Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.

Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.

The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells.

B cell antigen receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to optimize selection for high-affinity B cells. In GCBC, BCR signals are constrained, but the mechanisms are not well understood. Here we describe a GC-specific, AKT-kinase-driven negative feedback loop that attenuates BCR signaling. Mass spectrometry revealed that AKT target activity was altered in GCBCs compared with naive B cells. Retargeting was linked to differential AKT T308 and S473 phosphorylation, in turn controlled by GC-specific upregulation of phosphoinositide-dependent protein kinase PDK1 and the phosphatase PTEN. In GCBCs, AKT preferentially targeted CSK, SHP-1 and HPK1, which are negative regulators of BCR signaling. We found that phosphorylation enhances enzymatic activity of these proteins, creating a negative feedback loop that dampens upstream BCR signaling. AKT inhibition relieved this negative feedback and enhanced activation of BCR-proximal kinase LYN, as well as downstream BCR signaling molecules in GCBCs.

Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice.

CD4+ T cells differentiate into subsets that promote immunity or minimize damage to the host. T helper 17 cells (Th17) are effector cells that function in inflammatory responses. T regulatory cells (Tregs) maintain tolerance and prevent autoimmunity by secreting immunosuppressive cytokines and expressing check point receptors. While the functions of Th17 and Treg cells are different, both cell fate trajectories require T cell receptor (TCR) and TGF-ß receptor (TGF-ßR) signals, and Th17 polarization requires an additional IL-6 receptor (IL-6R) signal. Utilizing high-resolution phosphoproteomics, we identified that both synergistic and additive interactions between TCR, TGF-ßR, and IL-6R shape kinase signaling networks to differentially regulate key pathways during the early phase of Treg versus Th17 induction. Quantitative biochemical analysis revealed that CD4+ T cells integrate receptor signals via SMAD3, which is a mediator of TGF-ßR signaling. Treg induction potentiates the formation of the canonical SMAD3/4 trimer to activate a negative feedback loop through kinases PKA and CSK to suppress TCR signaling, phosphatidylinositol metabolism, and mTOR signaling. IL-6R signaling activates STAT3 to bind SMAD3 and block formation of the SMAD3/4 trimer during the early phase of Th17 induction, which leads to elevated TCR and PI3K signaling. These data provide a biochemical mechanism by which CD4+ T cells integrate TCR, TGF-ß, and IL-6 signals via generation of alternate SMAD3 complexes that control the development of early signaling networks to potentiate the choice of Treg versus Th17 cell fate.

Transforming growth factor ß (TGF-ß) receptor signaling regulates kinase networks and phosphatidylinositol metabolism during T-cell activation.

The cytokine content in tissue microenvironments shapes the functional capacity of a T cell. This capacity depends on the integration of extracellular signaling through multiple receptors, including the T-cell receptor (TCR), co-receptors, and cytokine receptors. Transforming growth factor ß (TGF-ß) signals through its cognate receptor, TGFßR, to SMAD family member proteins and contributes to the generation of a transcriptional program that promotes regulatory T-cell differentiation. In addition to transcription, here we identified specific signaling networks that are regulated by TGFßR. Using an array of biochemical approaches, including immunoblotting, kinase assays, immunoprecipitation, and flow cytometry, we found that TGFßR signaling promotes the formation of a SMAD3/4-protein kinase A (PKA) complex that activates C-terminal Src kinase (CSK) and thereby down-regulates kinases involved in proximal TCR activation. Additionally, TGFßR signaling potentiated CSK phosphorylation of the P85 subunit in the P85-P110 phosphoinositide 3-kinase (PI3K) heterodimer, which reduced PI3K activity and down-regulated the activation of proteins that require phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) for their activation. Moreover, TGFßR-mediated disruption of the P85-P110 interaction enabled P85 binding to a lipid phosphatase, phosphatase and tensin homolog (PTEN), aiding in the maintenance of PTEN abundance and thereby promoting elevated PtdIns(4,5)P2 levels in response to TGFßR signaling. Taken together, these results highlight that TGF-ß influences the trajectory of early T-cell activation by altering PI3K activity and PtdIns levels.

Cutting Edge: TCR Signal Strength Regulates Acetyl-CoA Metabolism via AKT.

