Loading...
Please wait, while we are loading the content...
Similar Documents
Cutting Edge: CD4CD25 Regulatory T Cells Contribute to Gender Differences in Susceptibility to Experimental Autoimmune Encephalomyelitis 1
| Content Provider | Semantic Scholar |
|---|---|
| Author | Reddy, Jay Waldner, Hanspeter Zhang, Xingmin Aaron Illes, Zsolt Wucherpfennig, Kai W. Sobel, Raymond A. Kuchroo, Vijay K. |
| Copyright Year | 2005 |
| Abstract | Female B10.S mice are highly resistant to proteolipid protein (PLP) 139–151-induced experimental autoimmune encephalomyelitis (EAE) and depletion of PLP 139–151reactive CD4+CD25+regulatory T (Treg) cells can slightly increase their EAE susceptibility. Although male B10.S mice are moderately susceptible to EAE, we report that depletion of Treg cells in male B10.S mice before immunization with PLP 139–151 renders them highly susceptible to severe EAE with more CNS neutrophil infiltrates than nondepleted controls. Increased susceptibility is associated with an enhanced PLP 139–151-specific T cell response and greater production of IFN-γ, IL-6, and IL-17. Male CD4+CD25+ effector cells depleted of Treg cells proliferate to a greater degree than those from females in response to either anti-CD3 or PLP 139–151. These data suggest that because of their capacity to regulate potent autoaggressive effector cells, Treg cells partly contribute to the resistance to autoimmunity in the male mice. Abbreviations: EAE, experimental autoimmune encephalomyelitis; Treg, regulatory T; PLP, proteolipid protein; wt, wild type; tg, transgenic; TMEV, Theiler’s murine encephalomyelitis virus; 7-AAD, 7-aminoactinomycin D; PMN, polymorphonuclear leukocyte 5591 digitalcommons.unl.edu 5592 R e d d y e t a l . i n T h e J o u r n a l o f I m m u n o l o g y 175 (2005) Proliferation assays To assess inhibitory effects of Treg cells, PLP 139–151-specific 5B6 tg T cells were fractionated into CD4+CD25+ and CD4+CD25cells by magnetic separation (Miltenyi Biotec). They were plated at a density of 1 x 106 cells/ml in the presence of APCs and PLP 139–151 (20 μg/ ml) for 2 days in complete DMEM. After pulsing with 1 mCi of [3H] thymidine (1 Ci = 37 GBq) for 16 h, proliferation was measured as counts per minute. CD3+ T cells enriched by negative selection (R&D Systems) from immunized B10.S wt mice were stimulated with PLP 139–151 in the presence of APCs. Response of CD4+CD25T cells to anti-CD3 (1 μg/ml, clone, 145-2C11; BD Pharmingen) was tested with CD11c+ cells as APCs. Flow cytometry To study cellular subsets in lymph nodes, various mAbs procured from BD Pharmingen. After staining, cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences); data were analyzed using FlowJo software (Tree Star). IAs tetramer staining Frequencies of Ag-specific T cells were determined by flow cytometry using IAs/PLP 139–151 and Theiler’s murine encephalomyelitis virus (TMEV) 70–86 tetramers (negative control) as described previously (15). Four-color analysis included anti-CD25/FITC (clone, 7D4), IAs tetramers (PE), 7-aminoactinomycinD( 7-AAD), and anti-CD4-allophycocyanin (clone, RM4-5). Percentages of tetramer+ cells in the live (7-AAD-) CD4+population were determined. Cytokine analysis CD3+ T cells were enriched from anti-CD25and control Ig-treated B10.S mice 10 days after immunization with PLP 139–151. They were stimulated with APCs loaded with PLP 139–151 (50 μg/ml) for 4–6 days. Supernatants on day 2 were tested for cytokines by ELISAs, and frequencies of cytokinesecreting cells were determined on day 4 by intracellular staining, as described previously (15). Real-time (TaqMan) RT-PCR Total RNA was extracted from CD4+CD25and CD4+CD2+ cells from a group of B10.S mice followed by treatment with DNase 1 (RNeasy kit; Qiagen). cDNA was synthesized using TaqMan RT reagents (Applied Biosystems). Fox P3 and GAPDH mRNA was measured by PCR in separate tubes in duplicates using probes labeled with FAM and VIC and with TAMRA as a quencher using TaqMan universal mix and the ABI Prism 7700 sequence detection system (Applied Biosystems). Primers and probe for GAPDH were purchased (Applied Biosystems). Fox P3 sequences were as described previously (17). A comparative threshold cycle (CT) was used to determine mRNA expression of Fox P3 and GAPDH relative to no-template control (calibrator). CT value was normalized for each sample using the formula: ΔCT = CT (Fox P3) CT (GAPDH), and the relative expression of Fox P3 was then calculated using the formula 2-CT. Histopathology Following induction of EAE, the mice were monitored for clinical signs of disease for 4 wk and scored as described previously (13). Mice were killed when their score reached ≥4 or when they began to recover. Brains and spinal cords were fixed in 10% phosphate-buffered formalin. Histologic disease was evaluated as previously described (13) and polymorphonuclear leukocyte (PMN) infiltration as described in Table I legend. Statistics Student’s t test was used to determine significance of data except for rank correlations between inflammatory foci and PMN scores, which were determined by Wilcoxon signed rank test. A value of p ≤ 0.05 was considered significant. Ta bl e I. C lin ic al a nd h is to lo gi c EA E in B 10 .S m ic e tr ea te d w ith a nt i-C D 25 o r c on tr ol A b a H is to pa th ol og y (m ea n ± SE M ) ( N o. o f I nfl am m at or y Le si on s) C lin ic al D is ea se b PM N (n eu tr op hi l) sc or e (% ) Tr ea tm en t In ci de nc e (% ) M ea n da y M ea n m ax iM en in ge s Pa re nc hy m a To ta l 3+ 2+ 1+ 0 of o ns et c m um s co re M al es Ra t I gG c on tr ol 3/ 12 (2 5) 14 .3 ± 1 .9 0. 25 ± 0 .1 10 .3 ± 4 .1 8. 3 ± 5. 6 18 .6 ± 9 .4 0 0 2/ 12 (1 6. 6) 10 /1 2 (8 3. 3) A nt i-C D 25 12 /1 4 (8 5. 7) 17 .3 ± 1 .3 1. 8 ± 0. 4 64 .3 ± 1 1. 8 60 .6 ± 1 2. 6 12 4. 9 ± 23 .2 2/ 14 (1 4. 3) 6/ 14 (4 2. 8) 2/ 14 (1 4. 3) 4/ 14 (2 8. 6) p va lu es 0. 00 1 0. 00 02 0. 00 08 0. 00 03 0. 00 04 Fe m al es Ra t I gG c on tr ol 0/ 8 (0 ) 0 0 2. 3 ± 1. 2 1. 6 ± 1. 1 3. 9 ± 2. 3 0 0 0 8/ 8 (1 00 ) A nt i-C D 25 3/ 9 (3 3. 3) 15 .0 ± 4 .6 1. 2 ± 0. 6 21 .0 ± 1 2. 4 23 .8 ± 1 5. 4 44 .8 ± 2 7. 7 1/ 9 (1 1. 1) 0 1/ 9 (1 1. 1) 7/ 9 (7 7. 8) a. N um be rs a re m ea n ± SE M . b. B 10 .S m al e or fe m al e m ic e w er e tr ea te d w ith a nt i-C D 25 o r r at Ig G A b on d ay s 5 a nd 3 p ri or to im m un iz at io n w ith P LP 1 39 –1 51 in C FA , a nd th e di se as e w as sc or ed a s d es cr ib ed in M at er ia ls an d M et ho ds a nd c om pa re d be tw ee n gr ou ps . c. Re pr es en ts o nl y m ic e th at sh ow ed cl in ic al d is ea se . P M N sc or e: 3 +, so lid , a bs ce ss -li ke fo ci w ith m os tly n eu tr op hi ls ; 2 +, m ul tip le fo ci w ith m ix tu re s o f n eu tr op hi ls a nd m on on uc le ar ce lls ; 1 +, fe w n eu tr op hi ls ; 0 , n o ne ut ro ph ils b ut w he n in fla m m at or y fo ci w er e pr es en t, th e in fil tr at es w er e pr ed om in an tly m on on uc le ar c el ls . t R e g C e l l s C o n t R i b u t e t o g e n d e R d i f f e R e n C e s i n eae 5593 Results and Discussion We report here that Treg cells partly contribute to gender differences in susceptibility to EAE. We previously showed that PLP 139–151-reactive Treg cells exist in the