Encephalitogenic T cells that stably express both T-bet and ROR gamma t consistently produce IFNgamma but have a spectrum of IL-17 profiles

Abstract

Th1/Th17 cells, secreting both IFNgamma and IL-17, are often associated with inflammatory pathology. We cloned and studied the cytokine phenotypes of MBP-specific, TCR-identical encephalitogenic CD4+ cells in relationship to Th1- and Th17-associated transcription factors T-bet and RORgammat. IFNgamma-producing cells could be sub-divided into those that are T-bet(+)/RORgammat(-) and those that are T-bet(+)/RORgammat(+). The latter comprises a spectrum of phenotypes, as defined by IL-17 production, and can be induced to up-regulate IL-23R with IL-12 or IL-23. The former, bona fide Th1 cells, lack IL-23R expression under all conditions. In vivo, T-bet(+)/RORgammat(-) and T-bet(+)/RORgammat(+) clones induce EAE equally well.

Figure 1

Profile of IFNγ and IL-17 secreted by 181 LN-derived clones (Line M10) and 29 CNS-derived clones (Line H). Levels of IFNγ and IL-17 in 40 hour supernatants of individual cloned T cells were quantitated by ELISA after first cloning lines derived from either BALB/c TCR-transgenic lymph nodes, 10 days after immunization, or CNS, at onset of clinical signs of EAE. Among LN-derived clones, 97 made IFNγ only, 19 made IL-17 only, and 65 made both cytokines. Among CNS-derived clones, 13 made IFNγ only, 8 made IL-17 only, and 8 made both cytokines.

Figure 2

Secreted levels of IFNγ and IL-17 by eight T cell clones, shown for first 3–5 rounds of restimulation after cloning. With the exception of the first round of stimulation, supernatants were collected and pooled from duplicate wells, 40 hours after addition of irradiated BALB spleen cells and MBP exon 2 peptide (10 μg/ml). Cytokines were quantitated in duplicate by ELISA, as detailed in Materials and Methods. Variance between duplicate wells was <20% for all positive samples. Intervals between rounds of stimulation were approximately 2 weeks.

Figure 3

Expression and modulation of IFNγ and IL-17 in double producing clones. A, Intracellular staining of IFNγ/IL-17-secreting clones M10.66 and M10.116. T cells were harvested 24 hours after activation with peptide antigen and irradiated BALB/c spleen cells, incubated with Brefeldin A for 2 hours, surface stained with FITC-CD4, followed by permeabilization and staining with APC-anti-IFNγ and PE-anti-IL-17 antibodies. Shown are percentages of CD4+ activated T cells staining with indicated anti-cytokine antibodies in each quadrant. B, Cytokine pre-treatment modulates IFNγ and IL-17 protein expression. IFNγ/IL-17-secreting clones were co-cultured with either TGFβ+IL-6 (5 and 30 ng/ml, respectively), IL-12 (10 ng/ml), IL-23 (50 ng/ml), or IL-21 (50 ng/ml) for seven days, harvested, washed, and added to irradiated BALB/c spleen cells without (left) or with 10 μg/ml antigen (right), together with the same exogenous cytokine used in pre-culture conditions. IFNγ and IL-17 secreted by 40 hours were quantitated by ELISA.

Figure 4

Expression of transcription factors T-bet, RORγt and their related target genes by clones with various IFNγ and IL-17 profiles. A, Expression of T-bet and RORγt in the panel of clones shown in Figure 2. Data is from resting clones. Ct values represent means from 3–5 independent experiments. Standard errors are <0.1. B, Expression of T-bet and RORγt and their target genes can be modulated in dual-positive clones by antigen, IL-12, and IL-23. Real time-PCR quantitation of gene expression in M10.66 and M10.116, cultured for 24 hours with irradiated spleen cells in the absence or presence of 10 μg/ml exon 2 peptide (open bars in right panel), and in the absence or presence of IL-12 (10 ng/ml), black bars or IL-23 (50 ng/ml), gray bars. Cultures were set up in parallel with those in Figure 3B. 18S rRNA was used to normalize Ct values; fold change is calculated as in Materials and Methods.

Figure 5

Modulation of gene expression induced by IL-12 and IL-23. Real-time PCR quantitation of gene expression in resting T cell clones (no antigen stimulation for ≥ 2 months), cultured with IL-12 (black bars) or IL-23 (gray bars) for 4 weeks. Fold changes were calculated as in Materials and methods, normalizing to 18S rRNA. Ct values >32 were assigned values of 32 for calculation purposes.

Figure 6

Intracellular detection of RORγt protein. Resting T cell clones were harvested from in vitro culture, permeabilized, and stained for RORγt protein using PE-anti-RORγt antibody (bold lines); PE-isotype control is also shown (thinner lines). The percentage of positive cells shown below histograms is calculated by subtracting the percentage positive with PE-isotype control from the percent positive with PE-RORγt antibody; mean fluorescence intensity of staining with anti- RORγt antibody is shown below histograms.

Figure 7

H & E staining of CNS sections from recipients of Th1 (Group I) and Th1/Th17 clones (Groups II and III). All mice were sacrificed at onset of neurological signs of disease; tissues from two mice in each group were harvested for histological analysis. M10.1 (Group I), Perivascular and parenchymal lymphocytic infiltration in cerebellar white matter (10×). Higher magnification (60×) shows mostly lymphocytes, some scattered macrophages. Meninges of lumbar spinal cord are focally infiltrated with lymphocytes; parenchyma has focal dense infiltrates (10×). Higher magnification (60×) shows dense meningeal infiltrate (right); parenchyma is infiltrated with lymphocytes and a few macrophages. M10.77 (Group II), Lateral medulla (cochlear nucleus) is densely infiltrated by macrophages (10×). Parenchyma has been replaced by sheets of confluent macrophages; notably few lymphocytes are observed (60×). In low lumbar spinal cord, there is dense infiltration of both meninges and parenchyma by lymphocytes (10×). Lymphocytes densely infiltrate meninges, and spinal cord white matter is densely infiltrated by macrophages (60×). M10.66 (Group III), Diffuse perivascular and parenchymal infiltration of lymphocytes and macrophages in cerebellar white matter (×10). Severe perivascular and parenchymal infiltration of cerebellar white matter with macrophages and lymphocytes (×60). Spinal cord white matter is infiltrated with macrophages and lymphocytes, extending from meninges and into parenchyma perivascularly (10×). On high magnification, very dense infiltration of lymphocytes are observed in meninges, and extending into parenchyma, where numerous macrophages are also observed (60×). M10.116 (Group III), Perivascular infiltration of lymphocytes in cerebellar white matter (10×). Lymphocytes and macrophages are observed infiltrating into parenchymal white matter of cerebellum (60×). Spinal cord is infiltrated with lymphocytes in sub-meningeal regions, and extending a short distance into parenchyma (10×). Lymphocytes and macrophages are observed in sub-meningeal and parenchymal regions of spinal cord (60×).

Figure 8

Real time PCR quantitation of genes expressed in CNS tissue (spinal cord and brainstem) of recipients of indicated T cell clones. CNS tissues were harvested on indicated days post-injection. Each bar corresponds to one recipient. Bars represent fold increase in indicated transcripts as compared with averaged expression in 4 normal BALB/c control mice, after normalization of each with GAPDH. Group IV includes one recipient each of clones M10.26, M10.28, M10.46, M10.49, and M10.52.