Maintenance Of CD8 Memory Research Articles

Effect of Chronic Endogenous Antigen Stimulation on CAR T Cell Persistence and Memory Formation

Adoptive transfer of T cells, genetically modified to express chimeric antigen receptors (CARs), targeting CD19 induce remission in 70-90% of pediatric patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL). Despite enthusiasm for the effectiveness of CAR therapy, emerging data indicate a substantial number of patients relapse, suggesting this is the next obstacle to be addressed as CAR T cell therapy is incorporated into standard treatment paradigms. Clinical observations have correlated the persistence of CAR T cells with achievement of long term remission and, ultimately, curative therapy. CD19 CAR T cells are continuously exposed to their cognate antigen in the form of newly formed B cell precursors. While CD19 CAR T cells can be identified in a subset of patients for months to years after infusion, it is currently unknown if the functionality and persistence of these CAR T cells are antigen independent like “classic” T cell memory. CAR T cell biology cannot be systematically evaluated in xenograft models. Thus, we evaluated the factors required for CAR T cell persistence in an immunocompetent, syngeneic murine model using CAR T cells expressing a second-generation anti-CD19 scfv/CD28/CD3ζ CAR to treat the pre-B ALL leukemia cell line E2aPbx. Mice treated with CD19 CAR T cells cleared established leukemia in a primary response and, up to several months later, re-expanded and cleared multiple re-challenges of E2aPbx. Furthermore, persistent CAR T cells from mice previously cured by CAR T cells were transferred into a leukemia bearing recipient and demonstrated superior expansion compared to primary CAR T cells (p=0.007) and mediated the complete clearance of established leukemia (p=0.001, survival relative mock). These data demonstrate there is a persistent, functional population of CAR T cells capable of re-expansion and clearance of secondary or tertiary leukemia similar to memory T cells. As these experiments evaluated CD19 CAR T cells in the presence of constant antigenic stimulation by CD19-expressing B cell precursors, the extent to which antigen exposure is required, for CAR T cell persistence, remains unclear. We established a model of CD19 replete (Ag+, WT) and deficient (Ag-, CD19KO) hosts to evaluate the role of antigen dependence of CAR T cell persistence. Results showed, CD8+ CAR T cells equally expanded during the primary immune response to leukemia regardless of the presence/absence of CD19 (p=0.90); however, there was higher expansion of CD4+ CAR T cells in Ag- recipients (p=0.031). Interestingly, in the absence of host-derived CD19+ B cells (Ag-) there was faster clearance of leukemia when compared to Ag+ mice at day 4 post infusion (p=0.015). However, CD19 deficient (Ag-) hosts had a complete absence of CD4+ CAR T cells at 45 days post infusion (P<0.001) and significantly reduced persistence of CD8+ CAR T cells after leukemia clearance compared to Ag+ recipients (Day 45 post infusion: P=0.007). The persistent CAR T cells in Ag+ recipients had a phenotype consistent with effector/effector memory cells and expressed elevated levels of exhaustion markers PD-1 (p=0.019), and Lag3(p=0.0005), but retained the ability to clear a secondary leukemia. In contrast, the residual CD8+ CAR T cells in Ag- hosts were unable to reject a re-challenge of leukemia (relative to mock p=ns), despite expressing fewer markers of exhaustion. Our data show, endogenous CD19 antigen is necessary for the persistence of CAR T cells and constant antigen stimulation results in a phenotypically “exhausted”, yet functional, population capable of protecting against re-challenge. To extend the persistence of the CAR T cells in the absence of antigen, we expanded the CAR T cell in IL15 instead of IL2 prior to infusion. IL15 has been previous shown to support the survival and maintenance of CD8 memory T cells. Exposure of CAR T cells to IL15 during in vitro expansion resulted in improved antigen independent persistence at day 45; however, persistence was still lower in Ag- hosts relative to Ag+ hosts (P=0.007). IL15 restored protection against leukemia re-challenge in both Ag+ and Ag- hosts with survival out to 150 days (p=ns). We conclude, under conditions mimicking clinical CAR T cell production, CAR T cell persistence and “memory” is dependent on constant antigen stimulation and CAR T cell persistence is not equivalent to “classic” T cell memory which is typified by persistence in the absence of antigen. DisclosuresNo relevant conflicts of interest to declare.

