Vandana Kalia

Cytotoxic CD8 T lymphocytes (CTL) are critical for the clearance of pathogenic cells via antigen-dependent cell lysis, thus providing protection against infectious diseases and cancers. CTLs represent one of the key targets of immunotherapies and vaccine design. While microarray and single-cell RNA sequencing of effector and memory CD8 T cells have identified several promising gene targets that may modulate CD8 T cell responses, their development is slowed down by the financial and time constraints related to the generation of germline knockout mice for further validation studies. Here we present a protocol for conducting efficient deletion of genes from activated effector, as well as resting naïve and memory primary murine CD8 T cells using RNP-based Crispr/Cas9 technology. This CRISPR modification of CD8 T cells was then adapted to study the effects of gene deletion in the context of acute memory differentiation as well as chronic exhaustion of CD8 T cells. To this end, we have titrated the necessary dose of antigen-specific CD8 T cells to study their differentiation in acute and chronic infections and confirmed the model by demonstrating rapid expansion of CRISPR mediated PD-1 ablated CD8 T cells in a chronic viral infection. Finally, by combining this methodology with a murine model of subcutaneous tumor challenge, this study provides a unique screening system for genes critical for mediating clearance of malignant tumor cells by CTLs. This study expands current technical capabilities for rapid evaluation of functional role of any candidate gene in CTL responses to infections and cancer through targeted gene deletion at any stage of CD8 T cell differentiation without the need for germline gene deletion mouse models.

Metabolic transitions between naïve, effector and memory T cell states are largely orchestrated by TCR, costimulatory and cytokine signals along with nutrient availability in the immune microenvironment. Treg cells have been shown to play a critical role in the effector-to-memory (E-M) transition of virus-specific CD8 T cells through regulation of proliferation and cytotoxic functional programs. However, the precise Treg-dependent metabolic changes that occur in the microniches and, the underlying molecular and cellular mediators of E-M transition remain undefined. Here we show that Treg cells promote the metabolic remodeling of memory-precursor effector CD8 T cells (MPEC) from aerobic glycolysis to fatty acid oxidation as they enter quiescence after antigen clearance. Our data implicate the anatomic microniche of the splenic white pulp as a site for Treg-MPEC interactions. We further show that optimal E-M metabolic transition requires regulation of effector CD4 T cells and inflammatory myeloid cells through inhibitory CTLA4 signals from Treg cells. Moreover, antagonism of inflammatory cytokine interferon-γ (IFN-γ) signals partially rescues the memory defects associated with absence of Treg cells. Together, these findings support a metabolic triad model of memory CD8 T cell differentiation where Treg-dependent regulation of inflammation from effector CD4 T cells promotes the transition of CD8 T cells from cytotoxic effector to quiescent memory metabolic programs. These studies define novel molecular targets that may be exploited to manipulate metabolism, migration and memory function during vaccination.

The miR-17∼92 microRNA cluster drives oncogenesis by inducing proliferation and survival of cancer cells. A similar pro-proliferative role of miR-17∼92 has also been identified in pathogen- and tumor-reactive T cells. However, the metabolic underpinnings of miR-17∼92-drivenT cell expansion and effector differentiation remain undefined. Using a murine model of conditional miR-17∼92 deletion or constitutive overexpression in viral antigen-specific CD4 or CD8 T cells, here we show that miR-17∼92 drives terminal differentiation of CD4 and CD8 T cells through sustained activation of mTOR and glycolytic bioenergetics pathways. Constitutively increased expression of miR-17∼92 led to a universal increase in the numbers of antigen-specific T follicular helper (T _FH ), T helper 1 (T _H1 ) and cytotoxic T lymphocyte (CTL) effector subsets. During early stages of T cell activation, miR-17∼92 overexpression was associated with increased glucose uptake and heightened glycolytic and oxidative metabolism to sustain increased proliferation in both CD4 and CD8 T cells. However, prolonged overexpression of miR-17∼92 led to a loss of polyfunctionality and compromised metabolic fitness (glycolysis and mitochondrial respiration) by the peak of the effector responses. These studies establish miR-17∼92 as a critical driver of T cell proliferation and effector differentiation through metabolic regulation of both effector CTL and CD4 T cell subsets (T _FH and T _H1 ) that are critical for combating viral infections. These studies form the basis for future manipulation of miR-17∼92 gene/metabolic regulatory network to commandeer T cell immunity during infection, vaccination or cancer immunotherapy.