TCR signaling activates kinases including AKT/mTOR that engage metabolic networks to support the energetic demands of a T cell during an immune response. It is realized that CD4+ T cell subsets have different metabolic requirements. Yet, how TCR signaling is coupled to the regulation of intermediate metabolites and how changes in metabolite flux contribute to T cell differentiation are less established. We find that TCR signaling regulates acetyl-CoA metabolism via AKT in murine CD4+ T cells. Weak TCR signals promote AKT-catalyzed phosphorylation and inhibition of citrate synthase, elevated acetyl-CoA levels, and hyperacetylation of mitochondrial proteins. Genetic knockdown of citrate synthase promotes increased nuclear acetyl-CoA levels, increased histone acetylation at the FOXP3 promotor and induction of FOXP3 transcription. These data identify a circuit between AKT signaling and acetyl-CoA metabolism regulated via TCR signal strength and that transient fluctuations in acetyl-CoA levels function in T cell fate decisions.

The Alzheimer's Disease-Associated Protein BACE1 Modulates T Cell Activation and Th17 Function.

ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is best known for its role in Alzheimer's disease amyloid plaque formation but also contributes to neurodegenerative processes triggered by CNS injury. In this article, we report that BACE1 is expressed in murine CD4+ T cells and regulates signaling through the TCR. BACE1-deficient T cells have reduced IL-17A expression under Th17 conditions and reduced CD73 expression in Th17 and inducible T regulatory cells. However, induction of the Th17 and T regulatory transcription factors RORγt and Foxp3 was unaffected. BACE1-deficient T cells showed impaired pathogenic function in experimental autoimmune encephalomyelitis. These data identify BACE1 as a novel regulator of T cell signaling pathways that impact autoimmune inflammatory T cell function.

The Lysophosphatidylcholine Transporter MFSD2A Is Essential for CD8+ Memory T Cell Maintenance and Secondary Response to Infection.

Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. In this study, we discovered that the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain containing 2A (MFSD2A) is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of LPC esterified to long chain fatty acids into activated CD8+ T cells, and MFSD2A-deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, which when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.

T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols.

T-cell receptor (TCR) signaling strength is a dominant factor regulating T-cell differentiation, thymic development, and cytokine signaling. The molecular mechanisms by which TCR signal strength is transduced to downstream signaling networks remains ill-defined. Using computational modeling, biochemical assays, and imaging flow cytometry, we found here that TCR signal strength differentially generates phosphatidylinositol species. Weak TCR signals generated elevated phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and reduced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, whereas strong TCR signals reduced PI(4,5)P2 and elevated PIP3 levels. A proteomics screen revealed that focal adhesion kinase bound PI(4,5)P2, biochemical assays disclosed that focal adhesion kinase is preferentially activated by weak TCR signals and is required for optimal Treg induction, and further biochemical experiments revealed how TCR signaling strength regulates AKT activation. Low PIP3 levels generated by weak TCR signals were sufficient to activate phosphoinositide-dependent kinase-1 to phosphorylate AKT on Thr-308 but insufficient to activate mTOR complex 2 (mTORC2), whereas elevated PIP3 levels generated by a strong TCR signal were required to activate mTORC2 to phosphorylate Ser-473 on AKT. Our results provide support for a model that links TCR signaling to mTORC2 activation via phosphoinositide 3-kinase signaling. Together, the findings in this work establish that T cells measure TCR signal strength by generating different levels of phosphatidylinositol species that engage alternate signaling networks to control cell fate decisions.

Histone acetyltransferase CBP is critical for conventional effector and memory T-cell differentiation in mice.

Compared with naïve T cells, memory CD8+ T cells have a transcriptional landscape and proteome that are optimized to generate a more rapid and robust response to secondary infection. Additionally, rewired kinase signal transduction pathways likely contribute to the superior recall response of memory CD8+ T cells, but this idea has not been experimentally confirmed. Herein, we utilized an MS approach to identify proteins that are phosphorylated on tyrosine residues in response to Listeria-induced T-cell receptor (TCR) stimulation in both naïve and memory CD8+ T cells from mice and separated by fluorescence- and flow cytometry-based cell sorting. This analysis identified substantial differences in tyrosine kinase signaling networks between naïve and memory CD8+ T cells. We also observed that an important axis in memory CD8+ T cells couples Janus kinase 2 (JAK2) hyperactivation to the phosphorylation of CREB-binding protein (CBP). Functionally, JAK2-catalyzed phosphorylation enabled CBP to bind with higher affinity to acetylated histone peptides, indicating a potential epigenetic mechanism that could contribute to rapid initiation of transcriptional programs in memory CD8+ T cells. Moreover, we found that CBP itself is essential for conventional effector and memory CD8+ T-cell formation. These results indicate how signaling pathways are altered to promote CD8+ memory cell formation and rapid responses to and protection from repeat infections.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

The Akt/mTOR pathway is a key driver of murine CD4+ T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions.

