Virus-Specific TILs May Muddle Diagnostics
The presence of CD8+ tumor-infiltrating lymphocytes (TILs) is correlated with a positive prognosis in cancer patients. Although it is often assumed that all T cells present in a tumor are tumor-associated Ag (TAA) specific, little is known about how infections (acute, latent, or cleared) may alter TIL populations. In this issue, Erkes et al. (p. 2979) examined T cell populations in tumors of mice acutely infected with either murine CMV (MCMV) or vaccinia virus (VacV) and found that activated virus-specific CD8+ T cells, independent of active viral replication, could migrate into cutaneous melanoma lesions alongside TAA-specific CD8+ T cells to comprise at least 20% of all CD8+ TILs. Unlike splenic TAA-specific CD8+ T cells, TAA-specific CD8+ T cells recovered from tumors of mice acutely infected with MCMV or VacV expressed PD-1 and were severely dysfunctional for cytokine production. Surprisingly, virus-specific CD8+ TILs, despite expressing PD-1, appeared to retain functionality when entering the tumor environment. Virus-specific TILs expressed significantly less PD-1 per cell than did TAA-specific TILs, and exhibited undetectable binding to PD-1 ligands. To determine whether the level of PD-1 expression on virus-specific TILs correlated with recent Ag exposure, the authors transplanted tumors into mice infected with MCMV 30 d prior, a time point at which MCMV-specific T cells in the circulation lacked PD-1, and injected the MCMV-derived M38 peptide directly into tumors. Compared with tumors that were injected with vehicle alone, MCMV-specific TILs exposed to M38 peptide uniformly expressed high levels of PD-1 and yet retained their function. Collectively, these data demonstrate that, depending on the individual’s immune status, functional, PD-1-expressing virus-specific CD8+ T cells can be present in tumors, potentially skewing the results of prognostic or diagnostic assays whose readouts are based upon the numbers of CD8 + TILs in tumors and their PD-1 expression patterns.
Tracing tTreg Transitions
Although accumulating evidence suggests that Foxp3+ cells can downregulate the expression of Foxp3 and undergo reprogramming into other T effector subsets, whether thymically-derived regulatory T cells (tTregs) are capable of downregulating Foxp3 remains controversial. In this issue, Zhang et al. (p. 2612) developed a mouse strain that allowed for identification and lineage tracing of tTregs by labeling them with Thy1.1. Only ∼ 1% of mature tTregs lost Foxp3 expression in secondary lymphoid organs, indicating that tTregs are stable under homeostatic conditions. Following adoptive transfer of tTregs into T cell lymphopenic TCR knockout mice, donor tTregs showed a dramatic loss of Thy1.1 and Foxp3 and increased expression of either IFN-γ by cells reprogrammed as Th1 cells, when recovered from the spleen, or CXCR5 by cells reprogrammed as follicular helper T cells, when recovered from Peyer’s patches. These data indicate that tTregs could be reprogrammed into other T effector subsets following TCR engagement during homestatic proliferation in the empty host. This reprogramming was also observed by monitoring Foxp3 expression in two functionally specialized Treg subpopulations, T follicular Tregs and T-bet–positive Tregs, that were derived from tTregs. Thy1.1+ tTregs possessed a bona fide Treg signature, whereas Thy1.1- cells, which were tTregs that lost Thy1.1 and Foxp3 expression, expressed genes involved in Treg activation, suggesting that Foxp3 instability was likely linked to Treg activation. Blockade of ICOS signaling or neutralization of IL-2 demonstrated that the signal switch from the IL-2 to ICOS signaling pathway during Treg activation induced Treg reprogramming, revealing how tTreg activation and stability are balanced. Finally, using a dual lineage tracing mouse model in which genetic tracing of Foxp3 and T-bet was simultaneously enabled, the authors demonstrated that T-bet expression by effector Tregs was unstable, and effector T-bet–positive Tregs that lost T-bet expression reverted to a CD25+CCR7+CD62L+ Treg phenotype and stabilized Foxp3 expression. Thus, this study demonstrates that although tTregs are a stable subset in steady state conditions, activation can drive Foxp3 instability and the switch to a functionally specialized effector population.
Self Help for Arthritis
Persistent inflammation with inadequate tissue repair is characteristic of rheumatoid arthritis (RA) and osteoarthritis (OA), but currently available treatment options rely heavily on pain management rather than strategies to repair cartilage defects. As recent studies have identified endogenous molecules, such as melanocortins, that can limit chondrocyte inflammation and cartilage degradation, Kaneva et al. (p. 2876) sought to identify additional endogenous molecules that may aid in resolution of joint inflammation and limit cartilage destruction. Using lung exudate from rats with pleurisy, chondrocytes stimulated with inflammatory exudate (collected 6 h after pleurisy induction) displayed typical catabolic activity as evidenced by decreased COL2A1 and ACAN and increased MMP13 mRNA expression. Resolving exudate (collected 24 h after pleurisy induction) was mostly inactive when added to chondrocytes alone, but stimulation of chondrocytes with both the resolving and the inflammatory exudate reduced MMP13 transcription by 20% and increased ACAN and COL2A1 transcription. Following gel filtration chromatography and proteomics analysis of the exudates, the authors selected three acute phase proteins, α1-antitrypsin (AAT), hemopexin (HX), and gelsolin (GSN), that were further analyzed for their possible relevance to chondroprotection. When added to chondrocytes stimulated with IL-1β, AAT and GSN improved COL2A1 and ACAN expression and reduced MMP13 expression, whereas HX was only effective at diminishing MMP13 expression. Addition of AAT, GSN, or HX to high-density 3D micromass cultures of chondrocytes reversed the effects of both IL-1β and osteoarthritis synovial fluid on IL-6, IL-8, and MMP-13 release and increased sulfated glycosaminoglycan deposition, which is an indication of improved cartilage health. Finally, when compared with untreated contralateral controls, intra-articular injection of GSN or AAT protected joint cartilage from erosion in mice with serum-induced inflammatory arthritis. This study identifies three proteins in resolving exudates that may be potential leads for drug discovery programs and demonstrates that anti-arthritic strategies may benefit from the exploitation of endogenous pathways that mediate the resolution of inflammation.
