Nat Immunol. 2020 Nov 23
doi: 10.1038/s41590-020-00818-9
Background
Effector T cells exhaustion, which leads to their short persistence, is an important factor limiting the efficacy of immunotherapy. Target therapies with small-molecule inhibitors have been used successfully as anticanceragents, some of which may be provided as tools to modulate immune responses. The mitogen-activated protein kinase (MAPK) pathway relays external mitogenic cues to the immune cells, leading to modulation of cellular differentiation and metabolic machinery that prepares the cells for effector functions. In the tumor microenvironment (TME), persistent mitogenic stimulation induces excessive division of effector cells, generating an exhausted phenotype that shows diminished effector functions and also compromises the generation of immune memory. Inhibition of MEK (MEKi) potentiates the antitumor effects of immunotherapy with checkpoint inhibitors, as well as adoptive T cell transfer therapy (ACT). These effects of MEKi are mediated, in part, by increasing tumor immunogenicity and modulation of the TME. An understanding of the immune modulatory mechanisms is essential to develop new and effective therapeutic strategies utilizing MEKi.
Methods
Mouse: four-to-six-week-old C57BL/6J mice (wild type) and Sirt3 knockout mice on C57BL/6J.
Results
1. MEKi enhances antitumor effects by preventing exhaustion and facilitating activation of effector CD8+ T cells in the TME.MEKi significantly enhanced the vaccine-specific antitumor response in both TC-1 and B16F10 models. A significant percentage of antigen-specific CD8+ T cells expressed granzyme B, indicating that MEKi leads to the expansion of functional antigen-specific effector cells in the TME. There is a significant decrease in CD8+ T cells expressing inhibitory receptors (CTLA4, LAG3 and TIM3), while an increase in CXCR3-, IL-2Rβ- and OX40-expressing activated CD8+ T cells in the TME of MEKi-treated mice. The expression of PD-1 on CD8+ T cells was not changed in MEKi-treated vaccinated mice compared to mice treated with vaccine alone, which is consistent with PD-1 being an activation marker. Together, these data show that MEK inhibition enhances tumor infiltration with the effector CD8+ T cells, prevents exhaustion and maintains effector CD8+ T cells in an activated state. 2. MEKi enhances, etabolic fitness in CD8+ T cells by upregulating mitochondrial function.The mitochondrial mass in CD8+ T cells in the TME was significantly increased in MEKi-treated vaccinated animals compared to control animals. Meanwhile, ex vivo, there was a discernibly higher number of mitochondria in pMel-1 MEKi-treated CD8+ T cells. Morphologically, MEKi-treated cells had compact mitochondria with tightly packed cristae, indicating an intact redox machinery and enhanced respiratory capacity, which was contrary to cells treated with gp100 alone, where mitochondria had diffused cristae. MEKi-treated CD8+ T cells had a significantly higher oxygen consumption rate (OCR)/maximal respiration and spare respiratory capacity (SRC), and lower extracellular acidification rates (ECAR). Increased expression of Cpt1a, a rate limiting enzyme of FAO, and increased uptake of BODIPY, a lipophilic probe that acts as an indicator of lipid uptake, showed enhanced uptake of FAs by MEKi-treated CD8+ T cells. There is also a marked decrease in the palmitate-mediated OCR and proliferation in MEKi-treated CD8+ T cells upon treatment with etomoxir. In line with in vitro data, MEKi treatment of vaccinated animals significantly enhanced FA-mediated metabolism without affecting the glucose uptake rates in the CD8+ T cells in the TME. Moreover, a significant increase in PGC1α, a protein involved in mitochondrial biogenesis confirmed that MEK inhibition enhances metabolism in CD8+ T cells.PCA and pairwise analysis revealed that MEKi-treated cells do not utilize glucose preferentially. Treatment of CD8+ T cells with gp100 alone led to an increase in lactic acid and a corresponding decrease in pyruvate compared to gp100 plus MEKi-treated cells. On the other hand, the overall levels of metabolites in the TCA cycle were less in MEKi-treated cells compared to untreated cells , indicating a quiescent nature for these cells. In line with the palmitate utilization assay the concentration of palmitic acid, as well as of various FA chains, was less in MEKi-treated cells, indicating a greater reliance of these cells on FA metabolism. These data clearly show that MEK inhibition differentially enhances the FA-mediated metabolism in CD8+ T cells.3. MEKi treatment induces TSCM in CD8+ T cells, which is distinct from Tcm and Tnaive.Enhanced metabolic fitness, high SRC and FAO are strong indicators of immune memory. CCR7 (associated with memory) upregulation and KLRG low expression (enabling memory generation) were found in CD8+ T cells from