14-3-3z sequesters cytosolic T-bet, upregulating IL-13 levels in TC2 and CD8+ lymphocytes from patients with scleroderma.

BACKGROUND: IL-13-producing CD8+ T cells have been implicated in the pathogenesis of type 2-driven inflammatory human conditions. We have shown that CD8+IL-13+ cells play a critical role in cutaneous fibrosis, the most characteristic feature of systemic sclerosis (SSc; scleroderma). However, the molecular mechanisms underlying production of IL-13 and other type 2 cytokines by CD8+ T cells remain unclear. OBJECTIVE: We sought to establish the molecular basis of IL-13 overproduction by CD8+ T cells from patients with SSc, focusing on T-bet modulation of GATA-3 activity, which we showed to underlie IL-13 overproduction in CD8+IL-13+ cells from patients with SSc. METHODS: Biochemical and biophysical methods were used to determine the expression and association of T-bet, GATA-3, and regulatory factors in CD8+ T cells isolated from the blood and lesional skin of patients with SSc with severe skin thickening. Chromatin immunoprecipitation analysis determined GATA-3 binding to the IL-13 promoter. ImageStream analysis and confocal microscopy visualized the subcellular localization of T-bet and GATA-3. Transcript levels were decreased by small interfering RNAs. RESULTS: Interaction of T-bet with the adaptor protein 14-3-3z in the cytosol of CD8+ T cells from patients with SSc reduces T-bet translocation into the nucleus and its ability to associate with GATA-3, allowing more GATA-3 to bind to the IL-13 promoter and inducing IL-13 upregulation. Strikingly, we show that this mechanism is also found during type 2 polarization of CD8+ T cells (TC2) from healthy donors. CONCLUSIONS: We identified a novel molecular mechanism underlying type 2 cytokine production by CD8+ T cells, revealing a more complete picture of the complex pathway leading to SSc disease pathogenesis.

Cutting Edge: Differential Regulation of PTEN by TCR, Akt, and FoxO1 Controls CD4+ T Cell Fate Decisions.

Signaling via the Akt/mammalian target of rapamycin pathway influences CD4(+) T cell differentiation; low levels favor regulatory T cell induction and high levels favor Th induction. Although the lipid phosphatase phosphatase and tensin homolog (PTEN) suppresses Akt activity, the control of PTEN activity is poorly studied in T cells. In this study, we identify multiple mechanisms that regulate PTEN expression. During Th induction, PTEN function is suppressed via lower mRNA levels, lower protein levels, and an increase in C-terminal phosphorylation. Conversely, during regulatory T cell induction, PTEN function is maintained through the stabilization of PTEN mRNA transcription and sustained protein levels. We demonstrate that differential Akt/mammalian target of rapamycin signaling regulates PTEN transcription via the FoxO1 transcription factor. A mathematical model that includes multiple modes of PTEN regulation recapitulates our experimental findings and demonstrates how several feedback loops determine differentiation outcomes. Collectively, this work provides novel mechanistic insights into how differential regulation of PTEN controls alternate CD4(+) T cell fate outcomes.

TCR scanning of peptide/MHC through complementary matching of receptor and ligand molecular flexibility.

Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear magnetic resonance to study TCR-pMHC binding, examining recognition of the QL9/H-2L(d) complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3ß loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3ß is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3ß and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with sufficient stability to enable T cell signaling.

Structural and dynamic control of T-cell receptor specificity, cross-reactivity, and binding mechanism.

