Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement.

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.

MEK inhibition reprograms CD8+ T lymphocytes into memory stem cells with potent antitumor effects.

Regenerative stem cell-like memory (TSCM) CD8+ T cells persist longer and produce stronger effector functions. We found that MEK1/2 inhibition (MEKi) induces TSCM that have naive phenotype with self-renewability, enhanced multipotency and proliferative capacity. This is achieved by delaying cell division and enhancing mitochondrial biogenesis and fatty acid oxidation, without affecting T cell receptor-mediated activation. DNA methylation profiling revealed that MEKi-induced TSCM cells exhibited plasticity and loci-specific profiles similar to bona fide TSCM isolated from healthy donors, with intermediate characteristics compared to naive and central memory T cells. Ex vivo, antigenic rechallenge of MEKi-treated CD8+ T cells showed stronger recall responses. This strategy generated T cells with higher efficacy for adoptive cell therapy. Moreover, MEKi treatment of tumor-bearing mice also showed strong immune-mediated antitumor effects. In conclusion, we show that MEKi leads to CD8+ T cell reprogramming into TSCM that acts as a reservoir for effector T cells with potent therapeutic characteristics.

Beta cell-specific CD8+ T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes.

The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.

Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

Epigenetic regulation of T cell adaptive immunity.

The conceptualization of adaptive immunity, founded on the observation of immunological memory, has served as the basis for modern vaccination and immunotherapy approaches. This fundamental concept has allowed immunologists to explore mechanisms that enable humoral and cellular lymphocytes to tailor immune response functions to a wide array of environmental insults and remain poised for future pathogenic encounters. Until recently, for T cells it has remained unclear how memory differentiation acquires and sustains a gene expression program that grants a cell with a capacity for a heightened recall response. Recent investigations into this critical question have identified epigenetic programs as a causal molecular mechanism governing T cell subset specification and immunological memory. Here, we outline the studies that have illustrated this concept and posit on how insights into T cell adaptive immunity can be applied to improve upon existing immunotherapies.

Stem, Effector, and Hybrid States of Memory CD8+ T Cells.

CD8+ T cell immunological memory of past antigen exposure can confer long-lived protection against infections or tumors. The fact that CD8+ memory T cells can have features of both naïve and effector cells has forced the field to struggle with several conceptual questions about the developmental origin of the cell and, consequently, the mechanism(s) that contribute to memory development. Here, we discuss recent conceptual advances in our understanding of memory T cell development that incorporate data describing a hybrid stem and/or effector state of differentiation. We theorize that the mechanisms involved in developing these cells could be mediated, in part, through epigenetic programs. Finally, we consider the potential therapeutic implications of inducing and/or utilizing such hybrid cells clinically.

Modulation of the IL-6 Receptor α Underlies GLI2-Mediated Regulation of Ig Secretion in Waldenström Macroglobulinemia Cells.

Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

A phase 1/2 clinical trial of invariant natural killer T cell therapy in moderate-severe acute respiratory distress syndrome.

Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials.

Antigen cross-presentation in young tumor-bearing hosts promotes CD8+ T cell terminal differentiation.

The immune system undergoes a progressive functional remodeling with age. Understanding how age bias shapes antitumor immunity is essential in designing effective immunotherapies, especially for pediatric patients. Here, we explore antitumor CD8+ T cell responses generated in young (prepubescent) and adult (presenescent) mice. Using an MHCI-deficient tumor model, we observed that tumor-reactive CD8+ T cells expanded in young tumor-bearing (TB) mice acquired a terminally differentiated phenotype characterized by overexpression of inhibitory receptors and the transcription factor Tox1. Furthermore, tumor-infiltrating CD8+ T cells from young tumors yielded a poor cytokine response compared with CD8+ T cells infiltrating adult tumors. Young migratory dendritic cells (migDCs) from the draining lymph nodes (dLNs), and mononuclear phagocytic cells (MPCs) infiltrating young tumors, were more competent in capturing and cross-presenting tumor antigen, leading to enhanced priming of CD8+ T cells in dLNs and their subsequent terminal differentiation in the tumors. Single-cell transcriptional profiling of tumor-infiltrating MPCs demonstrated that young MPCs are polarized toward an inflammatory, effector phenotype. Consistent with our observations in young versus adult TB mice, analysis of immune infiltrates from pediatric solid tumors showed a correlation between tumor-infiltrating CD8+ T cells with an exhaustion phenotype and the frequency of PD-L1-expressing monocytes/macrophages. Collectively, these data indicate that a young tissue microenvironment contributes to the generation of an immune response skewed toward a less pliable terminal effector state, thus narrowing the window for immunotherapeutic interventions.

