Engraftment Fitness of Human Central Memory-Derived Effector CD8+ T Cells In Immunodeficient Mice
Authors: Wang Xiuli , Carolina Berger , Riddell Stanley R. , Wong ChingLam W , Forman Stephen , Jensen Michael C 
Published: October 2019

Abstract 1019

Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of T cell transfer as a therapy for patients. But the potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. The repertoire of memory T cells is heterogeneous with respect to phenotypic, functional, and epigenetic attributes. Memory T cells are divided into sub-populations of 1) effector memory (TEM) cells that distribute to tissue beds and exhibit immediate cytolytic effector functioning, and 2) central memory (TCM) cells that home to lymph nodes based on CD62L/CCR7 expression and are capable of extensive proliferative activity upon re-encountering antigen. Thus the cell-intrinsic programming of distinct memory T cell subtypes, such as TEM and TCM, likely dictate divergent fates of their derived effector cells. To address this important issue, a clear functional dichotomy between TCM- and TEM-derived CD8+ CTLs was recently delineated in a nonhuman primate model, where it was found that virus-specific CD8+ CTL clones derived from TCM, but not TEM precursors, establish persistent and functional memory following adoptive transfer. Here, we extended these studies to human effector T cells using CMV as antigen model system to investigate the engraftment of human CMVpp65-specific CD8+ effector T cells derived in vitro from either sort purified CD45RO+CD62L+ TCM or CD45RO+CD62L- TEM precursors in NOD/Scid IL-2RγCnull (NOG) mice. TCM-derived effector cells (TE(CM)) and TEM-derived effector cells (TE(EM)) were adoptively transferred (i.v) into NOG mice reconstituted with human IL-15 and T cell levels in circulation were evaluated at different time points by FACS. 20% CD8+ TE(CM) and 3% CD8+ TE(EM) were detected on day 14. Then after, engraftment of the CD8+ TE(CM) remained at a steady state of approx 2% of circulating mononuclear cells for 100 days while TE(EM) remained at or below the level of detection, indicating that TE(CM) were superior in their ability to engraft in response to IL-15 as compared to TE(EM) after adoptive transfer (P<0.05). The long-term (100 days) persisting CD8+ TE(CM), harvested from primary recipient mice were found to be capable of engrafting secondary recipients. TcR Vβ analysis of persisting cells demonstrated that CD8+ TE(CM) engraftment was polyclonal, suggesting that homeostatic engraftment fitness is a general feature of these cells.

To delineate the mechanism(s) by which TE(CM) exhibit superior in vivo engraftment, TE(CM) and TE(EM) were first labeled with CFSE before in vivo administration. CFSE profiles appear that the TE(EM) proliferated more extensively than TE(CM) early after adoptive transfer as indicated by the percent of cells which diluted CFSE on day 9 (i.e., 80% vs. only 25%, respectively). However, using D2R cleavage as a measure of caspase activity as a surrogate for apoptosis, 5.8% of engrafting TE(CM) were positive for activated caspase activity compared to 31.6% of TE(EM), suggesting that in NOG mice both CD8+ TE(CM) and TE(EM) proliferate in response to IL-15 whereas TE(CM) are intrinsically resistant to caspase activation and apoptosis.

We also evaluated the antigen specific responsiveness of engrafted cells. Weekly infusions of irradiated pp65+/A2+ LCL as antigen significantly augmented the levels of circulating CD8+ TE(CM) as compared to no antigen stimulation (P<0.05), whereas CD8+ TE(EM) did not respond to antigen challenge. Moreover, when CMVpp65 specific CD8+ TE(CM) or TE(EM) were infused into CMVpp65+ tumor bearing mice, tumor cells progressed in mice receiving TE(EM) at a rate similar to untreated control mice over a ten day observation period, whereas TE(CM) significantly controlled tumor progression (P<0.05), indicating that CD8+ TE(CM) but not TE(EM) are able to mediate an anti-tumor response. Together these studies confirm that human CD8+ effector T cells derived from TCM precursors are capable of persistence after infusion, can proliferate in in vivo in response to antigen, can mediate an anti-viral or anti tumor response, and are likely the preferred T cells for antigen specific anti-tumor adoptive T cell therapy .

Disclosures:

No relevant conflicts of interest to declare.