Fig 1.
IL-10 production by Tregs is synergistically enhanced by CD4+FoxP3-CD45RbLoCD44+CD62L- T cells.
Tregs and various CD4+ T cell subsets were cultured in vitro with plate-bound anti-CD3ε antibody for 3 days, unless otherwise specified, to measure their cytokine responses by ELISA. (A) Comparison of mono- and co-cultures of magnetic bead purified (M) CD4+ Tconv and CD4+CD25+ Tr cells vs. flow sorted (Fl) CD4+CD25+ Tr cells. (B) Time course of cytokine production from co-cultures of flow-sorted Tconv and CD25+ Tregs. (C) Co-cultures of flow-sorted 50k Tconv and varied Tregs at indicated ratios. (D) 1:1 Cultures of flow sorted CD4+FoxP3+ Tregs and CD4+FoxP3-CD45RbHi/Lo cells, showing IL-10, IFNγ, and IL-2 production by ELISA. (E) 1:1 cultures of CD4+CD25+FoxP3+ Tregs and CD4+CD25-FoxP3-CD62L+CD44+ (Tcm), CD4+CD25-FoxP3-CD62L-CD44+ (Tem), and CD4+CD25-FoxP3-CD62L+CD44- (Tn) cells. (F) 1:1 cultures of CD4+CD25-FoxP3+ Tregs and Tcm, Tem, or Tn cells. (G) 1:1 cultures of CD4+FoxP3+ Tregs and CD4+FoxP3-CD45RbLoCD62L-CD44+ (RbLoTem) or CD4+FoxP3- CD45RbHiCD62L+CD44- (RbHiTn) cells. * = p<0.05; # = p>0.05. P value calculated from Student’s T-Test. Error bars show mean with SEM. For A-B and D-F, n = 3 experiments. For C, n = 6 experiments. For G, n = 4 experiments.
Table 1.
Lung inflammation clinical score rubric.
Fig 2.
Cytokine synergy of RbLoTem and FoxP3+ Tregs is limited to IL-10.
RbLoTem and RbHiTn cells were stimulated alone or in co-culture as before for 3 days to determine cytokine and chemokine secretion in relation to the mRNA transcriptomes using multiple Luminex. Statistical comparisons were made based on differences between the sum of the FoxP3+ Treg and RbLoTem individual responses and the corresponding co-culture. Shown are immune regulation-associated proteins with significant change in co-culture (A), other cytokine and chemokines with statistically significant differences in co-culture (B), and molecules not significantly different in co-culture (C). * = p<0.05; # = p>0.05. P value calculated from Student’s T-Test. Error bars show mean with SEM. n = 3 experiments for all panels.
Fig 3.
T cell synergy is mediated by a soluble factor.
Cells were flow sorted, stimulated with anti-CD3ε antibody for 3 days, and treated as indicated. ELISA measurements of IL-10 concentration in culture supernatant from FoxP3+ Tregs co-cultured with IL-10-knockout RbLoTem (A), fixed RbLoTem or RbHiTn cells (B) or the change in IL-10 in the supernatant from cultured FoxP3+ Tregs supplemented with conditioned media from RbLoTem, RbHiTn or FoxP3+ Tregs stimulated previously for 3 days as before (C). Conditioned media from activated RbLoTem or RbHiTn cells were administered to HDM-challenged IL-10-GFP reporter mice intranasally and compared to resting and HDM-challenged mice without conditioned media. Mice were sacrificed and the BALf was analyzed by flow cytometry, showing percent IL-10+ among CD4+ cells in representative histograms (D) as well as replicates and the total CD4+ cell counts (E). ELISA measurements of IFNγ concentration in culture supernatants from FoxP3+ Tregs, WT RbLoTem, or both cells in co-culture with anti-CD3ε stimulation for 4 days (F). * = p<0.05; # = p>0.05. P value calculated from Student’s T-Test. Error bars show mean with SEM. For A-C, n = 3 to 6 experiments. For E-F, n = 3 experiments.
Fig 4.
RbLoTem and RbHiTn transcriptomes reveal potential crosstalk mediators.
RbLoTem and RbHiTn cells were stimulated alone as before for 3 days, and RNA was isolated for deep sequencing and whole transcriptome analysis. Genes were arranged according to the log2 of the RbLoTem to RbHiTn ratio of expression, using the calculated RPKM values. Candidates were narrowed by excluding all membrane proteins, proteins known to exist only in the nucleus or cytoplasm, and those showing at least 16-fold increase in RbLoTem cells compared to RbHiTn cells. n = 2 per cell type.
Fig 5.
IL-4 and FoxP3+ Treg IL-4Rα (CD124) mediates Treg IL-10 production.
FoxP3+ Tregs and RbLoTem were stimulated in co-culture as before for 3 days with the addition of cytokine (A) or receptor (B) neutralizing antibodies to identify the mediator of IL-10 synergy. (C) To determine whether IL-4 was acting on RbLoTem cells or FoxP3+ Tregs, (C) RbLoTem cells were incubated with CD124 neutralizing antibody, washed, then co-cultured with fresh FoxP3+ Tregs as before (C); and then compared to pre-incubating FoxP3+ Tregs with CD124 antibody followed by co-culture with RbLoTem cells (D). FoxP3+ Tregs were stimulated as before in monoculture supplemented with either conditioned media from stimulated RbLoTem cells (E) or various concentrations, denoted in ng/mL, of recombinant IL-2 or IL-4 (F-H) with and without neutralizing IL-4 or CD124 antibodies (E-H). (I-J) FoxP3+ Tregs were cultured with and without αCD3ε stimulation, as well as with and without RbLoTem cells. At day 3, cells were harvested and stained for CD124 expression, and analyzed by geometric mean fluorescence intensity (I) and the percent CD124 positive among FoxP3+ Tregs (J). * = p<0.05; # = p>0.05. P value calculated from Student’s T-Test, with comparisons to control unless specified. Error bars show mean with SEM. For A-H, n = 3 to 6 experiments. For I-J, n = 6 experiments.
Fig 6.
RbLoTem cells reverse lung inflammation in an IL-4-dependent fashion.
Mice were given 60,000 of anti-CD3ε-stimulated WT or IL-4ko RbLoTem cells on day 7 of acute HDM-induced asthma to test their ability to reverse inflammatory pathology. The total number of infiltrating cells (A), the clinical score (see Table 1) (B), normalized cellular differentials from BALf harvests (C), and tissue pathology (D) by immunofluorescence staining for EpCAM (green) and MPO (red) from representative sections of pulmonary tissue are shown. * = p<0.05; # = p>0.05. P value calculated from an unpaired Student’s T-Test. Error bars show mean with SEM. n = 6 to 14 individual mice total per group, across two asthma trials, as indicated on the White Blood Cell counts in panel C.
Fig 7.
Lung inflammation, remodeling and mucus production is reversed by RbLoTem cells in an IL-4-dependent fashion.
Representative lung sections from n = 6 mice at 4x and 10x magnification from the mice described in Fig 6 were stained with H&E (top), TriChrome (middle) and PAS (bottom) to assess cellular infiltration, tissue remodeling/collagen deposition and mucus production respectively in HDM-challenged mice receiving 60,000 activated WT or IL-4ko RbLoTem cells.