Tolerance induced by anti-DNA Ig peptide in (NZB × NZW)F₁ lupus mice impinges on the resistance of effector T cells to suppression by regulatory T cells

Abstract

We have previously shown that immune tolerance induced by the anti-DNA Ig peptide pCons in (NZB × NZW)F₁ (NZB/W) lupus mice prolonged survival of treated animals and delayed the appearance of autoantibodies and glomerulonephritis. Part of the protection conferred by pCons could be ascribed to the induction of regulatory T cells (T_Reg) that suppressed the production of anti-DNA antibodies in a p38 MAPK-dependent fashion. Here we show that another effect of pCons in the induction of immune tolerance in NZB/W lupus mice is the facilitation of effector T cell suppression by T_Reg. These new findings indicate that pCons exerts protective effects in NZB/W lupus mice by differentially modulating the activity of different T cell subsets, implying new considerations in the design of T_Reg–based approaches to modulate T cell autoreactivity in SLE.

1. Introduction

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by the presence of dysregulated mechanisms of immune tolerance that include autoantibodies, autoreactive CD4⁺ T cells, and reduced numbers and/or function of regulatory T cells (T_Reg) that suppress CD4⁺CD25⁻Foxp3⁻effector T cells (T_Eff) [1]. Although it has long been held that the inability of T_Reg to control autoimmune reactivity in SLE could be either secondary to their decreased frequency or abnormal suppressive capacity [2], some studies have suggested that another possibility could be that T_Eff can become resistant to T_Reg-mediated suppression [3-5]. This phenomenon prompted our interest in revisiting the role of T_Reg in the mechanisms that control immune tolerance in SLE. In particular, we had previously shown that the treatment of (NZB × NZW)F₁ (NZB/W) lupus-prone mice with the anti-DNA Ig peptide pCons effectively delayed lupus-like disease and prolonged mice survival [6]. We also showed that part of the protection had to be ascribed to pCons induced T_Reg that suppressed autoimmune responses in a p38-dependent fashion [7]. However, the effects of pCons on T_Eff remained elusive. Here we report that the threshold for T_Eff suppression by T_Reg in untreated NZB/W mice is lowered by pCons in a p38-independent fashion, and this favors the inhibition of autoimmune reactivity. These findings can have implications for the design of targeted interventions in the inhibition of autoimmune T cell reactivity in SLE.

2. Materials and Methods

2.1 Mice

Female NZB/W mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and treated according to the National Institutes of Health guidelines for the use of experimental animals, with the approval of the Institutional Animal Research Committee.

2.2 Peptides

pCons has T cell determinants from different J558 V_H regions of NZB/W anti-dsDNA Ig, while the negative control peptide pNeg has similar MHC binding motifs but is does not activate CD4⁺ T cells [6]. Peptides were synthesized at Chiron Mimotopes (San Diego, CA), purified to a single peak by HPLC, and analyzed by mass spectroscopy for expected amino acid content before use.

2.3 Tolerance induction and cell preparation

For tolerance induction, 10- to 12-wk-old NZB/W mice received a single i.v. dose of 1 mg pCons dissolved in saline [6]. Control mice received either a similar amount of pNeg or saline. Ten days after treatment, single cell suspensions of splenocytes were prepared by passing cells through a sterile wire mesh. After lysis of red blood cells with ACK lysing buffer (Sigma-Aldrich, St. Louis, MO), cells were centrifuged, washed and resuspended in HL-1 medium (Lonza, Walkerville, MD). T_Reg or T_Eff were purified from splenocytes using the CD4⁺CD25⁺ Regulatory T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions.

2.4 p38 inhibition

The p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and negative control molecule 4-ethyl-2 (p-methoxyphenyl)-5-(4^/ -pyridyl)-IH-imidazole (SB202474) were purchased from Calbiochem (San Diego, CA) and dissolved in saline. For p38 inhibition in vivo, mice were injected i.p. daily for 2 wk with 2 mg/kg SB203580 or SB202474 or equal volumes of saline [7].