MEKi-treated animals. Meanwhile, there is a significant increase in the numbers of Tcm cells and a significant increase in Tnaive cells, and these cells were also expressing high levels of memory markers (CD95 and CCR7). TSCM cells are characterized by a naïve-like phenotype (CD62L+CD44-) with high expression of Sca1 and lower mitochondrial potential. Following MEKi treatment, there is a significant increased numbers of Sca1+CD62L+CD44- and TMRMlo CD62L+CD44-CD8+ T cells. Furthermore, the expression of IL-2Rβ on the naive-like cells, which acts as a survival factor for lymphocytes, a further indication of stem-like characteristics, was significantly increased following MEKi treatment. Hence, these data indicate that MEKi treatment results in a substantial enrichment of CD8+ TSCM in the TME. To confirm that TSCM (CD45RA+CCR7+CD95+) cells are distinct from Tnaive (CD45RA+CCR7-CD95-) and TCM(CD45RA-CCR7+) cells, the whole-genome and loci-specific methylation profiles of various FACS-sorted human CD8+ T cell phenotypes is examined. The heat map analysis revealed a distinct methylation profile of MEKi-induced TSCM cells. By comparing the CpG DNA methylation of key genes dynamically regulated during T cell memory development (Ifng, Prf1 and Tcf7) across three cell populations, the researchers found the methylation at these gene loci in TSCM cells was also distinct from Tnaive cells, which had a relatively similar degree of CpG demethylation in the Tcf7 locus, and demonstrated nearly full methylation of Ifng and Prf1 loci, which is reflective of their stem-like characteristic (Tcf7) and lack of effector programming (Ifng, Prf1). Hence, the TSCM cells generated after MEKi possessed the characteristics of an intermediate state between Tnaive and TCM cells. These results confirm that CD8+ T cells, when treated with MEKi, become TSCM cells having high self-renewal potential, multipotency and higher proliferation ability, and are distinct from Tnaive and TCM cells.4. MEKi induces TSCM by inhibiting cell cycle progression and differentiation during TCR-mediated cell priming. MEKi inhibits the cell cycle in early phases, while allowing proper antigen-mediated activation to generate naive-like antigen-experienced TSCM cells. Indeed, the analysis of different generations of proliferation clearly showed that generations 1 and 2 had increased numbers of TSCM cells (Sca1hi) when compared to generation 3 after MEKi treatment. Furthermore, without MEKi, the majority of the cells differentiated to TEM while MEK inhibition enriched the TSCM in early generations, inhibiting their further differentiation into TCM and TEM. This indicates that CD8+ T cell activation and its commitment to the cycle of differentiation are independent events. Hence, MEK1/2 inhibition holds CD8+ T cells in the earliest stages of the differentiation cycle without affecting the effector or activation status of the cells.5. MEKi-mediated inhibition of cell cycle progression and metabolic enhancement is necessary for TSCM generation. MEKi-induced TSCM CD8+ T cells have higher recall response and superior antitumor activity after ACT of tumors.MEKi-treated CD8+ T cells show strong effector functions during recall responses and that MEKi-induced TSCM cells contribute substantially to these responses. Reduced levels of mTORC1 are associated with memory generation while upregulated mTORC1 is required for generation of robust effector functions. We observed a reduction in the levels of p-mTORC1 in MEKi-treated CD8+ T cells during initial cell activation and TSCM generation, with an upregulated expression of p-mTORC1 in MEKi-treated CD8+ T cells following antigenic rechallenge, showing that, initially, mTORC1 is inhibited reversibly and available for recall responses. In conclusion, these results indicate that MEKi treatment induces CD8+ TSCM cells by a concerted effect on cell cycle, metabolism and cell activation, which have superior antitumor activity with prolonged persistence in the TME and ACT settings.
Conclusions
This article reported an effective strategy for reprograming naive CD8+ T cells into TSCM through inhibition of MEK1/2 signaling. They found that MEK1/2 inhibition generated exceedingly functional CD8+ TSCM cells with high FAO-mediated metabolic fitness, self-renew ability, high proliferation, multipotent capacity and antigen recall responses. This resulted in generation of potent effector CD8+ T cells with prolonged survival and superior antitumor activity. Thus, utilizing MEK1/2 SMIs represents a major opportunity to enhance the efficacy of immunotherapeutic strategies. This includes a strategy to enhance the antitumor efficacy of ex vivo expanded or engineered T cells for ACT; as well as in vivo use of these inhibitors to augment the effects of immune modulatory agents, especially those that induce, stabilize and enhance T cell memory.