Surojit Sarkar

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.

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a range of affinities for tumor antigens while suppressing regulatory T cells. Computational protein design has enabled the precise tailoring of proteins to meet specific needs. Here, we report a de novo designed IL-21 mimic, 21h10, with high stability and signaling potency in humans and mice. In murine and ex vivo human organotypic tumor models, 21h10 showed robust antitumor activity, with more prolonged signaling and stronger antitumor activity than native IL-21. 21h10 induced pancreatitis that could be mitigated by TNF blockade without compromising antitumor efficacy. Although antidrug antibodies to 21h10 formed, they were not neutralizing. 21h10 induced highly cytotoxic T cells with a range of affinities, robustly expanding intratumoral low-affinity cytotoxic T cells and driving high expression of IFN-γ and granzyme B compared with native IL-21, while increasing the frequency of IFN-γ ⁺ T helper 1 cells and reducing regulatory T cells. The full human-mouse cross-reactivity, high stability and potency, and low-affinity antitumor responses support the translational potential of 21h10.

Stem-like progenitor exhausted CD8 T cells are critical for maintaining long-term resistance during chronic infections and cancer, and represent an important checkpoint blockade immunotherapy target for functional reinvigoration and disease control. Hence, there is vigorous interest in understanding the ontogenesis of TCF-1 ^Hi stem-like exhausted CD8 T cells, and in defining the signals that promote their development. Here, we show that virus-specific TCF1 ^Hi GzmB ^Lo CD8 T cells develop during early stages of chronic viral infection, and are progressively deprogrammed from the stem-like lineage towards terminal differentiation by strong IL-2 signals. In vivo fate-tracking studies show that strong T cell-intrinsic IL-2 signals through the high affinity heterotrimeric IL-2Rα/β/γ receptor drive skewed development of terminally differentiated TCF-1 ^Lo GzmB ^Hi cells, which largely die in chronic antigenic environment. In contrast, tempered IL-2 signals through the intermediate affinity IL-2Rβ/γ heterodimer, or delayed priming in diminished IL-2 milieu support preferential development of TCF-1 ^Hi stem-like exhausted CD8 T cells, capable of long-term persistence and potent responsiveness to PD-1 therapy in later stages of chronic viral infection. In human tumors as well, single cell RNA-seq analyses of tumor infiltrating lymphocytes from melanoma, human papillomavirus+ head and neck cancer and lung cancer patients revealed an inverse relationship between IL-2 signaling signature and T cell stemness. Moreover, melanoma patients with enriched IL-2 signaling signature showed poor responses to checkpoint blockade immunotherapy. Collectively, these findings bear relevance to clinical immunotherapies of chronic viral infections and cancers, and support exogenous IL-2 signal manipulation prior to therapy as a strategy for augmenting long-term clinical outcomes. ONE SENTENCE SUMMARY: Programming of exhausted T cell fates by differential IL-2

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.

Robust T cell responses are crucial for effective anti-tumor responses and often dictate patient survival. However, in the context of solid tumors, both endogenous T cell responses and current adoptive T cell therapies are impeded by the immunosuppressive tumor microenvironment (TME). A multitude of inhibitory signals, suppressive immune cells, metabolites, hypoxic conditions and limiting nutrients are believed to render the TME non-conducive to sustaining productive T cell responses. In this study we conducted an in-depth phenotypic and functional comparison of tumor-specific T cells and tumor-nonspecific bystander memory T cells within the same TME. Using two distinct TCR transgenic and solid-tumor models, our data demonstrate that despite exposure to the same cell-extrinsic factors of the TME, the tumor-nonspecific bystander CD8 T cells retain the complete panoply of memory markers, and do not share the same exhaustive phenotype as tumor-reactive T cells. Compared to tumor-specific T cells, bystander memory CD8 T cells in the TME also retain functional effector cytokine production capabilities in response to ex vivo cognate antigenic stimulation. Consistent with these results, bystander memory T cells isolated from tumors showed enhanced recall responses to secondary bacterial challenge in a T cell transplant model. Importantly, the tumor-resident bystander memory cells could also efficiently utilize the available resources within the TME to elaborate in situ recall effector functions following intra-tumoral peptide antigen injection. Additionally, CRISPR-Cas9 gene deletion studies showed that CXCR3 was critical for the trafficking of both tumor antigen-specific and bystander memory T cells to solid tumors. Collectively, these findings that T cells can persist and retain their functionality in distinct solid tumor environments in the absence of cognate antigenic stimulation, support the notion that persistent antigenic signaling is the central driver of T cell exhaustion within the TME. These studies bear implications for programming more efficacious TCR- and CAR-T cells with augmented therapeutic efficacy and longevity through regulation of antigen and chemokine receptors.

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