Christopher D. Chien

CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity

Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL.

Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD

Acute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4(+) CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.

Synergism between CpG-Containing Oligodeoxynucleotides and IL-2 Causes Dramatic Enhancement of Vaccine-Elicited CD8+ T Cell Responses

Novel anticancer vaccination regimens that can elicit large numbers of Ag-specific T cells are needed. When we administered therapeutic vaccines containing the MHC class I-presented self-peptide tyrosinase-related protein (TRP)-2180–188 and CpG-containing oligodeoxynucleotides (CpG ODN) to mice, growth of the TRP-2-expressing B16F1 melanoma was not inhibited compared with growth in mice that received control vaccinations. When we added systemic IL-2 to the TRP-2180–188 plus CpG ODN vaccines, growth of B16F1 was inhibited in a CD8-dependent, epitope-specific manner. Vaccines containing TRP-2180–188 without CpG ODN did not cause epitope-specific tumor growth inhibition when administered with IL-2. The antitumor efficacy of the different regimens correlated with their ability to elicit TRP-2180–188-specific CD8+ T cell responses. When we administered TRP-2180–188 plus CpG ODN-containing vaccines with systemic IL-2, 18.2% of CD8+ T cells were specific for TRP-2180–188. Identical TRP-2180–188 plus CpG ODN vaccines given without IL-2 elicited a TRP-2180–188-specific CD8+ T cell response of only 1.1% of CD8+ T cells. Vaccines containing TRP-2180–188 without CpG ODN elicited TRP-2180–188-specific responses of 2.8% of CD8+ T cells when administered with IL-2. There was up to a 221-fold increase in the absolute number of TRP-2180–188-specific CD8+ T cells when IL-2 was added to TRP-2180–188 plus CpG ODN-containing vaccines. Peptide plus CpG ODN vaccines administered with IL-2 generated epitope-specific CD8+ T cells by a mechanism that depended on endogenous IL-6. This is the first report of synergism between CpG ODN and IL-2. This synergism caused a striking increase in vaccine-elicited CD8+ T cells and led to epitope-specific antitumor immunity.

TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance

TCR engagement induces CD8 CAR T cell exhaustion and poor in vivo functionality in mice. A tale of two receptors Although there has been an explosion of research on chimeric antigen receptor (CAR) T cell therapy, most preclinical studies use transduced human T cells in immunodeficient mice so that the clinical products can be evaluated. However, the endogenous T cell receptors (TCRs) on the human cells are unable to be activated by peptides presented in mouse MHC. To determine how CAR activity is affected by TCR engagement, Yang et al. used a CAR targeting CD19 and multiple types of transgenic T cells in immunocompetent mice. Exposure of CD8 CAR T cells to the antigen recognized by the TCR led to T cell exhaustion, apoptosis, and lack of efficacy; this phenomenon was not observed for CD4 CAR T cells. Their findings demonstrate how considering T cell biology could further improve CAR T cell therapy. Chimeric antigen receptor (CAR)–expressing T cells induce durable remissions in patients with relapsed/refractory B cell malignancies. CARs are synthetic constructs that, when introduced into mature T cells, confer a second, non–major histocompatibility complex–restricted specificity in addition to the endogenous T cell receptor (TCR). The implications of TCR activation on CAR T cell efficacy has not been well defined. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T cell therapy for pre–B cell acute lymphoblastic leukemia in which the CAR is introduced into T cells with known TCR specificity, we demonstrate loss of CD8 CAR T cell efficacy associated with T cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T cells demonstrate equivalent cytotoxicity to CD8 CAR T cells and, in contrast, retain in vivo efficacy despite TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CD8 CAR T cells upon dual receptor stimulation compared to CD4 CAR T cells and indicate inherent differences between CD4 and CD8 CAR T cells in the use of T cell–associated signaling pathways. These results provide insights into important aspects of CAR T cell immune biology and indicate opportunities to rationally design CAR constructs to optimize clinical efficacy.

Murine Models of Acute Leukemia: Important Tools in Current Pediatric Leukemia Research

Leukemia remains the most common diagnosis in pediatric oncology and, despite dramatic progress in upfront therapy, is also the most common cause of cancer-related death in children. Much of the initial improvement in outcomes for acute lymphoblastic leukemia (ALL) was due to identification of cytotoxic agents that are active against leukemia followed by the recognition that combination of these cytotoxic agents and prolonged therapy are essential for cure. Recent data demonstrating lack of progress in patients for whom standard chemotherapy fails suggests that the ability to improve outcome for these children will not be dramatically impacted through more intensive or newer cytotoxic agents. Thus, much of the recent research focus has been in the area of improving our understanding of the genetics and the biology of leukemia. Although in vitro studies remain critical, given the complexity of a living system and the increasing recognition of the contribution of leukemia extrinsic factors such as the bone marrow microenvironment, in vivo models have provided important insights. The murine systems that are used can be broadly categorized into syngeneic models in which a murine leukemia can be studied in immunologically intact hosts and xenograft models where human leukemias are studied in highly immunocompromised murine hosts. Both of these systems have limitations such that neither can be used exclusively to study all aspects of leukemia biology and therapeutics for humans. This review will describe the various ALL model systems that have been developed as well as discuss the advantages and disadvantages inherent to these systems that make each particularly suitable for specific types of studies.