2.5 Western blot

Total cell lysates from sorted T_Eff were obtained in 50 mM HEPES (pH 7.5), 250 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM sodium fluoride, 1mM sodium orthovanadate, 2μg/ml aprotinin, 2μg/ml leupeptin, 2μg/ml pepstatin. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (BioRad Laboratories, Hercules, CA). Membranes were blocked in 5% nonfat milk/PBS, 0.5% Tween 20 (PBST) at 4°C for 2 h, and then incubated with anti-phospho Abs from Cell Signaling Technology (Danvers, MA) (anti-p-ZAP-70^Tyr319, anti-p-ERK1/2^{Thr202/Tyr204}, anti-p-STAT1^Ser727, anti-p-STAT3^Ser727, anti-p-STAT6^Tyr641, anti-p-SAPK^{Thr183/Tyr185}, anti-p-p38^{Thr180/Tyr182}) or Santa Cruz Biotechnology (Santa Cruz, CA) (anti-p-JNK^{Thr183/Tyr185}, anti-p-p38^Tyr182). After wash in PBST, membranes were incubated with peroxidase-conjugated secondary Ab. After additional wash, peroxidase activity was detected with the ECL system (Amersham, Piscataway, NJ) or Femto system (Thermo Scientific, Rockford, IL). Membranes were subsequently stripped and reprobed with Ab for the corresponding non-phosphorylated protein purchased from Cell Signaling Technology: anti-ZAP-70, anti-p27^kip1, anti-ERK1/2, anti-STAT1, anti-STAT3, anti-STAT6, anti-SAPK and anti-p38, or Santa Cruz Biotechnology: anti-JNK, anti-p38, before peroxidase detection using secondary Ab. Finally, membranes were stripped for final reprobing with anti-β-actin Ab (Cell Signaling) to determine protein equivalency. Exposed films were quantified by densitometric analysis using ScionImage program (Scion Corporation, Frederick, MD).

2.6 Suppression assay

After cell sorting, T_Eff were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA) by incubation in 0.5 μM CFSE at 37°C for 10 min, then placed in HL-1/5% FCS and incubated in ice for 5 minutes before extensive washes. Mixtures of CFSE-labeled T_Reg and T_Eff at ratios of 1:1 or 1:4 or T_Eff alone were plated in round-bottom 96-well plates and stimulated with Dynabeads mouse anti-CD3/CD28 Ab (Invitrogen) at a ratio of 0.5 bead/cell. Since T_Reg:T_Eff ratios of 1:1 or 1:4 ratios gave comparable results, the manuscript only reports data on 1:1 ratios. Flow cytometry was performed after three days using a FACSCalibur™ instrument (BD Biosciences, San Jose, CA) and analysis was done using FlowJo software (Tree Star Inc., Ashland, OR). Cells were analyzed for CFSE dilution and percent suppression was determined based on the percentage of dividing CFSE-labeled cells in the coculture as compared with CFSE-labeled cells cultured alone.

2.7 Statistical analyses

Statistical analyses were done using Prism 4 software (GraphPad, La Jolla, CA). Comparisons between two groups were done by Mann-Whitney U test, and ANOVA for more than two groups. P<0.05 was considered statistically significant.

3. Results

3.1 Effects of pCons on T_Eff pathways

To study the effects of pCons on T_Eff, we analyzed molecular pathways related to cell cycle, anergy and T cell receptor signaling in sorted T_Eff from pCons-treated animals versus controls. No differences were observed in the activation of ZAP-70, p27, ERK, STAT1, STAT3, STAT6, JNK, SAPK and p38 in T_Eff from tolerized mice and controls (Fig.1).

Figure 1. Signaling pathways in T_Eff after tolerization with pCons.

Western blots for phosphorylated (p-) and non-phosphorylated ZAP-70, p27, ERK, STAT1, STAT3, STAT6, JNK, SAPK and p38 in sorted T_Eff from mice tolerized with pCons and control mice receiving pNeg (saline gave identical results, not shown). Graphs show the densitometric quantitation of each protein to its non-phosphorylated form. One representative experiment of four is shown.

3.2 pCons facilitates T_Eff suppression by T_Reg

Although intracellular signaling in the pathways tested in T_Eff was not influenced by pCons, the suppression of T_Eff by T_Reg was more effective in pCons-tolerized mice as compared to mock-treated controls (Fig. 2). Since it has been shown that T_Eff can acquire resistance to T_reg suppression in autoimmune conditions including SLE [2-4], we tested the possibility that pCons could modulate this aspect of the mechanisms of T_Reg-mediated suppression in NZB/W mice. In cocultures of CFSE-labeled T_Eff plus T_Reg from pCons-tolerized or control (pNeg-treated) mice, T_Reg more effectively suppressed T_Eff from pCons-tolerized than from control mock-treated mice, whether the Treg were derived from either tolerized or control NZB/W mice (Fig. 2). Conversely, T_Eff from tolerized mice were suppressed more than T_Eff from control mice independently of whether T_Reg were derived from pCons-treated or control mice (Fig. 2). Thus, pCons increased the sensitivity of T_Eff to T_Reg suppression in NZB/W mice. The observed effects were not due to altered T_Eff responsiveness after peptide treatment, since proliferative responses of T_Eff after polyclonal stimulation were similar between control and pCons-tolerized mice (Fig. S1).