Diet-Induced Obesity Impairs Outcomes and Induces Multi-Factorial Deficiencies in Effector T Cell Responses Following Anti-CTLA-4 Combinatorial Immunotherapy in Renal Tumor-Bearing Mice.

Associations between modifiable factors and the efficacy of cancer immunotherapies remain uncertain. We found previously that diet-induced obesity (DIO) reduces the efficacy of an immunotherapy consisting of adenovirus-encoded TRAIL plus CpG oligonucleotide (AdT/CpG) in mice with renal tumors. To eliminate confounding effects of diet and determine whether outcomes could be improved in DIO mice, we evaluated AdT/CpG combined with anti-CTLA-4 in diet-matched, obese-resistant (OB-RES) versus DIO tumor-bearing mice. Therapy-treated OB-RES mice displayed effective renal tumor control and sustained CD4+ and CD8+ T cell responses. In contrast, therapy-treated DIO mice exhibited progressive tumor outgrowth and blunted T cell responses, characterized by reduced intratumoral frequencies of IFNγ+ CD4+ and CD8+ T cells. Weak effector T cell responses in therapy-treated DIO mice were accompanied by low intratumoral concentrations of the T cell chemoattractant CCL5, heightened concentrations of pro-tumorigenic GM-CSF, and impaired proliferative capacity of CD44+CD8+ T cells in tumor-draining lymph nodes. Our findings demonstrate that in lean mice with renal tumors, combining in situ T cell priming upstream of anti-CTLA-4 enhances outcomes versus anti-CTLA-4 alone. However, host obesity is associated with heightened immunotherapy resistance, characterized by multi-factorial deficiencies in effector CD4+ and CD8+ T cell responses that extend beyond the tumor microenvironment.

CD19-CAR T cells undergo exhaustion DNA methylation programming in patients with acute lymphoblastic leukemia.

CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8+ CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated DNA methylation programming, indicating that efforts to prevent this process may be an attractive approach to improve CAR T cell efficacy.

Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined. Here, we report that exhaustion of human CAR T cells occurs through an epigenetic repression of the T cell's multipotent developmental potential. Deletion of the de novo DNA methyltransferase 3 alpha (DNMT3A) in T cells expressing first- or second-generation CARs universally preserved the cells' ability to proliferate and mount an antitumor response during prolonged tumor exposure. The increased functionality of the exhaustion-resistant DNMT3A knockout CAR T cells was coupled to an up-regulation of interleukin-10, and genome-wide DNA methylation profiling defined an atlas of genes targeted for epigenetic silencing. This atlas provides a molecular definition of CAR T cell exhaustion, which includes many transcriptional regulators that limit the "stemness" of immune cells, including CD28, CCR7, TCF7, and LEF1. Last, we demonstrate that this epigenetically regulated multipotency program is firmly coupled to the clinical outcome of prior CAR T cell therapies. These data document the critical role epigenetic mechanisms play in limiting the fate potential of human T cells and provide a road map for leveraging this information for improving CAR T cell efficacy.

The Antidiabetic Agent Acarbose Improves Anti-PD-1 and Rapamycin Efficacy in Preclinical Renal Cancer.

Although immune checkpoint inhibitors and targeted therapeutics have changed the landscape of treatment for renal cell carcinoma (RCC), most patients do not experience significant clinical benefits. Emerging preclinical studies report that nutrition-based interventions and glucose-regulating agents can improve therapeutic efficacy. However, the impact of such agents on therapeutic efficacy in metastatic kidney cancer remains unclear. Here, we examined acarbose, an alpha-glucosidase inhibitor and antidiabetic agent, in a preclinical model of metastatic kidney cancer. We found that acarbose blunted postprandial blood glucose elevations in lean, nondiabetic mice and impeded the growth of orthotopic renal tumors, an outcome that was reversed by exogenous glucose administration. Delayed renal tumor outgrowth in mice on acarbose occurred in a CD8 T cell-dependent manner. Tumors from these mice exhibited increased frequencies of CD8 T cells that retained production of IFNγ, TNFα, perforin, and granzyme B. Combining acarbose with either anti-PD-1 or the mammalian target of rapamycin inhibitor, rapamycin, significantly reduced lung metastases relative to control mice on the same therapies. Our findings in mice suggest that combining acarbose with current RCC therapeutics may improve outcomes, warranting further study to determine whether acarbose can achieve similar responses in advanced RCC patients in a safe and likely cost-effective manner.

Obesity induces limited changes to systemic and local immune profiles in treatment-naive human clear cell renal cell carcinoma.