Over the past two decades, structural biology has shown how T-cell receptors engage peptide/major histocompatibility complex (MHC) complexes and provided insight into the mechanisms underlying antigen specificity and cross-reactivity. Here we review and contextualize our contributions, which have emphasized the influence of structural changes and molecular flexibility. A repeated observation is the presence of conformational melding, in which the T-cell receptor (TCR), peptide, and in some cases, MHC protein cooperatively adjust in order for recognition to proceed. The structural changes reflect the intrinsic dynamics of the unligated proteins. Characterization of the dynamics of unligated TCR shows how binding loop motion can influence TCR cross-reactivity as well as specificity towards peptide and MHC. Examination of peptide dynamics indicates not only peptide-specific variation but also a peptide dependence to MHC flexibility. This latter point emphasizes that the TCR engages a composite peptide/MHC surface and that physically the receptor makes little distinction between the peptide and MHC. Much additional evidence for this can be found within the database of available structures, including our observations of a peptide dependence to the TCR binding mode and structural compensations for altered interatomic interactions, in which lost TCR-peptide interactions are replaced with TCR-MHC interactions. The lack of a hard-coded physical distinction between peptide and MHC has implications not only for specificity and cross-reactivity but also the mechanisms underlying MHC restriction as well as attempts to modulate and control TCR recognition.

Peptide modulation of class I major histocompatibility complex protein molecular flexibility and the implications for immune recognition.

T cells use the αß T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the "altered self" mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.

Cutting edge: Evidence for a dynamically driven T cell signaling mechanism.

T cells use the αß TCR to bind peptides presented by MHC proteins (pMHC) on APCs. Formation of a TCR-pMHC complex initiates T cell signaling via a poorly understood process, potentially involving changes in oligomeric state, altered interactions with CD3 subunits, and mechanical stress. These mechanisms could be facilitated by binding-induced changes in the TCR, but the nature and extent of any such alterations are unclear. Using hydrogen/deuterium exchange, we demonstrate that ligation globally rigidifies the TCR, which via entropic and packing effects will promote associations with neighboring proteins and enhance the stability of existing complexes. TCR regions implicated in lateral associations and signaling are particularly affected. Computational modeling demonstrated a high degree of dynamic coupling between the TCR constant and variable domains that is dampened upon ligation. These results raise the possibility that TCR triggering could involve a dynamically driven, allosteric mechanism.

An immunology primer for computational modelers.

The immune system is designed to protect an organism from infection and damage caused by a pathogen. A successful immune response requires the coordinated function of multiple cell types and molecules in the innate and adaptive immune systems. Given the complexity of the immune system, it would be advantageous to build computational models to better understand immune responses and develop models to better guide the design of immunotherapies. Often, researchers with strong quantitative backgrounds do not have formal training in immunology. Therefore, the goal of this review article is to provide a brief primer on cellular immunology that is geared for computational modelers.

Modeling the T cell immune response: a fascinating challenge.

The immune system is designed to protect the organism from infection and to repair damaged tissue. An effective response requires recognition of the threat, the appropriate effector mechanism to clear the pathogen and a return to homeostasis with minimal damage to self-tissues. T cells play a central role in orchestrating the immune response at all stages of the response and have been the subject of intense study by both experimental immunologists and modelers. This review examines some of the more critical questions in T cell biology and describes the latest attempts to address those questions using approaches that combine mathematical modeling and experiments.

AMPK Drives both Glycolytic and Oxidative Metabolism in Murine and Human T Cells During Graft-versus-host Disease.

Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process involving the cellular energy sensor AMP-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia (GVL) effects. In the current studies, murine AMPK KO T cells decreased oxidative metabolism at early timepoints post-transplant and lacked a compensatory increase in glycolysis following inhibition of the electron transport chain. Immunoprecipitation using an antibody specific to phosphorylated targets of AMPK determined that AMPK modified interactions of several glycolytic enzymes including aldolase, enolase, pyruvate kinase M (PKM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enzyme assays indicated impaired aldolase and GAPDH activity in AMPK KO T cells. Importantly, these changes in glycolysis correlated with both an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma (IFNγ) upon antigenic re-stimulation and a decrease in the total number of donor CD4 T cells recovered at later time points post-transplant. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following expansion in vivo. GVHD results also mirrored those of the murine model, with reduced CD4/CD8 ratios and a significant improvement in disease severity. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells and endorse further study of AMPK inhibition as a potential clinical target for future GVHD therapies.

Role of the CCL5 and Its Receptor, CCR5, in the Genesis of Aldosterone-Induced Hypertension, Vascular Dysfunction, and End-Organ Damage.

BACKGROUND: Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown. METHODS: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction. RESULTS: Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5+/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5-/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NFκB (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NFκB, or CCR5. CONCLUSIONS: Our data demonstrate that CCL5/CCR5, through activation of NFκB and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess.