IL-2 targeted to CD8+ T cells promotes robust effector T cell responses and potent antitumor immunity.

IL-2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, while others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL-2 to CD8+ T cells, which are key anti-tumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, CD8 cis-targeted IL-2 that demonstrates over 500-fold preference for CD8+ T cells over NK and Treg cells, which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL-2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+ T cells in primates. In mice, an AB248 surrogate demonstrated superior anti-tumor activity and enhanced tolerability as compared to an untargeted IL-2RBy agonist. Efficacy was associated with expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings.

CD8-targeted IL2 unleashes tumor-specific immunity in human cancer tissue by reviving the dysfunctional T cell pool.

Tumor-specific CD8+ T cells are key effectors of antitumor immunity but are often rendered dysfunctional in the tumor microenvironment. Immune checkpoint blockade can restore antitumor T cell function in some patients, however most do not respond to this therapy, often despite T cell infiltration in their tumors. We here explored a CD8-targeted IL2 fusion molecule (CD8-IL2) to selectively reactivate intratumoral CD8+ T cells in patient-derived tumor fragments. Treatment with CD8-IL2 broadly armed intratumoral CD8+ T cells with enhanced effector capacity, thereby specifically enabling reinvigoration of the dysfunctional T cell pool to elicit potent immune activity. Notably, the revival of dysfunctional T cells to mediate effector activity by CD8-IL2 depended on simultaneous antigen recognition and was quantitatively and qualitatively superior to that achieved by PD-1 blockade. Finally, CD8-IL2 was able to functionally reinvigorate T cells in tumors resistant to anti-PD-1, underscoring its potential as a novel treatment strategy for cancer patients.

Halofuginone-induced amino acid starvation regulates Stat3-dependent Th17 effector function and reduces established autoimmune inflammation.

The IL-23 pathway is genetically linked to autoimmune disease in humans and is required for pathogenic Th17 cell function in mice. However, because IL-23R-expressing mature Th17 cells are rare and poorly defined in mice at steady-state, little is known about IL-23 signaling. In this study, we show that the endogenous CCR6(+) memory T cell compartment present in peripheral lymphoid organs of unmanipulated mice expresses Il23r ex vivo, displays marked proinflammatory responses to IL-23 stimulation in vitro, and is capable of transferring experimental autoimmune encephalomyelitis. The prolyl-tRNA synthetase inhibitor halofuginone blocks IL-23-induced Stat3 phosphorylation and IL-23-dependent proinflammatory cytokine expression in endogenous CCR6(+) Th17 cells via activation of the amino acid starvation response (AAR) pathway. In vivo, halofuginone shows therapeutic efficacy in experimental autoimmune encephalomyelitis, reducing both established disease progression and local Th17 cell effector function within the CNS. Mechanistically, AAR activation impairs Stat3 responses downstream of multiple cytokine receptors via selective, posttranscriptional suppression of Stat3 protein levels. Thus, our study reveals latent pathogenic functions of endogenous Th17 cells that are regulated by both IL-23 and AAR pathways and identifies a novel regulatory pathway targeting Stat3 that may underlie selective immune regulation by the AAR.

Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids.

IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease.

Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V.

Follicular helper T cells (Tfh cells) are the major producers of interleukin-4 (IL-4) in secondary lymphoid organs where humoral immune responses develop. Il4 regulation in Tfh cells appears distinct from the classical T helper 2 (Th2) cell pathway, but the underlying molecular mechanisms remain largely unknown. We found that hypersensitivity site V (HS V; also known as CNS2), a 3' enhancer in the Il4 locus, is essential for IL-4 production by Tfh cells. Mice lacking HS V display marked defects in type 2 humoral immune responses, as evidenced by abrogated IgE and sharply reduced IgG1 production in vivo. In contrast, effector Th2 cells that are involved in tissue responses were far less dependent on HS V. HS V facilitated removal of repressive chromatin marks during Th2 and Tfh cell differentiation and increased accessibility of the Il4 promoter. Thus, Tfh and Th2 cells utilize distinct but overlapping molecular mechanisms to regulate Il4, a finding with important implications for understanding the molecular basis of allergic diseases.

Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells.

Human memory T cells (T(M) cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. These T cell lineages are almost exclusively CCR6(+) and are important mediators of chronic inflammation and autoimmunity. However, little is known about the mechanisms controlling IL-17/IL-22 expression in memory Th17/Th22 subsets. We show that common γ chain (γc)-using cytokines, namely IL-2, IL-7, and IL-15, potently induce Th17-signature cytokine expression (Il17a, Il17f, Il22, and Il26) in CCR6(+), but not CCR6(-), T(M) cells, even in CCR6(+) cells lacking IL-17 expression ex vivo. Inhibition of phosphoinositide 3-kinase (PI-3K) or Akt signaling selectively prevents Th17 cytokine induction by γc-cytokines, as does ectopic expression of the transcription factors FOXO1 or KLF2, which are repressed by PI-3K signaling. These results indicate that Th17 cytokines are tuned by PI-3K signaling in CCR6(+) T(M) cells, which may contribute to chronic or autoimmune inflammation. Furthermore, these findings suggest that ex vivo analysis of IL-17 expression may greatly underestimate the frequency and pathogenic potential of the human Th17 compartment.

Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs.

Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

Regulation of gene expression in peripheral T cells by Runx transcription factors.

Members of the Runx family of transcription factors, Runx1-3, are essential regulators of the immune system: a deficiency in one of the members, Runx1, results in complete ablation of hematopoiesis, and all three Runx proteins play important, nonredundant roles in immune system development and function. Here, we review gene regulation by Runx proteins in T lymphocytes, with a focus on their recently emerging roles in the development and function of peripheral CD4+ and CD8+ T lineages.

Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.

Cell differentiation involves activation and silencing of lineage-specific genes. Here we show that the transcription factor Runx3 is induced in T helper type 1 (T(H)1) cells in a T-bet-dependent manner, and that both transcription factors T-bet and Runx3 are required for maximal production of interferon-gamma (IFN-gamma) and silencing of the gene encoding interleukin 4 (Il4) in T(H)1 cells. T-bet does not repress Il4 in Runx3-deficient T(H)2 cells, but coexpression of Runx3 and T-bet induces potent repression in those cells. Both T-bet and Runx3 bind to the Ifng promoter and the Il4 silencer, and deletion of the silencer decreases the sensitivity of Il4 to repression by either factor. Our data indicate that cytokine gene expression in T(H)1 cells may be controlled by a feed-forward regulatory circuit in which T-bet induces Runx3 and then 'partners' with Runx3 to direct lineage-specific gene activation and silencing.

Deletion of a conserved Il4 silencer impairs T helper type 1-mediated immunity.

Helper T cell differentiation involves silencing as well as activation of gene expression. We have identified a conserved silencer of the gene encoding interleukin 4 (Il4) marked by DNase I hypersensitivity (HS IV) and permissive chromatin structure in all helper T cells. Deletion of HS IV increased Il4 and Il13 transcription by naive T cells and led to T helper type 2 skewing in vitro. HS IV controlled Il4 silencing during T helper type 1 differentiation, as HS IV-deficient T helper type 1 cells that expressed interferon-gamma also produced abundant interleukin 4 in vitro and in vivo. Despite mounting a vigorous interferon-gamma response, HS IV-deficient mice were more susceptible to Leishmania major infection than were wild-type littermate control mice, showing a critical function for Il4 silencing in T helper type 1-mediated immunity.