Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic

A20 upregulation during treated HIV disease is associated with intestinal epithelial cell recovery and function

<div><p>Untreated Human Immunodeficiency Virus (HIV) infection is characterized by intestinal epithelial barrier dysfunction and chronic inflammation, related features that are attenuated to variable degrees by suppressive antiretroviral therapy (ART). Specific mediators of intestinal epithelial cell (IEC) dysfunction and restoration during HIV disease and treatment have yet to be identified. We studied IECs isolated from intestinal biopsies by RNAseq and found that mRNA levels for the ubiquitin-modifying enzyme, A20, are upregulated in ART-treated individuals and are positively correlated with markers of epithelial function (e.g., <i>CTNNB</i>, <i>CLDN4</i>, and <i>TJP1</i>). In a murine intestinal organoid model, A20 expression was suppressed by interferon-alpha (IFNα), which is highly expressed during HIV viremia and induces IFN-mediated signaling. Notably, A20 deletion rendered intestinal organoids more susceptible to cell death and inhibition of barrier-related genes mediated by interferon-gamma (IFNγ), a cytokine also present at elevated levels during untreated infection. Furthermore, A20 specifically restricted expression of IL-17A-induced inflammatory genes in organoids. Finally, ART-suppressed chronically infected individuals treated with pegylated IFNα2a for five weeks demonstrated reduced expression of A20 in peripheral blood mononuclear cells. Our results are thus consistent with a model in which enhanced type I interferons suppress A20 levels, leading to IFNγ-mediated dysfunction. As such, variation in A20 expression during the course of HIV infection could underlie both the development of epithelial dysfunction before the initiation of ART and the recovery of intestinal epithelial integrity thereafter.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/Clinical Trial NCT00594880" target="_blank">Clinical Trial NCT00594880</a></p></div

A20 upregulation during treated HIV disease is associated with intestinal epithelial cell recovery and function - Fig 2

<p><b>Intestinal epithelial A20 is downregulated after exposure to IFN</b>α (<b>A</b>) Protein levels of A20, measured by western blot, in murine organoids after treatment with indicated dose of IFNα for 48 hours. (<b>B</b>) Quantification of A20 levels normalized to GAPDH in three independent experiments conducted as in (a). Statistical significance was assessed using a t-test. * P<0.05, **P≤0.01, ***P≤0.001.</p

IEC expression pattern from ART-treated participants is consistent with upregulated A20 (<i>TNFAIP3</i>) levels.

<p>(<b>A</b>) Pairwise comparison of IEC gene expression between viremic and ART-treated participants by RNAseq as analyzed by DESeq2 workflow. Each dot represents a sequenced gene. The right side of the plot indicates relative enrichment in ART-treated relative to viremic individuals, whereas the left side indicates the inverse, enrichment in viremics relative to ART-treated participants. Purple signifies greater than two-fold change expression relative to the comparator in either direction. Red indicates a significant p-value (<0.05) after false discovery rate correction. Green indicates fulfillment of both of these criteria. Genes of particular interest are highlighted in bold. (<b>B</b>) Spearman correlation of A20 transcript levels and <i>NFKBIA</i> expression, as measured by RNAseq, in ART-treated participants (n = 19). Spearman correlation of A20 mRNA or <i>NFKBIA</i> gene expression, as indicated on the x-axis, to transcript levels of (<b>C</b>) proliferation-associated β-catenin, <i>CTNNB1</i>, or the tight junction genes (<b>D</b>) <i>CLDN4</i> and (<b>E</b>) <i>TJP1</i>.</p

Clinical characteristics of the cohort.

<p>Clinical characteristics of the cohort.</p

Five week pegylated-IFNα2a immunotherapy of ART-suppressed HIV-infected individuals leads to reduction in A20 expression.

<p>(<b>A</b>) Schematic showing duration and frequency of pegylated-IFNα2a administration in participants. Transcript levels of (<b>B</b>) IFN-stimulated gene ISG15 and (<b>C</b>) A20 in PBMCs at baseline and after five weeks of IFNα treatment as assessed by qPCR. Statistical significance was determined by paired t-test. * P<0.05, ****P≤0.0001.</p

A20 modulation of epithelial function during HIV infection and treatment.

<p>Model demonstrating proposed relationship of A20 and relevant cytokines (Type I IFNs, IFNγ, and IL-17A) in the context of untreated (viremic) (left) and ART-treated (right) HIV disease.</p

A20 deletion enhances the cytotoxic effects of IFNα and IFNγ.

<p>(<b>A</b>) Quantification of cell viability by CellTiter Glo 3D assay in organoid cultures after stimulation with three doses of rIFNγ, rIFNα, or rIL-17A for 24 hours. Significance of Cre+ or Cre- was assessed separately by ANOVA. Stars above data points represent statistical significance of cytokine treatment relative to the media control within either the Cre+ (red) or Cre- (black) line after post-hoc comparisons were made by t-test, correcting for multiple comparisons by Scheffe method. Stars above brackets indicate a significant difference between A20-sufficient (Cre-, black) and A20-deficient (Cre+, red) culture within the indicated media condition by t-test. *P<0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. (<b>B</b>) Representative confocal images of propidium iodide (PI)-stained organoids treated for 24 hours with 4OHT followed by 10 ng/ml of indicated cytokine for 24 hours. Two organoids representing the range of PI staining are shown for each condition.</p