Zippers Hold Neutrophils Together See article p.80

TIGIT Tightens Treg Stability See article p.145

New Twists in Cytokine Regulation See article p.217

Seeking Skin T Cells in Their Natural Habitat See article p.386

The rare primary immunodeficiency disease neutrophil-specific granule deficiency (SGD) is caused by defects in the gene encoding CCAAT/enhancer binding protein-ε (C/EBPε) and is characterized by neutrophil defects that promote susceptibility to bacterial infections. C/EBPε is a granulocyte-specific transcription factor that binds to DNA via its basic leucine zipper (bZIP) domain and is key to terminal differentiation of neutrophils and expression of genes in these cells’ specific granules. In this issue, Wada et al. (p. 80) identify a germline-encoded 2-aa deletion (ΔRS) in the bZIP domain of C/EBPε in a patient with SGD and characterize how this mutation results in neutrophil dysfunction. Similar to a previously documented case of SGD, this patient had neutrophils with bilobed nuclei and an absence of cytoplasmic granules, and these cells also expressed CD14, suggesting aberrant differentiation toward the monocyte lineage. Unlike the frameshift mutations in the C/EBPε gene observed in two other SGD patients, the ΔRS mutation did not result in impaired C/EBPε expression or nuclear localization; however, all three mutant proteins exhibited significantly decreased transcriptional activity. Interestingly, the ΔRS mutant protein, but not the frameshift mutants, bound normally to DNA. It was also able to homodimerize normally and form heterodimers with wild-type C/EBPε. However, relative to the wild-type protein, the ΔRS mutant had an impaired ability to bind to the transcription factors Gata1 and PU.1, resulting in impaired transcription of eosinophil major basic protein. These data suggest that the ΔRS mutation causes pathology by impairing cooperative transcriptional activation, a finding that advances our understanding both of the pathogenesis of SGD and the normal activity of C/EBPε.

In the intestine, chronic stimulation of monocytes/macrophages through nucleotide-binding oligomerization domain 2 (NOD2) leads to cytokine downregulation, helping to maintain intestinal immune homeostasis. Zheng et al. (p. 217) have now found that the transcriptional repressors Twist1 and Twist2, which have been shown to regulate inflammation, act cooperatively to downregulate cytokine production in human monocyte-derived macrophages (MDMs) stimulated via NOD2 in either an acute or chronic manner. Chronic NOD2 stimulation increased Twist1 and Twist 2 expression in normal MDMs, but not in MDMs from Crohn’s disease patients bearing a loss-of-function NOD2 allele, and this upregulation required autocrine IL-10 and TGF-β production. Twist1/Twist2 affected cytokine expression not only by directly binding to cytokine gene promoters, but also by upregulating the transcriptional repressors c-Maf and Bmi1. Forced upregulation of these transcriptional repressors in Twist-deficient MDMs restored cytokine downregulation following chronic NOD2 stimulation. The Twist proteins also mediated the downregulated expression and reduced promoter binding of the transcriptional activators ATF4, C/EBPα, Runx1, and Runx2 observed after chronic NOD2 stimulation of MDMs. Chronic stimulation of several TLRs also induced upregulation of Twist1/Twist2 and activation of the same pathways observed with NOD2 stimulation, and NOD2 stimulation synergized with activation of these TLRs to augment Twist-dependent activity. Thus, Twist molecules play a major role in controlling cytokine expression following chronic stimulation of pattern recognition receptors.

Due to logistical constraints, many studies in humans evaluate immune responses based on circulating PBMCs. However, the immune system is highly compartmentalized, and distinct subsets of cells differentially localize to the blood, lymphoid organs, and peripheral tissues. In this issue, Tatovic et al. (p. 386) used ultrasound-guided fine-needle aspiration to examine the relationship between T cell populations in skin-draining lymph nodes (LN) and those found in circulation, both in baseline conditions and in response to Mycobacterium tuberculosis purified protein derivative (PPD). Through fine-needle aspiration, 1–10 × 106 white cells were collected from the LN cortex and, somewhat unsurprisingly, were found to have a lower proportion of CD45RACCR7 effector memory T cells than did PBMCs. At baseline, more CD4+ and CD8+ T cells expressed the activation marker CD69 in the LNs than in the blood. PBMCs were collected and LN cells were aspirated before and after PPD inoculation, and these cells were then stimulated with PPD in vitro and tested for IFN-γ production by ELISPOT. Although the prechallenge baseline for PPD reactivity was higher in PBMCs than LN cells, there was no increase in IFN-γ production detected in PBMCs after PPD administration. In contrast, there was a significant increase in IFN-γ production by LN cells 2 d after PPD challenge. These results demonstrate how ultrasound-guided fine-needle aspiration can be used to monitor tissue-specific immune responses, which may not always be accurately assessed by peripheral sampling.

Regulatory T cells (Tregs) mediate immunosupression that is essential to dampening deleterious immune responses, such as those that occur in autoimmune diseases. Similar to other types of T cells, some Treg subsets exhibit a degree of functional plasticity, which could be detrimental to their potential therapeutic use. With the hope of defining phenotypic markers to distinguish lineage-stable from plastic human Treg subsets, Fuhrman et al. (p. 145) investigated the involvement of a recently described immunomodulatory axis between the activating receptor CD226 and coinhibitory receptor T cell Ig and ITIM domain (TIGIT), two receptors that compete for binding to the same ligand. Phenotypic and transcriptional analyses of human Treg and T conventional cell (Tconv) subsets revealed that Treg populations expressing TIGIT, but not CD226, had increased suppressive capacity and reduced inflammatory cytokine production, were refractive to expansion in vitro, and exhibited demethylation at the FOXP3–Treg-specific demethylated region (TSDR), a trait characteristic of lineage-stable FOXP3-expressing Tregs. TIGIT was also expressed more frequently in thymic-derived than in peripherally induced Treg and Tconv populations. In contrast, CD226 was highly expressed on Tconv populations, including memory T, Th1, and Th17 cell populations. CD226 expression by Tregs was also associated with production of the inflammatory cytokine IFN-γ and decreased demethylation at the TSDR (but not decreased suppressive capacity), suggesting that Tregs expressing CD226 exhibited a higher degree of heterogeneity than those expressing TIGIT. Together, these data suggest that Treg subsets expressing TIGIT are more likely to represent lineage-stable populations than those expressing CD226. These surface markers provide a means to enrich for lineage-stable Tregs that have the potential for both experimental and therapeutic use.