Intrinsic and extrinsic drivers of Treg fragility in the tumor microenvironment

Regulatory T cells (Tregs) are required to maintain immune homeostasis through suppressive mechanisms. Characterized by the transcription factor Foxp3, they exert function in a variety of ways, including producing adenosine, and secreting suppressive cytokines. While mandatory for prevention of autoimmunity, Tregs contribute to cancer progression by suppressing the anti-tumor immune response. Depletion of the entire Treg pool is not a viable option for therapy, as mice and humans lacking this population succumb to systemic autoimmunity. Therefore, it is critical to uncover novel mechanisms to target Treg function specifically within the tumor microenvironment (TME) and determine means by which they can in turn be regulated or counter-regulated. Neuropilin-1 (Nrp1) is required to maintain Treg function and stability within the TME through binding of Semaphorin-4a, and when deleted from Tregs, mice show significantly reduced tumor growth or clearance (Appendix A). However, Nrp1 is dispensable for Treg function in the periphery, making this an attractive target in cancer immunotherapy. Interestingly, Nrp1–/– Tregs maintain Foxp3 expression but lose suppressive function, resulting in Treg ‘fragility’, a term I have coined to describe this phenomenon (Chapter 3). Rather, these cells secrete the pro-inflammatory cytokine IFNγ and cripple surrounding WT Tregs, leading to infectious fragility and enhanced anti-tumor immunity. Strikingly, IFNγ-mediated Treg fragility is required for response to PD-1 blockade in mice, and seems to be partially responsible for response to other immunotherapies, including antibodies and vaccines (Chapter 4). Nrp1 also prevents Treg fragility by acting as a driver of Treg metabolic plasticity within the tumor microenvironment. Indeed, while WT Tregs are distinct metabolically in order to survive in a nutrient-depleted environment within the tumor, Nrp1–/– Tregs are dependent on glycolysis (Chapter 5). This is thought to be due to an increase in Hif1α which supports glycolysis and increases IFNγ in Tregs. Lastly, I have shown that Nrp1 supports Treg stability through maintaining hypomethylation at the Foxp3 locus in the tumor microenvironment, as Nrp1–/– Tregs are hypermethylated, but maintain Foxp3 expression (Appendix B). Taken together, my findings have uncovered new mechanisms that drive Treg fragility in tumors that are critical for response to immunotherapy

Elucidating the role of Neuropilin-1 in intra-tumoral regulatory T cell stability

Regulatory T cells (Tregs) play an integral role in the adaptive immune system through suppression of self-reactive immune responses in order to prevent autoimmunity and maintain homeostasis. However, they are deleterious in cancer through suppression of the anti-tumor immune response. In fact, we show that deletion of 50% of Tregs results in normal tumor growth. Therefore, it is advantageous to understand the role of Tregs in the tumor microenvironment in order to create targeted cancer therapies. Our lab has shown that the Neuropilin-1 (Nrp1) pathway is required for Treg stability in the tumor microenvironment, but is disposable for maintaining immune homeostasis in the periphery, identifying it as a prime therapeutic target.\ud \ud In order to further understand the role of Nrp1-deficient Tregs intratumorally, we constructed a competitive environment by utilizing Foxp3, which is located on the X chromosome, and as a result of X-inactivation, female Foxp3 Cre-YFP heterozygous mice are cellular heterozygotes. We generated Nrp1 L/L Foxp3 Cre-YFP/+ heterozygous mice comprised of 50% WT Tregs and 50% Nrp1-deficient Tregs. Surprisingly, when given B16 melanoma, heterozygous mice phenocopy Nrp1 L/L Foxp3 Cre-YFP homozygous mice (Figure 1A). This suggests that Nrp1-deficient T regs are playing an active role in shifting the anti-tumor immune response by destabilizing surrounding WT T regs as determined by DNA methylation status (Figure 1B). Neither WT nor Nrp1-deficient Tregs in the tumor from Nrp1 L/L Foxp3 Cre-YFP/+ mice can suppress in a standard microsuppression assay ex vivo, unlike WT Tregs from Foxp3 Cre-YFP mice. Through various co-culture experiments, we revealed that destabilization of WT Tregs is possibly due to a soluble factor derived from Nrp1-deficient Tregs. Our data revealed that Nrp1-deficient Tregs produce large amounts of IFNγ in the tumor microenvironment. Indeed, when treated with IFNγ, WT T regs lose suppressive capacity. In order to uncover potential novel pathways leading to this phenotype, we are performing global transcript studies using RNASeq. Overall, we have shown that Nrp1 is required for intratumoral Treg stability, and in its absence, there is an alteration in the tumor microenvironment, leading to an enhanced anti-tumor immune response. These studies uncover a novel potential target for future cancer immunotherapies that preserves peripheral immune health

Combination immunotherapy: Where do we go from here?

The remarkable clinical success of cancer immunotherapies targeting the checkpoint receptors CTLA-4 and PD-1 has generated considerable excitement and emboldened efforts to build on this important foundation. Research efforts are now focused on understanding the mechanism of action of these immunotherapies, identifying new inhibitory mechanisms that could be targeted to achieve responses in patients with refractory cancers, and developing approaches that might exhibit efficacy against “immunologically inert” tumors. The outstanding challenges in moving forward are developing reliable strategies for determining which patients will respond optimally to a given immunotherapy, and what combination of immunotherapies and conventional therapies will prove beneficial against each tumor type. These issues were discussed in a one-day workshop at the SITC meeting in November 2014

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3. © 2013 Nature America, Inc. All rights reserved

Metabolic support of regulatory T cells by lactic acid

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumor microenvironment (TME) promotes the recruitment, differentiation, and activity of these cells1,2. Tumor cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumor for metabolites and impairs their function4–6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9–11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Lactic acid also contributes directly to epigenetic modifications through histone lactylation which may support the expression of Treg cell signature genes. Deletion of MCT1—a lactate transporter—in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumor growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use ‘alternative’ metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumors avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells

The physiological basis and mechanistic requirement for the high immunoreceptor tyrosine activation motifs (ITAM) multiplicity of the T cell receptor (TCR)-CD3 complex remains obscure. Here we show that while low TCR-CD3 ITAM multiplicity is sufficient to engage canonical TCR-induced signaling events that lead to cytokine secretion, high TCR-CD3 ITAM multiplicity is required for TCR-driven proliferation. This is dependent on compact immunological synapse formation, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate Notch1 recruitment and activation and ultimately c-Myc-induced proliferation. Analogous mechanistic events are also required to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and co-ordinated by the multiplicity of phosphorylated TCR-CD3 ITAMs