Figure 2. pCons reduces T_Eff resistance to suppression by T_Reg in NZB/W lupus mice.

CFSE-labeled T_Eff (T_E) were cocultured with T_Reg (T_R) from pCons-tolerized (_pC) or pNeg-treated control (_pN) NZB/W mice in the presence of CD3/CD28 Ab for 3 days before flow cytometry. Representative (A) and cumulative (B) results including the percent of T_Eff suppression by T_Reg (C). *P<0.004; **P<0.009; ***P<0.007.

3.3 pCons effects on T_Eff resistance are p38-independent

We previously showed that a modulation of p38 activity in T_Reg contributed to the protection induced by pCons in NZB/W mice [7]. Although here we did not find differences in major signaling events (Fig. 1) or T_Eff proliferation (Fig. S1) after pCons-induced tolerance, it could still be possible that p38 might influence T_Eff activity. To address this possibility, NZB/W mice were injected with p38 inhibitor SB203580 or with control SB202474 or saline for 14 days. On day 7, mice were tolerized with pCons or left untreated, and on day 15 T_Eff and T_Reg were isolated for functional studies. The proliferation of T_Eff from mice treated with SB203580 or SB202474 (or saline, not shown) was similar when T_Eff were suppressed by T_Reg from mice treated with SB203580 or SB202474 (Fig. 3), suggesting that the increased sensitivity of T_Eff to T_Reg suppression after pCons treatment was independent of the p38 pathway in T_Eff .

Figure 3. pCons reduces T_Eff suppression by T_Reg in a p38-independent fashion.

Groups of 7-8 NZB/W mice each were injected daily with the p38 inhibitor SB203580 (_p38-inh) or control SB202474 (_p38-c) for 14 days. On day 7, mice were tolerized with pCons or given control pNeg. After one week, ex vivo sorted T_Eff (T_E) were CFSE-labeled and cocultured with T_Reg (T_R) in the presence of CD3/CD28 Ab for 3 days before flow cytometry. Representative (A) and cumulative data of T_Eff suppression by T_Reg (B-C). *P<0.002 by ANOVA.

4. Discussion

In SLE, dysregulated T cell responses include an inadequate control of the activity of T_Eff by T_Reg [8]. Functional deficits and/or abnormal numbers of T_Eff and T_Reg have both been proposed as mechanisms that contribute to the loss peripheral immune tolerance in SLE [2]. For example, lupus T_Eff could differentiate into pathogenic T cell subsets and proliferate excessively [9], or T_Reg could be numerically reduced and/or be functionally deficient (and thus favor T cell autoreactivity) [10]. The findings reported in this manuscript might explain the conflicting data in the literature that reported abnormal or normal T_Reg number and/or function in SLE [2], since T_Eff resistance to suppression by T_Reg could influence outcomes. Importantly, our results also show that T_Eff resistance to T_Reg suppression can be modulated by the induction of immune tolerance. We acknowledge that additional factors that influence T_Reg suppression and T_Eff susceptibility, such distinct TCR ligands or cytokines or environmental factors, could all contribute to the T_Reg suppression of T_Eff [11-14]. Yet the finding that pCons operates at multiple levels that include a modulation of T_Eff resistance to suppression imply that a manipulation of T_Reg in therapeutic settings might be complicated by the responsiveness of target cells. For example, if T_Eff resistance occurred in the setting of a numeric increase of T_Reg., those T_Reg might not be clinically effective. Conversely, the induction of a reduced threshold of T_eff suppression by T_Reg (such as after tolerance), could result in beneficial effects on the disease progression and outcome.

Highlights.

• We studied the effects of the tolerogenic peptide pCons on T_Eff in NZB/W lupus mice

• We found that pCons decreased the resistance of T_Eff to suppression by T_Reg

• The findings are relevant in the design of T_Reg-based therapies in SLE

Abbreviations

SLE

systemic lupus erythematosus

T_Eff

T effector cells

T_Eff

T regulatory cells