Absence of STAT1 in donor-derived plasmacytoid dendritic cells results in increased STAT3 and attenuates murine GVHD

Selective targeting of non-T cells, including antigen-presenting cells (APCs), is a potential strategy to prevent graft-versus-host-disease (GVHD) but to maintain graft-versus-tumor (GVT) effects. Because type I and II interferons signal through signal transducer and activator of transcription-1 (STAT1), and contribute to activation of APCs after allogeneic bone marrow transplant (alloBMT), we examined whether the absence of STAT1 in donor APCs could prevent GVHD while preserving immune competence. Transplantation of STAT1(-/-) bone marrow (BM) prevented GVHD induced by STAT1(+/+) T cells, leading to expansion of B220(+) cells and regulatory T cells. STAT1(-/-) BM also preserved GVT activity and enhanced overall survival of tumor-challenged mice in the setting of GVHD. Furthermore, recipients of allogeneic STAT1(-/-) BM demonstrated increased CD9(-)Siglec H(hi) plasmacytoid dendritic cells (pDCs), and depletion of pDCs after STAT1(-/-) BM transplantation prevented GVHD resistance. STAT1(-/-) pDCs were found to produce decreased free radicals, IFNα, and interleukin (IL)-12, and increased IL-10. Additionally, STAT1(-/-) pDCs that were isolated after alloBMT showed increased gene expression of S100A8 and S100A9, and transplantation of S100A9(-/-) BM reduced GVHD-free survival. Finally, elevated STAT3 was found in STAT1(-/-) pDCs isolated after alloBMT. We conclude that interfering with interferon signaling in APCs such as pDCs provides a novel approach to regulate the GVHD/GVT axis.

The functional interplay between systemic cancer and the hematopoietic stem cell niche

Hematopoietic cells are increasingly recognized as playing key roles in tumor growth and metastatic progression. Although many studies have focused on the functional interaction of hematopoietic cells with tumor cells, few have examined the regulation of hematopoiesis by the hematopoietic stem cell (HSC) niche in the setting of cancer. Hematopoiesis occurs primarily in the bone marrow, and processes including expansion, mobilization, and differentiation of hematopoietic progenitors are tightly regulated by the specialized stem cell niche. Loss of niche components or the ability of stem cells to localize to the stem cell niche relieves HSCs of the restrictions imposed under normal homeostasis. In this review, we discuss how tumor-derived factors and therapeutic interventions disrupt structural and regulatory properties of the stem cell niche, resulting in niche invasion by hematopoietic malignancies, extramedullary hematopoiesis, myeloid skewing by peripheral tissue microenvironments, and lymphopenia. The key regulatory roles played by the bone marrow niche in hematopoiesis has implications for therapy-related toxicity and the successful development of immune-based therapies for cancer.

Abstract 2372: Inflammatory cytokine induced TSLP from bone marrow niches contributes to relapse of high risk TSLPR overexpressing acute lymphoblastic leukemia

Although 5 year survival rates are near 90%, leukemia is the most common cause of death from cancer in children with treatment failure primarily caused by relapse. A subset of acute lymphoblastic leukemia (ALL) patients that have an increased risk of relapse and consequently poorer overall survival have leukemia that overexpresses thymic stromal lymphopoietin receptor (TSLPR/CRLF2). However, it remains unclear what role Thymic Stromal Lymphopoietin (TLSP) signaling may play in increasing the risk of relapse. We hypothesize that TSLP/TSLPR axis may support progression of ALL by providing a signal through overexpressed TSLPR, which would sensitize the leukemia to low levels of the cytokine in bone marrow (BM) niches and promote the survival of the leukemia. We have created a high TSLPR expressing leukemia model (TSLPRhigh) through retroviral transduction of a transplantable syngeneic mouse ALL model (TSLPRlow) in which leukemia progression can be examined. This TSLPRhigh leukemia has expression of TSLPR comparable to what is found on patient derived ALL that naturally overexpress TSLPR and the receptor is functional as we see increased phosphorylation of STAT5 protein in response to TSLP stimulation. When ALL lines were injected into mice, we observed an 8 fold difference in the percentage of TSLPRhigh vs. TSLPRlow ALL in the BM 5 days after injection, where leukemia accounted for less than 4% of the BM corresponding to an early stage of ALL progression, whereas overall leukemia progression was not significantly different. This implies that the TSLP axis is most critical at low leukemic burden when bone marrow niches are intact and not disrupted by high levels of ALL in the BM. TSLPRlow and TSLPRhigh expressing cells show no significant difference in growth rate in vitro or in vivo, however when we treated ALL cells with chemotherapy in the absence or presence of TSLP we found that TSLP signaling significantly decreased the number of apoptotic cells in TSLPRhigh but not TSLPRlow ALL. These data suggest that high TSLPR expression sensitizes leukemia cells to TSLP to promote survival and not growth of ALL. This is significant because we believe that TSLP is likely to be expressed at low levels in the BM under normal circumstances. Interestingly, we have evidence using a nestin positive murine BM stromal cell line and stromal cells derived from fresh murine BM that TSLP mRNA and protein is induced by IL-1α and TNF-α stimulation. We further have found that cytarabine treatment of TSLPRhigh cells leads to induction of both IL-1α and TNF-α suggesting that chemotherapeutic treatment of ALL could potentially provide an unintended advantage to TSLPR overexpressing ALL. Based on this data, we postulate that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse in TSLPR overexpressing ALL particularly in the context of standard therapy. Citation Format: Christopher D. Chien, Sang Nguyen, Haiying Qin, Terry J. Fry. Inflammatory cytokine induced TSLP from bone marrow niches contributes to relapse of high risk TSLPR overexpressing acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2372. doi:10.1158/1538-7445.AM2015-2372