Investigations in Immunological Tolerance

Abstract

The vertebrate immune system has evolved the remarkable ability to restrain collateral damage to healthy tissues and cells, while being able to mount, in an individually-tailored and highly precise manner, anti-pathogen immune responses. This immunological homeostasis entails strict maintenance of peripheral tolerance, which depends on the intact development and function of a population of CD4+CD25+ regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (Foxp3). It is now appreciated that many human diseases are immune-dysregulatory in nature and may evolve as a consequence of impaired Treg cell function and/or development, especially in the context of sustained inflammation leading to Foxp3 destabilization. Surprisingly, Foxp3-deficient Treg (ΔTreg) cells retain a canonical core Treg cell transcriptome but acquire a subverted metabolic profile similar to that of effector T cells, which contributes to their regulatory degeneration. However, the recent demonstration that ΔTreg cells can be functionally “reprogrammed” by virtue of targeted metabolic perturbations has led to the conceptually novel motivation to therapeutically manipulate ΔTreg cell metabolism for the purposes of tolerance induction in the settings of autoimmunity.

In this work, we first investigated the functional consequences of restoring defective ΔTreg cell fatty acid oxidation (FAO) and its impact on the overall metabolic dysregulation in Foxp3 deficiency. Metabolomic analysis of ΔTreg cells revealed a profound reduction in levels of carnitine, a non-essential amino acid requisite for the transportation of activated long chain fatty acids into the mitochondrial matrix for β-oxidation. Consistent with a diminished contribution of FAO to the dysregulated oxygen consumption rate (OCR) of ΔTreg cells, carnitine deficiency was associated with heightened intracellular fatty acid concentrations. Supplementation of L-carnitine in-vivo did not extend survival of Foxp3-deficient mice despite reducing frequencies of splenic effector-memory CD4+ and CD8+ T lymphocytes. ΔTreg cells sorted from L-carnitine treated animals however demonstrated a striking reduction in their OCR, suggesting L-carnitine treatment in-vivo was sufficient to partially normalize the dysregulated oxidative phosphorylation (OXPHOS) of ΔTreg cells. Consistent with a carnitine intrinsic defect in the dysregulation of FAO in ΔTreg cells, treatment of Foxp3-deficient mice with the AMP-activated protein kinase activator metformin failed to recapitulate the effects of L-carnitine supplementation. Addition of L-carnitine to ΔTreg cells in-vitro augmented their OCR, indicating that L-carnitine supplementation was sufficient to increase the contribution of FAO to the OXPHOS capacities of ΔTreg cells. Collectively, these studies contribute to the functional delineation of metabolic dysregulation in ΔTreg cells and provide a rationale for combinatorial metabolic interventions aimed at restoring defective FAO of ΔTreg cells in Foxp3 deficiency. In our second investigation, we studied a patient with a novel mutation in the interleukin 2 receptor (IL-2R) alpha chain (CD25) gene (IL2RA) presenting with overlapping features of FOXP3 deficiency. We identified a homozygous nonsense mutation (c.246G>A) in exon 2 of IL2RA, leading to CD25 deficiency characterized by impaired T lymphocyte signal transducer and activator of transcription 5 (STAT5) responsiveness. Surprisingly, we observed increased frequencies of CD4+ but not CD8+ T lymphocytes in the peripheral blood, with massive expansion of the CXCR5+PD-1+ T follicular helper (TFH) pool. Augmentation of the TFH compartment was associated with high CXCR3 expression, indicative of T helper 1 cell-like skewing. Consistent with the massive peripheral TFH cell expansion, serum analysis revealed a marked increase in levels of circulating IgG and IgM antibodies against autologous antigens. Flow cytometry studies uncovered profound dysregulation in the naïve and memory T cell compartments with increased frequencies of effector and central memory T cell subsets in the peripheral blood. Although present at lower frequencies, Treg cells displayed a profound reduction in expression levels of canonical Treg cell markers including Helios, CTLA-4, and FOXP3. Taken together, these studies identify for the first-time dysregulation of the TFH compartment and provide a novel impetus for investigations into putative follicular regulatory T cell dysfunction in the setting of CD25 deficiency.