downregulate both Cxcr3 and Klrg1 while upregulating exhaustion markers PD-1 and Lag-3. We show that antigen-specific T cell infiltration into PDA is Cxcr3 independent. Further, Cxcr3-deficiency results in enhanced antigen-specific T cell IFNγ production in primary tumors, suggesting that Cxcr3 promotes loss of effector function. Ultimately, however, Cxcr3 was critical for mitigating cancer cell dissemination following immunotherapy with CD40 agonist + anti-PD-L1 or T cell receptor engineered T cell therapy targeting mesothelin. In the absence of Cxcr3, splenic Klrg1 + GzmB + antitumor T cells wain while pancreatic cancer disseminates suggesting a role for these cells in eliminating circulating metastatic tumor cells. Intratumoral myeloid cells are poised to produce Cxcl10, whereas splenic DC subsets produce Cxcl9 following immunotherapy supporting differential roles for these chemokines on T cell differentiation. Together, our study supports that Cxcr3 mitigates tumor cell dissemination by impacting peripheral T cell fate rather than intratumoral T cell trafficking.

BackgroundAchieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express a mesothelin-specific T cell receptor (TCRMsln) accumulate in autochthonous PDA, mediate therapeutic antitumor activity, but fail to eradicate tumors in part due to acquisition of a dysfunctional exhausted T cell state.MethodsHere, we investigated the role of immune checkpoints in mediating TCR engineered T cell dysfunction in a genetically engineered PDA mouse model. The fate of engineered T cells that were either deficient in PD-1, or transferred concurrent with antibodies blocking PD-L1 and/or additional immune checkpoints, were tracked to evaluate persistence, functionality, and antitumor activity at day 8 and day 28 post infusion. We performed RNAseq on engineered T cells isolated from tumors and compared differentially expressed genes to prototypical endogenous exhausted T cells.ResultsPD-L1 pathway blockade and/or simultaneous blockade of multiple coinhibitory receptors during adoptive cell therapy was insufficient to prevent engineered T cell dysfunction in autochthonous PDA yet resulted in subclinical activity in the lung, without enhancing anti-tumor immunity. Gene expression analysis revealed that ex vivo TCR engineered T cells markedly differed from in vivo primed endogenous effector T cells which can respond to immune checkpoint inhibitors. Early after transfer, intratumoral TCR engineered T cells acquired a similar molecular program to prototypical exhausted T cells that arise during chronic viral infection, but the molecular programs later diverged. Intratumoral engineered T cells exhibited decreased effector and cell cycle genes and were refractory to TCR signaling.ConclusionsAbrogation of PD-1 signaling is not sufficient to overcome TCR engineered T cell dysfunction in PDA. Our study suggests that contributions by both the differentiation pathways induced during the ex vivo T cell engineering process and intratumoral suppressive mechanisms render engineered T cells dysfunctional and resistant to rescue by blockade of immune checkpoints.

Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (TRM) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating TRM in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that TRM establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific TRM., Irreversible electroporation (IRE), a soft-tissue ablation technique used for tumour ablation, has been suggested to promote systemic immune responses. Here the authors show that IRE, followed by anti-CTLA-4 blockade, elicits the expansion of tumor antigen-specific CD8+ T cells and is associated with tissue residency and improved anti-tumor immune response in a preclinical model of prostate cancer.

