Abstract
Neutrophils Change with the Times See article p. 458
Old Genes Find a New Purpose See article p. 501
Switching Akt Substrate Specificity Shifts Splicing See article p. 589
Mycobacterium tuberculosis Clearance Trumped by T Cell IL-10 See article p. 613
Biased BCRs in Celiac Disease See article p. 782
Neutrophils Change with the Times
Neutrophils can mediate both proinflammatory and immunosuppressive responses, and conflicting data have implicated these cells in both promoting and restraining autoimmunity in systemic lupus erythematosus. In this issue, Bird et al. (p. 458) sought to clarify the in vivo effects of neutrophils on adaptive immunity in the NZB/W mouse model of lupus. In these mice, neutrophils infiltrated the spleen as disease progressed and were found in contact with T and B lymphocytes in the white pulp, preferentially associating with T cells earlier in disease and B cells later in disease. RNA sequencing analysis at three time points during disease (representing early, mid, and advanced disease) demonstrated that the transcriptional program of neutrophils in the spleen changed significantly during lupus progression. Between 14 and 20 wk, expression of factors promoting B cell autoreactivity decreased, while proresolving and anti-inflammatory mediators increased. In contrast, between 20 and 26 wk, genes associated with inhibition of inflammation were downregulated, while genes associated with neutrophil activation were upregulated. In agreement with these data suggesting that neutrophils might restrain lupus early, but not late, during disease progression, continuous depletion of neutrophils using an anti-Ly6G Ab from 25–30 wk of age did not alter lupus progression, relative to treatment with an isotype control. In contrast, anti-Ly6G–mediated neutrophil depletion from 14–26 wk of age resulted in increases in splenomegaly, proteinuria, germinal center formation, and production of anti-dsDNA autoantibodies. Relative to control Ab treatment, mice treated with anti-Ly6G early in disease also had increased numbers of germinal center B cells and T follicular helper cells. Taken together, these data suggest that neutrophils play a protective role in murine lupus early during disease development, but that this protective influence is lost later in disease. Thus, the stage of disease progression should be taken into account in the development of treatments targeting neutrophils in lupus.
Mycobacterium tuberculosis Clearance Trumped by T Cell IL-10
Control and clearance of Mycobacterium tuberculosis infection is dependent on CD4+ T cells and cytokines such as IL-12, IFN-γ, and TNF. Conversely, the immunosuppressive cytokine IL-10, which has been shown to be elevated in the pleural fluid of patients with active pulmonary tuberculosis, has a detrimental role during infection by limiting host protective immune responses. To date, the specific cellular source of IL-10 during M. tuberculosis infection remains unclear and, in this issue, Moreira-Teixeira et al. (p. 613) used IL-10 reporter mice to demonstrate that during early infection (days 1–14), Ly6C+ monocytes accounted for up to 70% of IL-10–producing cells in the lungs, whereas at later stages of infection (day 21 onwards), monocytes and T cells accounted for 50% and 25% of IL-10–producing cells, respectively. Bacterial growth in the lungs 60 d postinfection was inhibited 70% in mice deficient in IL-10 compared with wild-type (WT) controls, an inhibition that selective ablation of Il10 in immune cell subsets showed is also present in mice deficient only in T cell–derived IL-10. IL-10–producing T cells in the lungs 21 d postinfection were primarily CD4+ and CD8+ T cells that expressed high levels of CD44 and Tbet and produced IFN-γ upon ex vivo stimulation, indicating that highly activated effector T cells were the major source of T cell–derived IL-10 during M. tuberculosis infection. Finally, when compared with infected WT controls, infected mice deficient in either IL-27 receptor α or the type I IFN receptor exhibited decreased bacterial loads in the lungs at 60 d postinfection and reduced IL-10 mRNA expression in CD4+ T cells at 28 d post infection, indicating that IL-10 expression during early infection was partially regulated by IL-27 and type I IFN. Taken together, these data identify the cellular source of IL-10 during M. tuberculosis infection and provide new insight into the factors regulating IL-10 production and the consequent suppression of protective immunity in tuberculosis.
Biased BCRs in Celiac Disease
The development of high-throughput sequencing (HTS) technologies has allowed analysis of BCR repertoires at the single-cell level, an important tool for driving a more complete understanding of humoral immunity. Roy et al. (p. 782) have now developed a strategy to analyze transglutaminase 2 (TG2)-specific BCRs in patients with celiac disease (CD), as this disease is strongly associated with the production of anti-TG2 IgA autoantibodies. Plasma cells (PCs) were isolated from duodenal biopsies from 10 CD patients, and single-cell HTS was used to obtain paired H chain V region (VH) and L chain V region (VL) sequence information from 1482 TG2-specific and 1421 non–TG2-specific PCs, focusing on unique clonotypes to avoid clonal expansion-related skewing of the data. In contrast to the λ L chain bias observed in other studies of autoantibodies, the κ L chain predominated in TG2-specific PCs, and further analysis suggested that some of the same TG2 epitopes could be recognized by both κ and λ L chain–containing Abs. VH and VL gene segments preferentially used to encode TG2-specific Abs were identified, and the most highly represented VH:VL pair was IGHV5-51:IGKV1-5. Specific VH:VL pairs, including IGHV5-51:IGKV1-5 and IGKV1-39 paired with a variety of VH segments, were found to have preferentially undergone clonal expansion in CD patients. Interestingly, relative to non–TG2-specific PCs, TG2-specific VH and VL sequences tended to have lower levels of mutation, with particularly low levels in PCs using the IGHV5-51 segment, suggesting unexpectedly low levels of somatic hypermutation. The authors further characterized the mutational pattern and CDR3 length in the TG2-specific IGHV5-51:IGKV1-5 pair, finding a strong preference for 14-aa H chain and 11-aa κ L chain CDR3 regions, along with a bias for inclusion of the IGKJ2 gene segment. This study of the clonal representation of autoantigen-specific paired VH and VL sequences from CD patients hints at the molecular mechanisms underlying the development of a humoral response in human disease.
Old Genes Find a New Purpose
Although allergic asthma is characterized by a Th2 response, the molecular mechanisms involved in regulating this response remain unclear. Previous studies have indicated that the Hox5 genes (Hoxa5, Hoxb5, Hoxc5) are important transcriptional regulatory factors in mesenchymal cell populations within the developing lung. Hox5 gene expression has also been detected in naive and stimulated T lymphocytes, leading Ptaschinski et al. (p. 501) to investigate a regulatory role for these genes during the development of allergic airway disease. The authors showed that Hox5 gene expression was upregulated in mouse lungs during the induction of allergic asthma. When compared with wild-type (WT) controls, mice harboring a compound loss of function mutation in Hox5 (Hox5AabbCc) showed exacerbated lung pathology and increased Gata3+CD4+ T cells in the lungs following asthma induction. Additionally, mesenteric lymph node cells from asthmatic Hox5AabbCc mice produced more IL-4, IL-5, and IL-13 than those from WT animals following ex vivo stimulation with Ag. Studies using bone marrow (BM) chimeric mice demonstrated that mice receiving Hox5AabbCc BM cells displayed increased pleural inflammation and activated CD4+ T cells in the lungs following induction of allergic asthma, regardless of whether the recipient was WT or Hox5AabbC, indicating that the enhanced pathology was mediated by Hox5 mutations in immune cells and was independent of mesenchymal cell Hox5 function. Consistent with these conclusions, only T cells grown in Th2 polarizing conditions showed increased Hox5 gene expression, and Hox5 deficiency further increased cell proliferation, Gata3 expression, and Th2 cytokine production when compared with WT controls. Finally, HOX5 proteins were found to bind directly to STAT6 binding sites in the Gata3 promoter and this interaction decreased Gata3 expression and IL-4 production. Taken together, these data demonstrate that Hox5 gene products, which have primarily been described as mesenchyme-associated transcription factors, can be redeployed for regulation of Th2 development.
Switching Akt Substrate Specificity Shifts Splicing
Differences in TCR signal strength have been shown to direct CD4+ T cell differentiation, with low dose Ag and low levels of Akt/mTOR activation promoting regulatory T (Treg) cell differentiation and higher Ag doses favoring induction of Th cells. In this issue, Hawse et al. (p. 589) sought to clarify the mechanism by which Akt signaling controls T cell fate decisions. Analysis of Akt phosphorylation following TCR engagement revealed that high dose stimulation resulted in phosphorylation of both Thr308 and Ser473, whereas low dose stimulation only triggered Thr308 phosphorylation, suggesting that downstream Akt activities might differ depending on the level of TCR stimulation. Indeed, mass spectrometric analysis of Akt substrates in CD4+ T cells stimulated with high or low dose anti-CD3 Ab identified 99 proteins phosphorylated by Akt, of which only 18 were phosphorylated under both stimulation conditions. Substrates unique to high or low dose stimulation included multiple proteins involved in RNA processing and splicing; in particular, heterogeneous ribonucleoprotein (hnRNP) A1 and hnRNP L were targeted by Akt following low dose (Treg cell–inducing) TCR stimulation. Further supporting a role for Akt in RNA splicing, treatment of CD4+ T cells with an Akt inhibitor altered the splicing of multiple proteins involved in TCR signaling, including CD45, Lck, Tec, Fyn, and CD247 (CD3ζ). Small interfering RNA–mediated knockdown of hnRNP A1 affected CD45 splicing similarly to Akt inhibition. Relative to controls, knockdown of hnRNP A1 or hnRNP L prior to low dose TCR stimulation impaired Treg cell differentiation and increased the representation of Th cells, indicating a role for these RNA splicing factors in the promotion of Treg cell differentiation. Thus, depending on the strength of TCR engagement, Akt may phosphorylate different substrates and regulate CD4+ T cell differentiation through the control of RNA splicing.