Clusterin Alleviates Asthmatic Stress See article p.2021

An Eyeful of Polyclonal Protection See article p.2109

Tregs Should Take Their Vitamins for Foxp3 Stability See article p.2119

Promiscuous T Cell Reactivity in Herpes See article p.2205

Allergic asthma is a chronic inflammatory respiratory disease characterized by airway constriction, a Th2-mediated immune response, and influx of inflammatory infiltrates into the airways. Oxidative stress resulting from exposure to allergens in the lungs can trigger an allergic immune response, likely through a mechanism, not yet fully elucidated, that involves bronchial epithelial cell (BEC) secretion of inflammatory cytokines and chemokines, including CCL20. In this issue, Hong et al. (p. 2021) identify a potential role for clusterin, a ubiquitously expressed secretory glycoprotein known to regulate oxidative stress, in modulating allergic airway responses, likely via regulation of CCL20 production. Using a house dust mite (HDM) model to induce allergic airway responses in clusterin knockout (Clu−/−) or clusterin heterozygous (Clu+/−) mice, the authors found increased inflammatory infiltrates in the lung and bronchoalveolar lavage fluid (BALF) relative to levels found in wild-type mice. The BALF of Clu−/− mice also exhibited increased IL-4, CCL20, CCL2, and CCL11 levels, suggestive of an exacerbation of the allergic Th2 immune response. Higher chemokine levels in the lungs of these mice were accompanied by increased numbers of inflammatory CD11b+CD103 dendritic cells and Ly6C+ monocytes, suggesting that these inflammatory cell populations may have been recruited to the lung in response to the increased concentration of chemokines. After HDM challenge, clusterin was found to dampen lung levels of malondialdehyde and 4-hydroxyl-2-nonenal, molecules associated with tissue injury and oxidative stress, respectively. Clusterin also regulated production of CCL20 and ROS by the BEC cell line BEAS-2B in vitro. Together, these data suggest that clusterin may modulate CCL20 production and subsequent recruitment of inflammatory dendritic cell populations to the lung in allergic airway responses by negatively regulating intracellular oxidative stress.

Regulatory T (Treg) cells are currently being investigated as a potential therapeutic for a wide range of autoimmune diseases. It has been demonstrated that autoantigen-specific Treg cells are most efficient for disease amelioration, but this is not always feasible due to unidentified autoantigens or technical limitations in obtaining sufficient numbers of Ag-specific Treg cells. In this issue, Grégoire et al. (p. 2109) examined the efficacy of prestimulated polyclonal Treg (poly-Treg) cells using a mouse model of uveitis based on the transfer of hemagglutinin (HA)-specific T effector (HA-Teff) cells into mice that express HA in the retina. As preparation of Treg cells for patient therapy is often conducted by magnetic bead enrichment of CD25high cells, mouse Treg cells were similarly purified, resulting in 70% purity of CD4+CD25+Foxp3+ cells. Poly-Treg cells administered intravenously to host mice were unable to protect against uveitis, even when preactivated. However, preactivated poly-Treg cells injected into the vitreous 3-4 d after disease induction were able to reduce clinical signs of uveitis, so all subsequent experiments involved poly-Treg cells transferred in situ. Unlike Ag-specific Treg cells, poly-Treg cells were not able to protect against disease induction upon HA-Teff cell rechallenge. Numbers of immune cells infiltrating the eyes were similar between PBS-treated and poly-Treg cell-treated mice, as was the production of host T cell cytokines. There was a slight increase in IL-10 production from host cells in poly-Treg cell-treated mice, and blocking IL-10 abrogated the protective effects of poly-Treg cell transfer. One of the many immunomodulatory effects of IL-10 is reduced production of reactive oxygen species (ROS), and poly-Treg cell injection resulted in reduced ROS production by microglia, dendritic cells, macrophages and lymphocytes. These results demonstrate that preactivated poly-Treg cells inhibit autoimmune uveitis by reducing ROS production, and similar strategies could be used in other autoimmiune conditions, eliminating the need for purification of autoantigen-specific Treg cells.

As members of the Alphaherpesvirinae subfamily, varicella zoster virus (VZV) and HSV-1 and HSV-2 have dozens of homologous genes, and boosting of Abs reactive to one virus by infection with another has been observed. Less is known about T cell cross-reactivity to these viruses, but it is possible that HSV-reactive T cells identified in seronegative individuals resulted from anti-VZV responses, prompting Jing et al. (p. 2205) to examine the HSV/VZV cross-reactive T cell population. Whole VZV and IL-2 were used to stimulate PBMC to create polyclonal VZV-reactive CD4+ T cell lines, which produced IFN-γ and IL-2 in response to HSV-1, even if the donor was not seropositive for HSV-1. Reciprocally, PBMCs from HSV-1 seropositive individuals selected for CD137 (4-1BB) positivity and stimulated with HSV-1 also reacted to whole VZV. The authors identified multiple cross-reactive homologs using proteome-covering recombinant protein sets with CD4+ T cells. These positive results were validated using peptide sets, which resulted in the identification of 11 peptide sets that stimulated cross-reactive CD4+ T cells. Interestingly, CD4+ T cells reactive to whole HSV-2 or HSV-2 peptides that were also cross-reactive to VZV proteins were identified in HSV seronegative individuals. Similar cross-reactivity was also detected in the CD8+ T cell population. In a VZV/HSV-1 seropositive individual, a proportion of HSV-1–stimulated CD8+ T cells also produced cytokines and killed target cells in response to VZV stimulation. HSV-1–driven CD8+ T cell lines were stimulated by about one tenth of VZV homologs of previously identified HSV-1 CD8+ T cell epitopes. Tetramer sorting of HSV+ or HSV/VZV+ CD8+ T cells followed by TCRB CDR3 sequencing determined that these responding cells were polyclonal. The identification of cross-reactive T cell epitopes in these viruses not only provides clues about the presence of Ag-specific T cells in seronegative individuals, but may also be useful for future vaccine development.

The differentiation of CD4+ T cell subsets is dependent on genetic programs, and stable expression of the transcription factor Foxp3 is important for maintenance of the regulatory T cell (Treg) phenotype. Even within Treg populations, different subsets such as thymus-derived Tregs (tTregs), peripheral Tregs (pTregs) and in vitro–generated induced Tregs (iTregs) have distinct phenotypes as well as genetic and epigenetic profiles, indicating that there may be different requirements for their differentiation. Foxp3 stability has been associated with demethylation of CpG motifs within the conserved noncoding sequence 2 (CNS2) intron. As a relationship between vitamin C and DNA demethylation has been observed, Sasidharan Nair et al. (p. 2119) examined how vitamin C affected the epigenetic regulation of Treg subsets. As previously reported, Foxp3 CNS2 CpG motifs were demethylated in tTregs and pTregs; however, these motifs in iTregs were methylated, suggesting that Foxp3 expression might be less stable. Treatment of iTregs with physiological levels of vitamin C almost completely demethylated CNS2 but not other Treg signature genes. This active demethylation of CNS2 in iTregs was shown to be dependent on the enzyme ten-eleven-translocation 2. Vitamin C treatment also boosted the suppressive activity of iTregs. Administration of a sodium-dependent vitamin C transporter inhibitor prevented CNS2 demethylation of pTregs in vivo and reduced the number of Foxp3+ cells following Ag immunization. These results demonstrate how environmental cues such as nutrition can affect epigenetic programming of cells, thereby impacting their differentiation and function.