Genocea’s ATLAS platform is an empirical bioassay that uses patient autologous immune cells to identify both true neoantigens and Inhibigens࣪ for inclusion in or exclusion from neoantigen-targeted vaccines and cell therapies, respectively. In ATLAS, patient-derived antigen-presenting cells (APCs) are pulsed with E. coli expressing individual mutations identified from the patient mutanome ± listeriolysin O, enabling interrogation of both CD8+ and CD4+ T cell recognition. True neoantigens induce T cell activation and cytokine release, while Inhibigens lead to a downregulation of T cell responses and thus can promote tumor growth. Previous ATLAS screening of CD8+ T cells from mice carrying B16F10 mouse melanoma tumors identified both neoantigens and Inhibigens. Upon therapeutic vaccination, adjuvanted neoantigens generated immunogenicity and anti-tumor efficacy1. In contrast, therapeutic vaccination with multiple ATLAS-identified Inhibigens, alone or in combination with an otherwise-protective vaccine, led to accelerated tumor growth, impaired T cell responses, and abrogated tumor immune infiltration. Our current study further explores the mechanism of Inhibigen-specific responses through adoptive transfer of vaccine-experienced T cells into tumor-bearing recipient mice, as well as through analysis of T cell gene expression. Additionally, in order to determine whether Inhibigen identification and treatment translates into pro-tumor effects universally across tumor models, we performed ATLAS screening on CD4+ and CD8+ T cells isolated from mice bearing orthotopic KPC pancreatic cancer. Out of 73 total non-synonymous mutations, we successfully identified 14 CD4+ and 15 CD8+ true neoantigens, and 16 CD4+ and 18 CD8+ Inhibigens. This is the first known comprehensive characterization of endogenous antigens in this model. Therapeutic administration of neoantigens as adjuvanted peptide vaccines in KPC tumor-bearing mice led to smaller tumor sizes and reduced ascites volumes, whereas Inhibigen vaccination accelerated tumor growth. Mouse studies are ongoing and additional data will be presented. Taken together, our data from human cancer patients and two mouse cancer models support the importance of appropriate neoantigen selection and Inhibigen identification and exclusion from cancer therapies. Genocea’s GEN-011 neoantigen-targeted peripheral T cell (NPT) therapy candidate, designed using ATLAS-identified neoantigens and omitting Inhibigens, is being evaluated in an ongoing clinical trial (NCT04596033). Continued exploration of mechanisms of action of Inhibigen-specific responses may reveal new paradigms of cancer immune evasion. 1H Lam et al, Cancer Discov 2021;11:1-18 Citation Format: Hanna S. Starobinets, Victoria L. DeVault, Zoe C. Schmiechen, Ebony A. Miller, Eduardo Cruz, Meagan R. Rollins, Adam L. Burrack, Stephanie J. Rinaldi, Julie Arnold, Emily Tjon, Kyle Gonzalez, Dimitry Lineker, Hubert Lam, Ingunn M. Stromnes, Jessica B. Flechtner. ATLAS-identified Inhibigen-specific responses accelerate tumor growth in mouse melanoma and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2088.

Pancreatic cancer is a particularly lethal malignancy that resists immunotherapy. In this study, using a preclinical pancreatic cancer murine model, we demonstrate a progressive decrease in IFN-γ and granzyme B and a concomitant increase in Tox and IL-10 in intratumoral tumor-specific T cells. Intratumoral myeloid cells produced elevated IL-27, a cytokine that correlates with poor patient outcome. Abrogating IL-27 signaling significantly decreased intratumoral Tox+ T cells and delayed tumor growth yet was not curative. Agonistic αCD40 decreased intratumoral IL-27–producing myeloid cells, decreased IL-10–producing intratumoral T cells, and promoted intratumoral Klrg1+Gzmb+ short-lived effector T cells. Combination agonistic αCD40+αPD-L1 cured 63% of tumor-bearing animals, promoted rejection following tumor rechallenge, and correlated with a 2-log increase in pancreas-residing tumor-specific T cells. Interfering with Ifngr1 expression in nontumor/host cells abrogated agonistic αCD40+αPD-L1 efficacy. In contrast, interfering with nontumor/host cell Tnfrsf1a led to cure in 100% of animals following agonistic αCD40+αPD-L1 and promoted the formation of circulating central memory T cells rather than long-lived effector T cells. In summary, we identify a mechanistic basis for T cell exhaustion in pancreatic cancer and a feasible clinical strategy to overcome it.

T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of human autologous dendritic cells from monocytes Basic Protocol 2: In vitro priming and expansion of human antigen-specific T cells Basic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing data Basic Protocol 4: Validation of T cell receptor expression and functionality in vitro Basic Protocol 5: Rapid expansion of T cell receptor-transduced T cells and human T cell clones.

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.