A major obstacle in current T cell-based immunotherapy against solid tumors is the widely observed T cell exhaustion and loss of functionality within the tumor microenvironment (TME). We, and others, have demonstrated that chronic antigenic signaling is the predominant factor that drives TCR tumor-reactive, and CAR T cell exhaustion in the TME. To combat this exhaustion, we have engineered CD19-targeted third generation CAR constructs to express pro-memory transcription factors associated with longevity, polyfunctionality and/or enhanced responsiveness to immune checkpoint blockade in cancer immunotherapy. Using an immunocompetent murine model of syngeneic solid tumors expressing truncated human-CD19 (hCD19t), we demonstrate that ectopic expression of pro-memory transcription factors in the 4^th generation transcription factor-CARs (TF-CARs) augments CAR T cell therapeutic efficacy against solid tumors. Amongst CAR T cells recovered from established solid tumors, the 4^th generation TF-CAR T cells retained a memory-precursor effector (MPEC) T cell like phenotype, while the 3^rd generation CAR T cells largely displayed a terminally exhausted phenotype (T_Ex). Importantly, the inclusion of select pro-memory transcription factors did not compromise CAR T cell proliferation or expression of the effector cytokines IFN-γ and TNFα following restimulation. In addition to superior control of primary tumors, the TF-CAR T cells instilled greater long-term protection from tumor relapse in mice, which cleared primary tumors (6/7) compared to 3rd generation CAR T cells (3/7). Collectively, these data present incorporation of pro-memory transcription factors as an attractive engineering strategy to intrinsically combat CAR T cell exhaustion in the TME and promote long-term CAR T cell memory and protection from tumor relapse. Citation Format: Steven J. Reed, Samantha J. Tower, Ryma Toumi, Vandana Kalia, Surojit Sarkar. Incorporation of pro-memory transcription factors in fourth generation CARs enhances CAR T cell immunotherapy against solid tumors in an immunocompetent murine model [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 5580.

PD-1 signals promote development of long-lived quiescent memory CD8 T cells by regulating the balance of glycolysis and fatty acid oxidation.

Interleukin-2 (IL-2) regulates key aspects of CD8 T cell biology–signaling through distinct pathways IL-2 triggers critical metabolic and transcriptional changes that lead to a spectrum of physiological outcomes such as cell survival, proliferation, and effector differentiation. In addition to driving effector differentiation, IL-2 signals are also critical for formation of long-lived CD8 T cell memory. This review discusses a model of rheostatic control of CD8 T cell effector and memory differentiation by IL-2, wherein the timing, duration, dose, and source of IL-2 signals are considered in fine-tuning the balance of key transcriptional regulators of cell fate.

1100 Background: Recent studies show strong correlation between tumor infiltrating lymphocytes (TILs) and triple-negative breast cancer (TNBC) patient survival. CD8+ T cells serve as a favorable prognostic marker for TNBC. In addition, other cells such as CD4+ T cells, macrophages, B cells, and Tregs also infiltrate tumors. In this study, we delineate a strong relationship between the cycling kinetics of proliferating cells in TNBCs and antitumor immune response. Methods: A multi-institutional study performed by our group has previously shown that KAMS (Ki67-Adjusted Mitotic Score) provides a measure of the cycling kinetics of proliferating tumor cells and robustly stratifies TNBC patients into slow cycling (low KAMS) cyclophosphamide-methotrexate-fluorouracil (CMF)-responsive and fast cycling (high KAMS) CMF-resistant subgroups. In this study, we reviewed clinical data from 124 CMF-treated TNBC patients from Nottingham Hospital and sought correlations between cycling kinetics (High/Low KAMS) and tumor infiltrating immune cells. Results: We found that slow cycling TNBCs had higher mean expression of tumor infiltrating immune cells than fast cycling TNBCs. Intratumoral CD68 (p = 0.003), CD3 (p = 0.006), CD20 (p = 0.01), FOXP3 (p = 0.01), and total numbers of intratumoral and stromal CD68 (p = 0.01) and CD3 (p = 0.03) expressing cells were found to be significantly higher in low KAMS tumors than in high KAMS tumors. Of these biomarkers, CD68 was significantly associated with patients’ breast cancer-specific survival (BCSS): (a) low KAMS, high CD68 TNBCs had better BCSS than low KAMS, low CD68 (p = 0.01) TNBCs, and (b) high KAMS, low CD68 cases had better BCSS than high KAMS, high CD68 cases. Conclusions: Our observation that there are more TILs in slow cycling TNBCs suggests that there may be a dynamic cross-regulation between cycling kinetics and antitumor immune response. From our surprising observation that CD68 exerts polar roles in low/high KAMS subgroups, we propose that distinctions in M1 and M2 macrophage subsets in slow and fast cycling TNBCs may correlate with distinct outcomes. In addition, metabolic competition between tumor and immune cells may determine the level and function of TILs.

An important question in memory T cell differentiation is understanding the mechanisms that regulate the generation of terminal effector cells that die following antigen clearance versus effector cells that give rise to the long‐lived pool of memory CD8 T cells. Using killer cell lectin‐like receptor G1 as a marker to effectively distinguish terminal effectors from memory precursors, we found that despite their diverse cell fates both subsets possess remarkably similar gene expression profiles and function as equally potent direct ex vivo killer cells, with similar ability for inflammatory cytokine production and granzyme B expression. Interestingly, in addition to these conventional effector CTL characteristics memory precursors also produced IL‐2, a property typically ascribed to naïve or memory cells; thus presenting them as a novel effector subset. On the other hand, terminal effector cells represent a “late leaving” subset of cells that continues to perceive stimulation towards the tail end of antigen clearance, and exhibits an enrichment of the “effector cell signature”. Importantly, curtailing stimulation during the later stages of infection enhanced the generation of long‐lived memory cells. Collectively, these data define a model of memory differentiation wherein effector CD8 T cells that are driven further down the differentiation pathway by continued stimulation towards the later stages of infection become metabolically and functionally unfit for memory lineage. These studies strongly support the classical decreasing potential model of memory differentiation, and further demonstrate that “late leaver” effector cells contribute less towards the long‐lived pool of memory CD8 T cells.