No Requirement of Trans Presentations of IL-15 for Human CD8 T Cell Proliferation

The trans presentation of IL-15 by cells expressing the specific high-affinity receptor α-chain (IL-15Rα) to cells expressing the signaling receptor β-chain and γ-chain is essential for the generation and maintenance of CD8 memory T cells, NK cells, and NKT cells in an in vivo mouse system. We have also demonstrated in vitro that cell-surface IL-15Rα on cells expressing all the receptor components present IL-15 to receptor β-chain/γ-chain coexpressed on the same cell surface (cis presentation). However, although mouse CD8 T cells express all the IL-15R components, they show no evidence of cis presentation. In this study, we demonstrate that increased expression of mouse IL-15Rα in mouse CD8 T cells by retrovirus-mediated gene transfer changes the ability of the T cell to use cis presentation on the cell surface, indicating that cis presentation requires high expression of mouse IL-15Rα on the cell surface. Using cell lines expressing human or mouse receptors, we demonstrate that cis presentation occurs more efficiently in the human receptor-ligand combination than in that of the mouse system. Moreover, we found that primary human CD8 T cells do not require trans presentation of human IL-15 in vitro. These findings raise the possibility that the maintenance and generation of memory CD8 T cells are achieved via distinct mechanisms in humans and mice. Therefore, careful study of the human immune system, rather than extrapolation from the murine model, is necessary to achieve more complete understanding of memory CD8 T cell development in humans.

Impaired T cell function and CD8 memory maintenance in a novel IL-7Rα knock-in mouse (B30)

Abstract Interleukin (IL)-7 is an essential growth factor for thymocyte development and survival of peripheral T cells, specifically CD8+ memory T cells. Current models such as IL-7 or IL-7 receptor alpha (IL-7Rα) knock-out mice have demonstrated the necessity of IL-7 signaling in these processes, but are unable to delineate the involvement of downstream pathways. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Rα Y449XXM motif in mice by knock-in mutagenesis (IL-7Rα449F). Thymocytes from these mice overcome a double negative (DN) stage developmental block that allows subsequent T cell maturation and peripheral migration. This is in stark contrast to the severe cytopenia seen in IL-7Rα−/− mice. These findings highlight the importance of IL-7Rα Y449-mediated signals during thymopoiesis, but also indicate a requirement for Y449-independent signals. Infection of IL-7Rα449F mice with the intracellular pathogen Listeria monocytogenes revealed that CD4 and CD8 T cells had different requirements for IL-7Rα signals. Strikingly, CD4 T cells failed to mount a detectable primary response to Listeria. Despite normal CD8 primary responses, maintenance of Listeria-specific CD8 memory was impaired in IL-7Rα449F mice. Furthermore, we show that Bcl-2 is IL-7Rα Y449-independent and insufficient for IL-7-mediated maintenance of CD8 memory. Research funded by the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research

Dendritic cell–T cell interactions in the generation and maintenance of CD8 T cell memory

Dendritic cells (DCs) are the critical antigen-presenting cells involved in initiating CD8 T cell responses to microbial and viral pathogens. Hence the generation of memory T cells from naïve T cells is intricately intertwined with DCs at every level. This review broadly addresses DC-CD8 T cell interactions that result in the generation and maintenance of CD8 memory T cells.

IL-15 is a growth factor and an activator of CD8 memory T cells.

Memory lymphocytes, arising from naïve lymphocytes after antigenic stimulation and being long-lived, are the cellular basis for immunological memory. Recent studies of CD8 T cells suggest that generation of CD8 memory T cells requires the engagement of T cell antigen receptors (TCR) with antigen, yet the maintenance of CD8 memory T cells appears to be dependent on cytokines, such as IL-15, independent of TCR. Although considerable progress has been made in understanding the molecular and cellular events of TCR-induced differentiation and proliferation in the past decade, less is known about the mechanisms of IL-15 action. From a kinetic and comparative analysis of the responses of memory phenotype CD8 T cells to IL-15 and TCR stimulation in vitro, we found that IL-15 and anti-CD3 induce highly similar responses in memory phenotype CD8 T cells as measured by general gene expression profiles, synthesis of effector molecules (IFNgamma, TNFbeta, granzyme B and perforin), induction of cytotoxicity, and cellular proliferation. These findings indicate that IL-15 is not only a growth factor but also an antigen-independent activator for CD8 memory T cells.