BackgroundPancreatic ductal adenocarcinoma (PDA) is a lethal malignancy characterized by a highly suppressive tumor microenvironment. Despite this, engineered T cell therapy has promise for effectively targeting PDA. To identify the underlying mechanisms of antigen-specific engineered T cell immunosuppression in PDA, we create novel TCR knock-in mouse models for a robust and standardized source of naïve mesothelin (Msln)-specific T cells.MethodsSpecifically, we integrate two murine mesothelin-specific TCRs into the physiologic Trac locus in primary murine T cells and zygotes using CRISPR/Cas9 and rAAV expressing the TCR DNA. Simultaneously using CRISPR/Cas9, Msln was disrupted to circumvent T cell tolerance.ResultsThis strategy resulted in the rapid generation of homozygous TCR Trac knock-in mice and with homozygous null mutations in Msln. In these TCR-exchanged (TRex) mice, most T cells expressed the 1045 (high affinity) or 7431 (low affinity) as determined by tetramer staining. TRex T cells exhibit a naïve phenotype and rapidly differentiate into effector T cells upon antigenic stimulation. While the high affinity 1045 TCR elicits function in CD4 T cells, the lower affinity 7431 T cells exhibit a higher functional avidity and less TCR downregulation when antigen is limiting. Historical TCR transgenic T cells, in which the TCR is randomly integrated into the genome, exhibit increased PD1, CD25, and CD69, decreased functionality, and a bias to CD25-Foxp3+ Treg as compared to T cells from TRex mice. Further, TCR Trac integration in primary T cells retain superior function following repetitive antigenic stimulations retrovirally transduced T cells. Adoptive transfer of 1045 TRex T cells significantly prolongs survival of mice bearing autochthonous PDA. When combined with a vaccine, 1045 TRex T cells cause involution of the fibroinflammatory tumor stroma.ConclusionsIn sum, we rapidly generate mice that physiologically express the desired TCR, circumventing the shortcomings of standard T cell engineering strategies and TCR transgenic models.Ethics ApprovalUniversity of Minnesota Institutional Animal Care and Use Committee approved all animal studies to Dr. Ingunn Stromnes (2005-38115A.) Generation of TCR knockin (KI) animals was performed in the Mouse Genetic Laboratory at the University of Minnesota.

Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that is resistant to conventional therapies including monotherapy using PD-1 or PD-L1 inhibition. Combination agonistic anti-CD40 and PD-1/PD-L1 blockade have clinical promise in advanced cancer patients including PDA. The underlying mechanism(s) driving the therapeutic effects of this combination are ill-defined. Here, we create a syngeneic PDA animal model and utilize various genetic tools to assess how CD40 agonist, PD-L1 blockade or the combination impact tumor antigen-specific T cells using fluorescently-labeled peptide:MHC tetramers and cells in the tumor microenvironment. Molecular analyses of tumor cell escape variants is also performed. Methods: We recently developed a high-throughput orthotopic syngeneic KPC pancreatic cancer mouse model that expresses a novel model neoantigen in B6 mice described in Burrack et al., Cell Reports, 2019. We create fluorescently labeled peptide:H-2Db tetramers to track the fate of endogenous pancreatic tumor-antigen specific CD8+ T cells over time. Here, we use this model alone or mixed at a 1:1 ratio of KPC tumor cells that do not express the neoantigen to examine how agnostic anti-CD40 (a single dose, clone FGK145), anti-PDL1 (3 doses, clone 10F.932), or the combination impact tumor growth in the pancreas over time using bioluminescent imaging and high-resolution ultrasound. We use multiparameter flow cytometry to investigate how anti-CD40 +/- PD-L1 blockade impacts the phenotype, longevity and functionality of tetramer-binding T cells over time. We assess how other immune cell lineages are altered systemically and in the tumor microenvironment by quantifying myeloid subpopulations, B cells, NK cells and regulatory T cells following therapy. We use Batf3-/- mice and XCR1VenusDTR mice to assess the role of conventional type I dendritic cells (cDC1s) on therapeutic efficacy. We employ both cytokine and chemokine reporter strains to identify how anti-CD40 +/- PD-L1 blockade impacts inflammatory gene expression in immune cells enriched the tumor microenvironment. We examine the persistence and location of tetramer-binding T cells in the pancreas, lung and liver of mice following tumor eradication. Additionally, we re-derive resistant tumor cells from mice and evaluate the integrity of MHC class I antigen processing and presentation pathways. Finally, single cell sequencing is performed to assess the traits of subpopulations of tumor-antigen specific T cells that correlate with enhanced antitumor activity following therapy. Results: We show that anti-CD40 or anti-PD-L1 monotherapy have significant yet transient antitumor effects in mice with neoantigen+ PDA with distinct effects on tumor specific T cells. Objective responses occur in 100% of the monotherapy treated mice and survival is significantly prolonged. However, tumors recur in 100% of these animals. Tumor escape variants defective in MHC class I protein and Tap1 gene expression following IFN-gamma treatment ultimately emerge. In contrast, combination agonistic anti-CD40 + PD-L1 blockade synergize therapeutically resulting in cures in 60% of the animals and formation of pancreas resident memory T cells that specifically bind tetramer and express CD49a and CD103 following tumor eradication. Mechanistically, the combination selectively expands conventional type 1 dendritic cells (cDC1s) in the spleens and tumors of tumor-bearing animals. cDC1s in PDA are CD11c+MHCII+ and express CD8, CD103 and Xcr1. Using Batf3-/- mice or an Xcr1venusDTR transient cDC1 depletion model, we demonstrate a striking dependency on cDC1s for therapeutic benefit with anti-CD40 or PD-L1 blockade. Unexpectedly, we find that the expansion of cDC1s in pancreatic tumor-bearing animals is partially dependent on Xcr1 expression by DCs. Anti-CD40+PD-L1 blockade significantly expand the number of tetramer-binding T cells that express KLRG1 in PDA. The tetramer-binding T cells remain PD-1+ yet have lower expression of Lag3 and have heightened polyfunctionality as measured by cytokine production. Further studies using chemokine and cytokine reporter models, we uncover key differences in how anti-CD40 and anti-PD-L1 impact inflammatory gene expression by antigen presenting cells in PDA. Finally, we demonstrate the requirement for tumor neoantigen expression for efficacy because in mice that have tumors containing a 50:50 mixture of neoantigen+ pancreatic tumor cells with neoantigen- pancreatic tumor cells, combination anti-CD40 + PD-L1 blockade results in elimination of predominantly those tumor cells that express the neoantigen. Further single cell sequencing data on how this combination impacts tumor-antigen specific T cell subpopulations as well as epitope spreading will be discussed. Conclusions: These findings reveal for the first time to our knowledge that anti-CD40 + PD-L1 blockade synergize via the expansion of cDC1s in pancreatic tumor-bearing animals. Instead of anti-CD40 promoting priming of neoantigen-specific T cells, we find that this combination promotes the systemic expansion and intratumoral accumulation of KLRG1+ tumor-specific T cells that eradicate PDA and form pancreas resident CD49a+CD103+ memory T cells. Citation Format: Adam L. Burrack, Meagan R. Rollins, Ellen J. Spartz, Jackson F. Raynor, Iris Wang, Jason Mitchell, Tsuneyasu Kaisho, Brian Fife, Ross Kedl, Stephen Shen, Ingunn M. Stromnes. Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG12.

