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Barrier tissues are sentinelled by CD4+ TRM cells with potent anti-microbial activities and considerable lineage plasticity. We hypothesized that local antigen presentation by lung epithelial cells (LECs) instruct CD4+ TRM cell activities. Pneumococcal infections in transgenic mice, flow- and spectral-cytometry, computational biology, and immunofluorescence were used to study this biology. All LECs including a novel alveolar surfactant protein C (SPC)low LEC were adept at antigen presentation. Temporal analysis of LECs for MHC-II and costimulatory/coinhibitory molecules revealed that airway club cells were T-cell stimulatory via CD40 while alveolar LECs expressed T-cell inhibitory PD-L1. This anatomical segregation of LEC antigen presentation correlated with deposition of CD4+ TRM cells around airways such that ablation of LEC MHC-II disrupted CD4+ TRM niches and blockade of CD40 signals prevented accumulation of CD4+ TRM cells. Recurrent memory recalls in absence of LEC MHC-II led to expansion of unconventional CD4+ TRM cells co-expressing classically incompatible lineage-defining transcription factors, changing their cytokine repertoire and leading to dysregulated immunity that phenocopied clinical features of checkpoint blockade therapy. Consequently, a tight correlation between MHC-II and PD-L1 was confirmed in mouse and human LECs. We discovered that LEC MHC-II functions in post-translational trafficking lockstep with PD-L1 to exert its restraints on TRM cell activities. Our results identify epithelial antigen presentation as critical instructors of CD4+ TRM cell locations, phenotypes and activities and establish epithelial-CD4+ TRM cell immunological synapses as key components of barrier immunity.

Obesity-associated Type 2 diabetes (T2D) is driven by chronic inflammation. A combinatorial Th17 cytokine profile characterizes and mathematically predicts T2D in people, but the mechanisms that generate the Th17 profile are not clear. We tested the possibility that anaerobic glycolysis, which fuels inflammatory cytokine production from multiple immune cell types, drives the T2D-associated Th17 profile. We showed that activated PBMCs and purified CD4+T cells from T2D subjects prefer anaerobic glucose metabolism to produce ATP regardless of fuel availability. Unexpectedly, glucose starvation did not abrogate the T2D-predictive Th17 profile. Gene expression array suggested that mitochondrial fatty acid uptake catalyzing protein CPT1a differentiated PBMCs from T2D and ND subjects. CPT1a inhibition by etomoxir strongly down-regulated the Th17 profile, surprisingly independent of CPT1a-mediated fatty acid oxidation (OXPHOS). These data suggest that OXPHOS-independent mitochondrial changes that are also glucose-independent support the Th17 profile in T2D. Analyses from T2D vs ND PBMCs showed a lower CACT:CPT1a protein ratio in T2D, indicating defects in lipid uptake and thus defects in lipid flux. We knocked down CACT protein and overloaded PBMCs from lean subjects with fatty acylcarnitines to mimic lipid uptake defects in T2D. 16C-fatty acylcarnitine, but not 6C- or 10C-derivatives, increased frequency of CD4+IL-17+T cells, and phenocopied the Th17 profile only in CACT knockdown cells. 16C-fatty acylcarnitine alone had no effect. We conclude that excessive long chain fatty acylcarnitine combines with dysfunctional mitochondria to support a T2D-associated Th17 profile largely independent of glycolysis.

Our previous work implicates γδ T cells as an inflammatory driver in ART-suppressed HIV infection and suggests that this unique T cell population serves distinct roles in the ‘inflamm-aging’ that occurs with and without virally controlled HIV. In this study, we honed our previous findings to determine the precise γδ T cell subsets that are driving inflammation with age +/− HIV infection. We performed in depth immunophenotyping of circulating γδ T cells via multi-color flow cytometry and analysis of plasma markers of inflammation, coagulation, and intestinal permeability from subjects of our HIV and Aging cohort, which includes ART-suppressed HIV+ individuals and matched uninfected controls stratified by age into younger (≤35yo) and older (≥50yo) groups. We found discrete differences in the combinational expression the inhibitory receptors (IRs) TIGIT, PD-1, and CD160 between the Vδ1+ and the Vδ2+ γδ T cell subsets with HIV infection and healthy aging. Also, we found that specific IR signatures of Vδ1+ and Vδ2+ γδ T cells correlated with plasma inflammatory markers; however, different connections between the plasma analytes were found for each of the two γδ T cell subsets. Taken together, these data indicate that Vδ1+ and Vδ2+ γδ T cells exist in distinct states and possess divergent roles in the ‘inflamm-aging’ found in aviremic HIV+ infection and with normal aging. Further investigation into the anatomical locations, ex vivo functions, and additional surface markers that define γδ T cell subsets may reveal novel therapeutic targets to abrogate the aberrant inflammation found with HIV infection, aging, and other inflammatory conditions.

Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated proinflammatory cytokine response remain poorly characterized. Bromodomain and extraterminal (BET) proteins are “readers” of histone acetylation marks, with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of brd2 lo mice, a BET protein hypomorph, to show that Brd2 is essential for proinflammatory cytokine production in macrophages. Studies that use small interfering RNA knockdown and a small-molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that Brd2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the “cytokine storm” in endotoxemic mice by reducing levels of IL-6 and TNF-α while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small-molecule inhibitors will benefit hyperinflammatory conditions associated with high levels of cytokine production.

Virologically suppressed HIV+ individuals have an increased risk and earlier onset of age-associated diseases such as cardiovascular atherosclerosis, cancer, and osteoporosis, referred to as HIV-Associated Non-AIDS conditions (HANAs). Whether HIV drives HANAs via same mechanisms as normal aging or other processes is unclear. We analyzed PBMC from 45 ART-suppressed HIV+ subjects (20 ≤35yo ‘younger’, 25 ≥50yo ‘older’) and 39 uninfected controls (20 younger, 19 older) with a 16-color flow cytometry panel that measures the inhibitory receptors (IRs) PD-1, TIGIT, CD160, TIM-3, and LAG-3 on CD4+ T cells, CD8+ T cells, NK cells, iNKT cells, and gd T cells. Bioinformatic (CITRUS) analysis revealed that gd T cell IR expression exclusively differentiated HIV+ subjects from controls. Also, a synergistic effect of HIV and aging on TIGIT+ and ≥ 2 IR gd T cell percentages was found. gd T cell functional profiling revealed a positive association between TIGIT expression and spontaneous release of granzymes A and B from HIV+ subjects but not controls. Using supervised multivariate modeling (Partial Least Squares discriminant analysis, PLS) of gd T IR signatures, gd T cell cytokine secretion, and inflammatory plasma analytes, all four subject groups (HIV+ and uninfected, ≤35 and ≥50yo) were effectively distinguished. Also, a statistically significant covariance between gd T cell IR signatures and levels of inflammatory plasma analytes, including IL-6, IL-1b and fibrinogen was found. Our data implicate gd T cells as a key inflammatory driver in ART-suppressed HIV infection and our PLS results suggest a divergence of ART-suppressed HIV inflammation and healthy aging processes, revealing differential “inflammaging” with and without HIV infection.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder associated with aberrant fibrosis of the skin and internal organs that can lead to organ failure and death in patients. With few treatment options many patients succumb to fibrosis of the lungs or kidneys, or due to vascular damage culminating in pulmonary disease; the 50 percent survival rate is only about 10 years. We collected PBMCs from 36 SSc patients that were not on immune-suppressants, along with 25 age-matched healthy controls. Using a 15 color flow cytometry panel our lab has observed increases in the inhibitory receptors PD-1, TIGIT, and Tim-3 on PBMCs from SSc patients. This increase in expression is cell type specific with PD-1 and TIGIT shown to be increased on defined T cell types including CD4+ T cells, both Treg and non-Treg subsets, as well as CD8+ T cells. Tim-3 was only statistically significantly increased on a mature subset of CD16+ CD56med natural killer cells. This increase was not seen for all inhibitory receptors. The expression of LAG-3 remained low across all cell types observed. This increase in inhibitory receptor expression was enhanced in patients with a high percent of Tregs within the CD4+ gate. In vitro blockade of these inhibitory receptors provided differential cytokine secretion in patients versus healthy subjects, suggesting a possible role in disease pathogenesis. The observation of increased inhibitory receptors in SSc as well as the ability to modulate cytokines by blocking the interaction of these receptors with their ligands could provide insights into possible immune modulation in patients.

Inhibitory Receptors (IRs) such as PD-1, TIM-3, CD160, and TIGIT are upregulated during activated, exhausted, and pathological immune cell states. How the combinational expression of multiple IRs on individual cells tracks with functional status and/or fate is largely unknown. Due to paucity of human samples, large panels for immune cell characterization are of a high value, and combining them with automated platforms of multi-dimensional single cell analysis would allow objective and comprehensive population characterization. We report an 18-parameter (16-color) fluorescent panel that allows IR signature analysis as well as cell sorting for functional studies, and adopt algorithmic methods previously used for mass cytometry into a novel platform to analyze fluorescent datasets in an unbiased analytical workflow. PBMC from diseased and healthy control subjects as well as tumor specimens were stained and analyzed on a FACSARIA cell sorter. For the automated analysis, the datasets were processed with t-SNE algorithm into a 2D map of phenotypic space that was subsequently clustered using SPADE. From the samples, a 32-plex combinational IR signature was determined for the CD4+ T, CD8+ T, NK, iNKT, and γδ T cell lineages. The results indicate novel combinational IR ‘hits’ for particular immune subsets and some immune subset IR signatures correlated with disease status. In conclusion, 18-parameter flow cytometry with automated analysis provides robust results from human blood and solid tissue samples. In combination with functional and other readouts, measurement of single immune cell IR signatures could provide new insight into activation, exhaustion, and pathogenic processes and potentially reveal novel therapeutic targets.