CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Indoleamine 2,3-dioxygenase 1 is essential for sustaining durable antibody responses.

Humoral immunity is essential for protection against pathogens, emphasized by the prevention of 2-3 million deaths worldwide annually by childhood immunizations. Long-term protective immunity is dependent on the continual production of neutralizing antibodies by the subset of long-lived plasma cells (LLPCs). LLPCs are not intrinsically long-lived, but require interaction with LLPC niche stromal cells for survival. However, it remains unclear which and how these interactions sustain LLPC survival and long-term humoral immunity. We now have found that the immunosuppressive enzyme indoleamine 2,3- dioxygenase 1 (IDO1) is required to sustain antibody responses and LLPC survival. Activation of IDO1 occurs upon the engagement of CD80/CD86 on the niche dendritic cells by CD28 on LLPC. Kynurenine, the product of IDO1 catabolism, activates the aryl hydrocarbon receptor in LLPC, reinforcing CD28 expression and survival signaling. These findings expand the immune function of IDO1 and uncover a novel pathway for sustaining LLPC survival and humoral immunity.

CD28 Regulates Metabolic Fitness for Long-Lived Plasma Cell Survival.

Durable humoral immunity against epidemic infectious disease requires the survival of long-lived plasma cells (LLPCs). LLPC longevity is dependent on metabolic programs distinct from short-lived plasma cells (SLPCs); however, the mechanistic basis for this difference is unclear. We have previously shown that CD28, the prototypic T cell costimulatory receptor, is expressed on both LLPCs and SLPCs but is essential only for LLPC survival. Here we show that CD28 transduces pro-survival signaling specifically in LLPCs through differential SLP76 expression. CD28 signaling in LLPCs increased glucose uptake, mitochondrial mass/respiration, and reactive oxygen species (ROS) production. Unexpectedly, CD28-mediated regulation of mitochondrial respiration, NF-κB activation, and survival was ROS dependent. IRF4, a target of NF-κB, was upregulated by CD28 activation in LLPCs and decreased IRF4 levels correlated with decreased glucose uptake, mitochondrial mass, ROS, and CD28-mediated survival. Altogether, these data demonstrate that CD28 signaling induces a ROS-dependent metabolic program required for LLPC survival.

Survival of Long-Lived Plasma Cells (LLPC): Piecing Together the Puzzle.

Durable humoral immunity is dependent upon the generation of antigen-specific antibody titers, produced by non-proliferating bone marrow resident long-lived plasma cells (LLPC). Longevity is the hallmark of LLPC, but why and how they survive and function for years after antigen exposure is only beginning to be understood. LLPC are not intrinsically long-lived; they require continuous signals from the LLPC niche to survive. Signals unique to LLPC survival (vs. PC survival in general) most notably include those that upregulate the anti-apoptotic factor Mcl-1 and activation of the CD28 receptor expressed on LLPC. Other potential factors include expression of BCMA, upregulation of the transcription factor ZBTB20, and upregulation of the enzyme ENPP1. Metabolic fitness is another key component of LLPC longevity, facilitating the diversion of glucose to generate pyruvate during times of stress to facilitate long term survival. A third major component of LLPC survival is the microenvironment/LLPC niche itself. Cellular partners such as stromal cells, dendritic cells, and T regulatory cells establish a niche for LLPC and drive survival signaling by expressing ligands such as CD80/CD86 for CD28 and producing soluble and stromal factors that contribute to LLPC longevity. These findings have led to the current paradigm wherein both intrinsic and extrinsic mechanisms are required for the survival of LLPC. Here we outline this diverse network of signals and highlight the mechanisms thought to regulate and promote the survival of LLPC. Understanding this network of signals has direct implications in increasing our basic understanding of plasma cell biology, but also in vaccine and therapeutic drug development to address the pathologies that can arise from this subset.

Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma.

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.