Tumor cells are capable of limiting antitumor CD8+ T cell responses through their cell surface expression of PD-L1. In addition to PD-1 expressed by CD8+ T cells, PD-L1 also binds to CD80 expressed by CD8+ T cells. The influence of the PD-L1/CD80 interaction on CD8+ T cell function has not been fully characterized, so we sought to investigate the impact of the PD-L1/CD80 interaction on PD-L1-induced apoptosis of activated CD8+ T cells. We found that CD8+ T cells that lacked CD80 expression got activated to the same extent as wild-type CD8+ T cells, but when cultured with anti-CD3 and PD-L1/Fc protein, activated CD8+ T cells that lacked CD80 expression survived better than activated wild-type CD8+ T cells. These findings indicate that PD-L1 induces apoptosis in activated CD8+ T cells in part by signaling through CD80. Thus, in the design and implementation of checkpoint blockade therapies that target PD-L1, it is essential that both binding partners for PD-L1, PD-1, and CD80 are considered.

Memory CD8+ T cells populate non-lymphoid tissues (NLT) and can protect against malignancies, but little is known about the establishment of tumor-specific tissue resident memory CD8+ T cells (TRM) by therapeutically initiated immune responses. We used a transplantable mouse model of prostate carcinoma and MHCI tetramers to track and quantify tumor-specific CD8+ T cells. Tumor challenge led to systemic expansion of endogenous, naïve tumor antigen-specific CD8+ T cells and established a small population of TRM (CD44hi, CD62Llo, CD69+, CD103+) in several NLTs. Previously, we demonstrated that debulking primary tumors using irreversible electroporation (IRE) induced expansion of tumor-specific T cells. We hypothesized that IRE would cooperate with checkpoint blockade to increase antigen-specific TRM and promote tumor clearance in vivo. Combination treatment with IRE followed by α-CTLA4 promoted a robust 50-fold expansion of proliferating (Ki67+) tumor-specific T cells in blood, a 10-fold increase of TRM in NLT, and was associated with complete remission of residual tumor. Conversely, monotherapy allowed tumor outgrowth and failed to boost T cell numbers, resulting in small populations of PD-1hi tumor-specific CD8+ T cells. Similarly, anti-PD-1 alone or following IRE was also ineffective, suggesting that combined IRE/α-CTLA4 therapy targets newly primed T cells. IRE also enhanced antigen presentation by XCR1+ dendritic cells, promoting T cell priming in regional lymph nodes. These results indicate that combining α-CTLA4 checkpoint immunotherapy with focal tumor ablation, such as IRE, capable of releasing high quantities and qualities of endogenous tumor antigens may serve as a potent in situ tumor vaccination strategy.