naive periphery of EAE-resistant B10.S female mice and that depletion of these in vivo before immunization with PLP 139–151 resulted in expansion of IFN-γproducing cells and induction of EAE in ~30% of mice (15). Because autoimmune diseases are more common in females than males, we hypothesized that there may be gender differences in the function of the naturally occurring CD4+CD25+ Treg cell subset. When we depleted Treg cells in vivo by administration of antiCD25 Ab in male B10.S mice before immunization, 85% of the mice (12 of 14) developed moderate to severe EAE, whereas only 3 of 12 control mice (25%) developed mild EAE, i.e., loss of tail tonicity (p ≤ 0.001) (Table I). In agreement with our previous results (15), a small number of female B10.S mice (three of nine, 33%) depleted of Treg cells but no control Abtreated mice developed disease, suggesting that genetic resistance to EAE in female B10.S mice does not lie entirely in the Treg population. In female B10.S mice, multiple factors may contribute to EAE resistance, i.e., Ag-specific defects in Th1 cytokine secretion and IL12-Rb2 expression (18, 19), a tendency to produce elevated amounts of Th2 cytokines (20), APCs (16) and the presence of a high proportion of Treg cells that react with PLP 139–151 tetramers in their naive periphery (15). Once males develop EAE following anti-CD25 treatment, there were no other clinical differences between males and females. Histologic disease in anti-CD25 Ab-treated male mice was significantly greater than in the controls (p ≤ 0.0003) (Table I), and there were more PMNs in CNS infiltrates (p ≤ 0.0004) (Table I, Figure 1). The histologic disease in males was also more severe than in anti-CD25-treated females (mean total inflammatory foci = 124.9 vs 44.8) (p ≤ 0.05). The disease severity in females was enhanced when compared with control mice but inflammatory foci contained fewer neutrophils (Table I, Figure 1b). To determine the mechanisms for greater incidence and severity of EAE in Treg-depleted male B10.S mice, we tested whether there are any differences in Treg cell function between male and female B10.S mice in vitro. We first determined that male B10.S mice have a higher percentage of CD3+ cells in their lymph nodes than age-matched female B10.S mice, but the percentage of Treg cells is lower in males (Table II). We then confirmed that Treg cells in male and female B10.S mice are equally effective in inhibiting expansion of effector cells using anti-CD3 as a stimulus (data not shown). FoxP3 mRNA, a marker for Treg cells, was also expressed in comparable amounts (Figure 2a). Therefore, we could identify no major differences in Treg populations between male and female B10S mice. We next tested whether Treg cells from male and female B10.S mice have any defect in their suppressive function in response to self-Ags using TCR tg mice specific to PLP 139– 151 (16). We found that Treg cells from male and female tg B10.S mice did not proliferate to PLP 139–151, whereas male and female effector cells responded equally well to PLP 139– 151. Treg cells inhibited the proliferative response of effector cells but there were differences; the inhibition of CD4 |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1024&context=vbsjayreddy |
| Alternate Webpage(s) | http://www.jimmunol.org/content/175/9/5591.full.pdf |
| Alternate Webpage(s) | http://www.direct-ms.org/pdf/ImmunityRegulation/Wucherpfennigregcellsgender.pdf |
| Alternate Webpage(s) | http://www.direct-ms.org/sites/default/files/Wucherpfennigregcellsgender.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
