Superantigen-induced T cell responses in acute rheumatic fever and chronic rheumatic heart disease patients

Abstract

CD4+ and CD8+ T cells from healthy donors, acute rheumatic fever (ARF) and chronic rheumatic heart disease (CRHD) patients responded variably to a superantigen from Streptococcus pyogenes--Streptococcal pyrogenic erythrogenic toxin A (SPE-A). In vitro culture of CD4+ T cells from ARF patients (CD4-ARF) with SPE-A exhibited a Th1 type of response as they produced high levels of IL-2, while CD4+ T cells from CRHD patients (CD4-RHD) secreted IL-4 and IL-10 in large amounts, i.e. Th2 type of cytokine profile. The skewing of human CD4+ T cells (in response to SPE-A stimulation) to Th1 or Th2 type reflects the role of the two subsets in a disorder with differing intensities at the two extremes of the spectrum. Moreover, the anergy induction experiments revealed that CD8-ARF and CD8-RHD undergo anergy (to different extents), whereas CD4+ T cells do not, in response to re-stimulation by SPE-A. These results initially demonstrate that both CD4+ and CD8+ T cells respond differentially to SPE-A, and hence it is an important observation with respect to the pathogenesis of ARF/CRHD. Anergy in CD8+ T cells in the presence of SPE-A in vitro goes a step further to show the clinical relevance of these cells and their possible role in suppression of the disease.

Fig 1

Dose–response curve. Proliferative response of CD4⁺ and CD8⁺ cells upon in vitro stimulation with Streptococcal pyrogenic erythrogenic toxin A (SPE-A). CD4⁺ or CD8⁺ T cells (2 × 10⁴) and 10⁵ adherent, mitomycin C-treated autologous population from healthy donors, were co-cultured with varying amounts of SPE-A (•, CD4; ▪, CD8), anti-CD3 (20 ng/ml) alone (ct/min 86 462 ± 5710), anti-CD3 (20 ng/ml) with anti-SPE-A (80 926 ± 6515), anti SPE-A (6 μg/ml) raised in rabbit (○), normal rabbit serum (1:500 dilution). Normal rabbit serum was unable to cause inhibition of SPE-A (data not shown). The cultures were pulsed with ³H-thymidine after 48 h, for 16 h and data are presented as mean ct/min ± s.d., where experiments were conducted in triplicates.

Fig 2

Effect of repeated dose of Streptococcal pyrogenic erythrogenic toxin A (SPE-A) on CD4⁺ T cells. CD4⁺ T cells (10⁵ cells/ml) from all the three groups under study were incubated initially with 50 ng/ml SPE-A for 16–20 h in the absence of any antigen presenting cells (APC) in a 24-well tissue culture plate. The cells were collected on Ficoll, washed and rested for 5 days. After rest, the cells were restimulated with SPE-A in the presence of autologous mitomycin C-treated APC and cell proliferation was measured by ³H-thymidine uptake (abscissa). Recombinant IL-2 along with APC and SPE-A was added to check for reversal of anergy. Controls: T cells alone (ct/min 437 ± 64), T cells + SPE-A (542 ± 72), T cells incubated with SPE-A and anti-SPE-A followed by addition of T cells + SPE-A + APC (ct/min 26 542 ± 2472) and normal cells + anti-CD3 (74 829 ± 3641), acute rheumatic fever (ARF)-CD4 T cells + anti-CD3 (92 461 ± 5212), chronic rheumatic heart disease (CRHD)-CD8 T cells + anti-CD3 (93 298 ± 4718) were included. Addition of anti-SPE-A to these anti-CD3 control wells did not bring any significant change in proliferation data. All the controls were treated in parallel to the test samples under similar conditions. Results are depicted as (ordinate): open blocks, stimulation with SPE-A in the presence of APC as done in Fig. 1; black blocks, restimulation as done for anergy induction; hatched blocks, response to exogenous IL-2. *P < 0.05, response in presence and absence of IL-2 in normal CD4⁺ T cells.

Fig 3

Induction of anergy in CD8⁺ T cells. CD8⁺ T cells (10⁵ cells/ml) from all three groups were cultured with Streptococcal pyrogenic erythrogenic toxin A (SPE-A; 50 ng/ml) in the absence of antigen-presenting cells (APC) for 16–20 h. These cells were collected on Ficoll, washed and rested for 5 days. Restimulation with SPE-A autologous mitomycin C-treated APC was measured by the amount of ³H-thymidine incorporated by proliferating cells (abscissa). Reversal of anergy was brought about by addition of APC, SPE-A and exogenous IL-2. T cells alone (ct/min 368 ± 52]; T cells + SPE-A (625 ± 48); T cells incubated with SPE-A and anti-SPE-A followed by T + SPE-A + APC (ct/min 204 22 ± 4214) and normal T cells + anti-CD3 (80 325 ± 3552); acute rheumatic fever (ARF)-CD8 T cells + anti-CD3 (84 293 ± 4881); chronic rheumatic heart disease (CRHD)-CD8 T cells + anti-CD3 (82 969 ± 5238). Addition of anti-SPE-A to the anti-CD3 control wells did not bring about any change in the proliferation data. Open blocks: stimulation with SPE-A ipo APC as done in Fig. 1; black blocks, restimulation as done for anergy induction; hatched blocks, response to exogenous IL-2. *P < 0.05, restimulation in ARF and CRHD compared with normal; **P < 0.01, response to exogenous IL-2 in normal and ARF; ***P < 0.01, comparison of response in absence and presence of IL-2 in ARF; ****P < 0.005, comparison of response in absence and presence of IL-2 in CRHD.