The endosteal niche is a component of the bone marrow microenvironment that can serve to protect hematological malignancies such as acute myeloid leukemia (AML) from standard chemotherapies such as cytarabine (Ara-C). Surviving AML cells harbored by this niche can eventually lead to relapse. The endosteal niche is rich in osteoblast lineage cells. U937 or KG1a AML cell lines were cultured with or without osteoblast lineage cells (MC3T3 or W-20-17 cell lines), challenged with doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM of Ara-C, and assayed for apoptosis via annexin-V staining and flow cytometry. Osteoblast lineage cells (MC3T3 or W-20-17 cell lines) were able to protect AML cells (U937 or KG1a cell lines) from Ara-C-induced apoptosis. Histone deacetylase inhibitors (HDACi) globally alter gene expression within cells. When we pre-treated osteoblast lineage cells (MC3T3) with the HDACi vorinostat (suberoylanilide hydroxamic acid, SAHA), it reduced the ability of the osteoblast lineage cells (MC3T3) to protect AML cells (U937) from Ara-C-induced apoptosis, which we have previously confirmed in the KG1a AML cell line as well. This indicates that osteoblast lineage cell-mediated protection of AML from Ara-C occurs via an HDACi sensitive mechanism. To begin to further explore the mechanism of action, we co-cultured AML cells (KG1a or U937) with and without osteoblast lineage cells (MC3T3) in the presence or absence of a transwell. We found that the presence of the transwell reduced the osteoblast lineage cell-mediated protection, indicating that osteoblast lineage cell-mediated protection of AML from Ara-C is cell contact dependent. Thus, osteoblast lineage cells can protect AML cells from Ara-C induced apoptosis, this protection can be reduced by pre-treatment of the osteoblast lineage cells with the HDACi vorinostat, and osteoblast lineage cell-mediated protection from Ara-C is cell contact dependent. These studies begin to characterize the mechanisms of osteoblast lineage cell-mediated protection of AML from Ara-C. Manipulating the protective properties of osteoblast lineage cells of the endosteal niche may help make AML cells more susceptible to chemotherapeutics. Therefore, developing combination therapies that target the protective mechanisms of osteoblast-lineage cells may help to further deplete the bone marrow microenvironment of AML cells and prevent relapse of disease. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Meagan R. Rollins, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblast-lineage cells protect AML cells from cytarabine-induced apoptosis via a mechanism sensitive to HDACi and reduced cell-cell contact [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2137.

T cell cytokine production was recently shown to depend not only on the T cell antigen receptor (TCR), but also on the chemokine receptor CXCR4. Our lab recently published that stimulation of the TCR results in the TCR associating with and transactivating CXCR4, which, in turn, activates a PREX-1/Rac-1 pathway that stabilizes cytokine mRNA. This pathway therefore normally significantly increases IL-2, IL-4, and IL-10 production by activated T cells. Here, we further characterize the molecular mechanisms responsible for the first step of this pathway: TCR-mediated TCR-CXCR4 complex formation. First, we used FRET and proximity ligation assay (PLA) to define the required structural motifs of CXCR4, showing that this pathway requires the CXCR4 cytoplasmic tail domain and phosphorylation of CXCR4-S339. Second, we showed that TCR-induced TCR-CXCR4 complex formation requires TCR-induced activity of Src family and ZAP-70 tyrosine kinases, as well as one or more Ser/Thre kinases. Third, we found that the G protein-coupled receptor kinase-2 (GRK2) is required both for TCR-induced phosphorylation of CXCR4-S339 and for the formation of TCR-CXCR4 complexes. Finally, we found that GRK2 is required for robust IL-2, IL-4, and IL-10 cytokine secretion by normal human T cells. Together these results identify a novel role for TCR-induced GRK-2 in inducing the TCR-CXCR4 complex formation that subsequently signals to increase TCR-mediated cytokine production. Targeting GRK2 or other mechanisms required for TCR-induced TCR-CXCR4 formation may therefore be therapeutically useful for limiting production of T cell cytokines in humans, for example, during graft versus host disease or immunotherapy.