The malate shuttle detoxifies ammonia in exhausted T cells by producing 2-ketoglutarate.

The malate shuttle is traditionally understood to maintain NAD+/NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8+ T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD+/NADH ratio and limited antiviral CD8+ T cell responses to chronic infection; however, increasing the NAD+/NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8+ T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8+ T cells is not to maintain the NAD+/NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8+ T cells during chronic infection.

Regulatory T cell-derived interleukin-15 promotes the diversity of immunological memory.

While the immunosuppressive function of regulatory T (Treg) cells has been extensively studied, their immune-supportive roles have been less well investigated. Using a lymphocytic choriomeningitis virus (LCMV) Armstrong infection mouse model, we found that Treg cell-derived interleukin (IL)-15 is required for long-term maintenance of the KLRG1+ IL-7Rα- CD62L- terminal effector memory CD8+ T (tTEM) cell subset, but dispensable for the suppressive function of Treg cells themselves. In contrast, deletion of Il15 from other sources, including myeloid cells and muscles, did not affect the composition of the memory CD8+ T cell pool. Our findings identify Treg cells as an essential IL-15 source maintaining tTEM cells and suggest that Treg cells promote the diversity of immunological memory.

Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine-mediated regulation of c-Fos.

CD4+ regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) and suppress the immune system. Whether and how metabolite availability in the TME influences Treg cell differentiation is not understood. Here, we measured 630 metabolites in the TME and found that serine and palmitic acid, substrates required for the synthesis of sphingolipids, were enriched. A serine-free diet or a deficiency in Sptlc2, the rate-limiting enzyme catalyzing sphingolipid synthesis, suppressed Treg cell accumulation and inhibited tumor growth. Sphinganine, an intermediate metabolite in sphingolipid synthesis, physically interacted with the transcription factor c-Fos. Sphinganine c-Fos interactions enhanced the genome-wide recruitment of c-Fos to regions near the transcription start sites of target genes including Pdcd1 (encoding PD-1), which promoted Pdcd1 transcription and increased inducible Treg cell differentiation in vitro in a PD-1-dependent manner. Thus, Sptlc2-mediated sphingolipid synthesis translates the extracellular information of metabolite availability into nuclear signals for Treg cell differentiation and limits antitumor immunity.

Rgs16 promotes antitumor CD8+ T cell exhaustion.

T cells become functionally exhausted in tumors, limiting T cell-based immunotherapies. Although several transcription factors regulating the exhausted T (Tex) cell differentiation are known, comparatively little is known about the regulators of Tex cell survival. Here, we reported that the regulator of G protein signaling 16 (Rgs-16) suppressed Tex cell survival in tumors. By performing lineage tracing using reporter mice in which mCherry marked Rgs16-expressing cells, we identified that Rgs16+CD8+ tumor-infiltrating lymphocytes (TILs) were terminally differentiated, expressed low levels of T cell factor 1 (Tcf1), and underwent apoptosis as early as 6 days after the onset of Rgs16 expression. Rgs16 deficiency inhibited CD8+ T cell apoptosis and promoted antitumor effector functions of CD8+ T cells. Furthermore, Rgs16 deficiency synergized with programmed cell death protein 1 (PD-1) blockade to enhance antitumor CD8+ T cell responses. Proteomics revealed that Rgs16 interacted with the scaffold protein IQGAP1, suppressed the recruitment of Ras and B-Raf, and inhibited Erk1 activation. Rgs16 deficiency enhanced antitumor CD8+ TIL survival in an Erk1-dependent manner. Loss of function of Erk1 decreased antitumor functions of Rgs16-deficient CD8+ T cells. RGS16 mRNA expression levels in CD8+ TILs of patients with melanoma negatively correlated with genes associated with T cell stemness, such as SELL, TCF7, and IL7R, and predicted low responses to PD-1 blockade. This study uncovers Rgs16 as an inhibitor of Tex cell survival in tumors and has implications for improving T cell-based immunotherapies.

No Evidence for Human Monocyte-Derived Macrophage Infection and Antibody-Mediated Enhancement of SARS-CoV-2 Infection.

Vaccines are essential to control the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and to protect the vulnerable population. However, one safety concern of vaccination is the possible development of antibody-dependent enhancement (ADE) of SARS-CoV-2 infection. The potential infection of Fc receptor bearing cells such as macrophages, would support continued virus replication and inflammatory responses, and thereby potentially worsen the clinical outcome of COVID-19. Here we demonstrate that SARS-CoV-2 and SARS-CoV neither infect human monocyte-derived macrophages (hMDM) nor induce inflammatory cytokines in these cells, in sharp contrast to Middle East respiratory syndrome (MERS) coronavirus and the common cold human coronavirus 229E. Furthermore, serum from convalescent COVID-19 patients neither induced enhancement of SARS-CoV-2 infection nor innate immune response in hMDM. Although, hMDM expressed angiotensin-converting enzyme 2, no or very low levels of transmembrane protease serine 2 were found. These results support the view that ADE may not be involved in the immunopathological processes associated with COVID-19, however, more studies are necessary to understand the potential contribution of antibodies-virus complexes with other cells expressing FcR receptors.

Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, a new member of the genus Betacoronavirus, is a pandemic virus, which has caused numerous fatalities, particularly in the elderly and persons with underlying morbidities. At present, there are no approved vaccines nor antiviral therapies available. The detection and quantification of SARS-CoV-2-neutralizing antibodies plays a crucial role in the assessment of the immune status of convalescent COVID-19 patients, evaluation of recombinant therapeutic antibodies, and the evaluation of novel vaccines. To detect SARS-CoV-2-neutralizing antibodies, classically, a virus-neutralization test has to be performed at biosafety level 3, considerably limiting the general use of this test. In the present work, a biosafety level 1 pseudotype virus assay based on a propagation-incompetent vesicular stomatitis virus (VSV) has been used to determine the neutralizing antibody titers in convalescent COVID-19 patients. The neutralization titers in serum of two independently analyzed patient cohorts were available within 18 h and correlated well with those obtained with a classical SARS-CoV-2 neutralization test (Pearson correlation coefficients of r = 0.929 and r = 0.939, respectively). Most convalescent COVID-19 patients had only low titers of neutralizing antibodies (ND50 < 320). The sera of convalescent COVID-19 patients also neutralized pseudotype virus displaying the SARS-CoV-1 spike protein on their surface, which is homologous to the SARS-CoV-2 spike protein. In summary, we report a robust virus-neutralization assay, which can be used at low biosafety level 1 to rapidly quantify SARS-CoV-2-neutralizing antibodies in convalescent COVID-19 patients and vaccinated individuals.