Understanding the effects of obesity on the immune profile of renal cell carcinoma (RCC) patients is critical, given the rising use of immunotherapies to treat advanced disease and recent reports of differential cancer immunotherapy outcomes with obesity. Here, we evaluated multiple immune parameters at the genetic, soluble protein, and cellular levels in peripheral blood and renal tumors from treatment-naive clear cell RCC (ccRCC) subjects (n = 69), to better understand the effects of host obesity (Body Mass Index "BMI" ≥ 30 kg/m2) in the absence of immunotherapy. Tumor-free donors (n = 38) with or without obesity were used as controls. In our ccRCC cohort, increasing BMI was associated with decreased percentages of circulating activated PD-1+CD8+ T cells, CD14+CD16neg classical monocytes, and Foxp3+ regulatory T cells (Tregs). Only CD14+CD16neg classical monocytes and Tregs were reduced when obesity was examined as a categorical variable. Obesity did not alter the percentages of circulating IFNγ+ CD8 T cells or IFNγ+, IL-4+, or IL-17A+ CD4 T cells in ccRCC subjects. Of 38 plasma proteins analyzed, six (CCL3, IL-1ß, IL-1RA, IL-10, IL-17, and TNFα) were upregulated specifically in ccRCC subjects with obesity versus tumor-free controls with obesity. IGFBP-1 was uniquely decreased in ccRCC subjects with obesity versus non-obese ccRCC subjects. Immunogenetic profiling of ccRCC tumors revealed that 93% of examined genes were equivalently expressed and no changes in cell type scores were found in stage-matched tumors from obesity category II/III versus normal weight (BMI ≥ 35 kg/m2 versus 18.5-24.9 kg/m2, respectively) subjects. Intratumoral PLGF and VEGF-A proteins were elevated in ccRCC subjects with obesity. Thus, in ccRCC patients with localized disease, obesity is not associated with widespread detrimental alterations in systemic or intratumoral immune profiles. The effects of combined obesity and immunotherapy administration on immune parameters remains to be determined.

Obesity diminishes response to PD-1-based immunotherapies in renal cancer.

BACKGROUND: Obesity is a major risk factor for renal cancer, yet our understanding of its effects on antitumor immunity and immunotherapy outcomes remains incomplete. Deciphering these associations is critical, given the growing clinical use of immune checkpoint inhibitors for metastatic disease and mounting evidence for an obesity paradox in the context of cancer immunotherapies, wherein obese patients with cancer have improved outcomes. METHODS: We investigated associations between host obesity and anti-programmed cell death (PD-1)-based outcomes in both renal cell carcinoma (RCC) subjects and orthotopic murine renal tumors. Overall survival (OS) and progression-free survival (PFS) were determined for advanced RCC subjects receiving standard of care anti-PD-1 who had ≥6 months of follow-up from treatment initiation (n=73). Renal tumor tissues were collected from treatment-naive subjects categorized as obese (body mass index, 'BMI' ≥30 kg/m2) or non-obese (BMI <30 kg/m2) undergoing partial or full nephrectomy (n=19) then used to evaluate the frequency and phenotype of intratumoral CD8+ T cells, including PD-1 status, by flow cytometry. In mice, antitumor immunity and excised renal tumor weights were evaluated ±administration of a combinatorial anti-PD-1 therapy. For a subset of murine renal tumors, immunophenotyping was performed by flow cytometry and immunogenetic profiles were evaluated via nanoString. RESULTS: With obesity, RCC patients receiving anti-PD-1 administration exhibited shorter PFS (p=0.0448) and OS (p=0.0288). Treatment-naive renal cancer subjects had decreased frequencies of tumor-infiltrating PD-1highCD8+ T cells, a finding recapitulated in our murine model. Following anti-PD-1-based immunotherapy, both lean and obese mice possessed distinct populations of treatment responders versus non-responders; however, obesity reduced the frequency of treatment responders (73% lean vs 44% obese). Tumors from lean and obese treatment responders displayed similar immunogenetic profiles, robust infiltration by PD-1int interferon (IFN)γ+CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC), yielding favorable CD44+CD8+ T cell to MDSC ratios. Neutralizing interleukin (IL)-1ß in obese mice improved treatment response rates to 58% and reduced MDSC accumulation in tumors. CONCLUSIONS: We find that obesity is associated with diminished efficacy of anti-PD-1-based therapies in renal cancer, due in part to increased inflammatory IL-1ß levels, highlighting the need for continued study of this critical issue.

Obesity Does Not Exacerbate the Protumorigenic Systemic Environment in Sarcoma Subjects.