The Shocking Truth about IL-17A
Exposure to bacterial superantigens (SAgs) causes toxic shock syndrome (TSS), a life-threatening systemic inflammatory response syndrome that involves overproduction of inflammatory mediators in a cytokine storm. To better understand the early phase of TSS in hopes of identifying therapeutic targets, Szabo et al. (p. 2805) investigated the potential involvement of the proinflammatory cytokine IL-17A, which often plays a role in protective antibacterial responses. Humanized HLA-DR4 transgenic (DR4tg) mice produced high levels of IL-17A as soon as 2 h after i.p. injection with bacterial SAgs through a mechanism requiring the presence of human MHC. The highest levels of IL-17A production were observed in intestinal intraepithelial lymphocytes and in the lamina propria, and the majority of the IL-17A-producing cells were found to be CD4+ effector memory T (TEM) cells. Treatment of human PBMCs with SAgs also caused the rapid generation of high levels of IL-17A, and the authors found that the mRNA encoding IL-17A produced after SAg exposure was much more stable than that encoding IFN-γ, which was also upregulated by SAgs. As in the humanized mouse model, the majority of the IL-17A-expressing cells in human PBMCs were CD4+ TEM cells, which made up a small fraction of total PBMCs. Within these PBMCs, distinct subsets of CD4+ T cells produced IL-17A or IFN-γ; coexpression of both cytokines was rare. Blockade of IL-17A in PBMC cultures using an anti–IL-17RA mAb resulted in downregulation of proinflammatory cytokines and chemokines and upregulation of anti-inflammatory cytokines, suggesting that IL-17A promotes inflammation and may have a pathogenic role in TSS. Indeed, treatment of SAg-injected DR4tg mice with an anti–IL-17A mAb significantly improved survival and reduced TSS pathology, including hepatic steatosis and intestinal damage, relative to that in mice given an isotype control. Thus, although IL-17A often plays a protective role in bacterial infections, it may be pathogenic early in TSS, and therapeutic targeting of IL-17A production by TEM cells might ameliorate tissue damage and consequent mortality in TSS patients.
Transformers Protect the Sea Urchin
The diverse Sp185/333 gene family of the California purple sea urchin, Strongylocentrotus purpuratus, is predicted to encode proteins that share common structural features and may, based on their expression patterns, mediate host defense, although the sequences of these genes are not similar to those that encode any known proteins. To begin to clarify the potential functional attributes of one of these proteins, Lun et al. (p. 2957) investigated the structure of rSp0032, which they propose renaming rSpTransformer-E1 (rSpTrf-E1) based on this study’s results. In agreement with predictions from bioinformatic analysis, rSpTrf-E1 was found through circular dichroism (CD) analysis to be an intrinsically disordered protein without well-defined secondary structure in steady state conditions. However, CD analysis indicated that the N-terminal rGly-rich and the C-terminal rHis-rich regions of rSpTrf-E1, when produced as separate polypeptides, had α helical components that were not predicted by bioinformatics. In the presence of SDS or 2,2,2-trifluorethanol (TFE), rSpTrf-E1 transformed from a disordered state to one with a high percentage of α helices. However, the structure of the N- and C-terminal domains differed depending on the buffer; that is, in SDS, the α helical content of the rGly-rich and rHis-rich domains increased, whereas in TFE, these domains shifted to a β strand structure. These indications of rSpTrf-E1 structural plasticity led the authors to speculate that this protein might adopt different structures when exposed to different ligands. Supporting the idea of ligand-induced conformational changes, rSpTrf-E1 took on an α-helical conformation in the presence of even low concentrations of LPS, which is known to interact with rSpTrf-E1, whereas the rGly-rich region shifted to a β strand structure and the rHis-rich region increased its α helical content. Taken together, these data suggest that rSpTrf-E1 may interact with a wide range of ligands via a high degree of conformational plasticity, and the authors suggest that this conformational variability, if found throughout this family of putative host defense proteins, could provide S. purpuratus with an immune system able to respond to a broad array of pathogens. Thus, an aspect of Pauling’s template theory of Ab conformation is now being revived from an unexpected source, the sea urchin SpTrf family.
- Copyright © 2017 by The American Association of Immunologists, Inc.