Sarcomas are a rare but fatal tumor type that accounts for <1% of adult solid malignancies and ~15% of childhood malignancies. Although the use of immunotherapy is being actively investigated for other solid tumors, advances in immunotherapy for sarcoma patients are lacking. To better understand the systemic immune environment in sarcoma patients, we performed a detailed multiplex analysis of serum cytokines, chemokines, and protumorigenic factors from treatment-naive subjects with localized, high-grade sarcoma. Because obesity is a major healthcare issue in the United States, we additionally examined the effects of obesity on serum protein profiles in our sarcoma subject cohort. We found that the systemic host environment is profoundly altered to favor tumor progression, with epidermal growth factor, angiopoietin-2, vascular endothelial growth factor A, IL-6, IL-8, and MIP-1ß all increased relative to tumor-free controls (all p < 0.05). Surprisingly, we found that obesity did not exacerbate this protumorigenic profile, as epidermal growth factor and IL-8 decreased with increasing subject body mass index (both p < 0.05 versus normal or overweight subjects). The Th2-related cytokines IL-4, IL-5, and IL-13 were also decreased in the presence of obesity. Thus, although the systemic environment in sarcoma subjects favors tumor progression, obesity does not further aggravate the production of protumorigenic factors.

Obesity as defined by waist circumference but not body mass index is associated with higher renal mass complexity.

OBJECTIVES: Obesity, typically defined as a body mass index (BMI)≥30kg/m2, is an established risk factor for renal cell carcinoma (RCC) but is paradoxically linked to less advanced disease at diagnosis and improved outcomes. However, BMI has inherent flaws, and alternate obesity-defining metrics that emphasize abdominal fat are available. We investigated 3 obesity-defining metrics, to better examine the associations of abdominal fat vs. generalized obesity with renal tumor stage, grade, or R.E.N.A.L. nephrometry score. METHODS AND MATERIALS: In a prospective cohort of 99 subjects with renal masses undergoing resection and no evidence of metastatic disease, obesity was assessed using 3 metrics: body mass index (BMI), radiographic waist circumference (WC), and retrorenal fat (RRF) pad distance. R.E.N.A.L. nephrometry scores were calculated based on preoperative CT or MRI. Univariate and multivariate analyses were performed to identify associations between obesity metrics and nephrometry score, tumor grade, and tumor stage. RESULTS: In the 99 subjects, surgery was partial nephrectomy in 51 and radical nephrectomy in 48. Pathology showed benign masses in 11 and RCC in 88 (of which 20 had stage T3 disease). WC was positively correlated with nephrometry score, even after controlling for age, sex, race, and diabetes status (P = 0.02), whereas BMI and RRF were not (P = 0.13, and P = 0.57, respectively). WC in stage T2/T3 subjects was higher than in subjects with benign masses (P = 0.03). In contrast, subjects with Fuhrman grade 1 and 2 tumors had higher BMI (P<0.01) and WC (P = 0.04) than subjects with grade 3 and 4 tumors. CONCLUSIONS: Our data suggest that obesity measured by WC, but not BMI or RRF, is associated with increased renal mass complexity. Tumor Fuhrman grade exhibited a different trend, with both high WC and BMI associated with lower-grade tumors. Our findings indicate that WC and BMI are not interchangeable obesity metrics. Further evaluation of RCC-specific outcomes using WC vs. BMI is warranted to better understand the complex relationship between general vs. abdominal obesity and RCC characteristics.

Obesity alters immune and metabolic profiles: New insight from obese-resistant mice on high-fat diet.

OBJECTIVE: Diet-induced obesity has been shown to alter immune function in mice, but distinguishing the effects of obesity from changes in diet composition is complicated. It was hypothesized that immunological differences would exist between diet-induced obese (DIO) and obese-resistant (OB-Res) mice fed the same high-fat diet (HFD). METHODS: BALB/c mice were fed either standard chow or HFD to generate lean or DIO and OB-Res mice, respectively. Resulting mice were analyzed for serum immunologic and metabolic profiles and cellular immune parameters. RESULTS: BALB/c mice on HFD were categorized as DIO or OB-Res, based on body weight versus lean controls. DIO mice were physiologically distinct from OB-Res mice, whose serum insulin, leptin, gastric inhibitory polypeptide, and eotaxin concentrations remained similar to lean controls. DIO mice had increased macrophage(+) crown-like structures in white adipose tissue, although macrophage percentages were unchanged from OB-Res and lean mice. DIO mice also had decreased splenic CD4(+) T cells, elevated serum GM-CSF, and increased splenic CD11c(+) dendritic cells, but impaired dendritic cell stimulatory capacity (P < 0.05 vs. lean controls). These parameters were unaltered in OB-Res mice versus lean controls. CONCLUSIONS: Diet-induced obesity results in alterations in immune and metabolic profiles that are distinct from effects caused by HFD alone.