Human CD4- 8- T cells are a distinctive immunoregulatory subset

Abstract

Human CD4(-)8(-) T cells are a minor subset quantitatively but potentially important in immunity because they are predominantly distributed at body surfaces, and their number and activities increase in autoimmune diseases and decrease with aging. Distinguishing characteristics of CD4(-)8(-) T cells are found to include a unique profile of cytokines, including Serpin E1, which is not generated by other T cells, MIF, and TGF-beta. At 2-5% of the total in mixtures with CD4 + CD8 T cells, CD4(-)8(-) T cells enhance the generation of IFN-gamma and IL-17 by up to 12- and 5-fold, respectively, without contributing either cytokine or affecting cytokine production by NK/NKT cells. CD4(-)8(-) T cell-derived MIF is their major enhancer and TGFbeta their principal inhibitor of CD4 and CD8 T cell cytokine production. Decreases in CD4(-)8(-) T cell effects may diminish protective immunity in aging, whereas increases may augment the severity of autoimmune diseases.

Figure 1.

CD4⁻8⁻ T-cell enhancement of generation of IFN-γ and IL-17, but not TGFβ, by mixed CD4 and CD8 T cells stimulated with anti-CD3 + anti-CD28 antibodies. Columns and bars depict mean ± sd results of studies of T cells from 4 healthy human donors, expressed as fold increase above the cytokine concentration attained by CD4⁻8⁻ T-cell-depleted mixtures of CD4 and CD8 T cells. Native T-cell mixture contained relative numbers of CD4, CD8, and CD4⁻8⁻ T cells normally present in the blood of each donor. Left-hand (open) and right-hand (shaded) bars of each pair show results for 2 and 3 d of incubation, respectively. CD4 + CD8 T cells depleted of CD4⁻8⁻ T cells and stimulated with 1 μg each of anti-CD3 + anti-CD28 antibodies achieved a range of concentrations of IFN-γ of 65–281 pg/ml on d 2 and 175–1076 pg/ml on d 3, IL-17 of 32–75 pg/ml on d 2 and 58–94 pg/ml on d 3, and TGFβ of 610–930 pg/ml on d 2 and 862–1215 pg/ml on d 3. ⁺P < 0.05; t test.

Figure 2.

Target-cell specificity of CD4⁻8⁻ T-cell enhancement of generation of IFN-γ. Columns and bars depict mean ± sd results of studies of cells from 3 healthy human donors, expressed as fold increase above the IFN-γ concentration attained by NK/NKT cell mixtures stimulated with 5 ng/ml of IL-2 and 2 ng/ml of IL-12, and CD4 and CD8 T cells stimulated with 2 μg each of anti-CD3 and anti-CD28 antibodies in the absence of CD4⁻8⁻ T cells. Left-hand (open) and right-hand (solid) bars of each pair show results on d 1 and 2 for NK/NKT cells, and d 2 and 3 for CD4 and CD8 T cells, respectively. Range of concentrations of IFN-γ achieved in the absence of CD4⁻8⁻ T cells on d 1 and 2 for NK/NKT cells were 21,250–29,300 and 75,200–80,350 pg/ml, respectively, and on d 2 and 3, 1650–2664 and 4030–4684 pg/ml for stimulated CD8 T cells, and 362–2315 and 1474–4861 pg/ml for stimulated CD4 T cells, respectively. ⁺P < 0.05, *P < 0.01, **P < 0.001; t test.

Figure 3.

Generation of immune cytokines by purified CD4⁻8⁻ T cells. Columns and bars depict mean ± sd results of studies of CD4⁻8⁻ T cells on d 1 and 2 or of CD4 + CD8 T cells on d 2 (bar 2P) from 5 healthy human donors, expressed as pg/ml/10⁶ T cells. Repetitive sets of 4 columns for CD4⁻8⁻ T cells on d 1 and 2 represent, in order from left to right: no stimulus, anti-CD3 + anti-CD28 antibodies, anti-CD3 + anti-CD161 antibodies, and IL-12 (5 ng/ml)+ IL-18 (50 ng/ml). Single 2P bar in each frame depicts values on d 2 for CD4 + CD8 T cells from 5 donors (IFN-γ and IL-17) or 3 donors (other cytokines) stimulated with anti-CD3 + anti-CD28 antibodies. *P < 0.01 vs. d 2 level for anti-CD3 + anti-CD28 antibody-stimulated CD4⁻8⁻ T cells. N for the Serpin E1 2P bar indicates not detectable.

Figure 4.

CD4⁻8⁻ T-cell-type cytokine enhancement of generation of IFN-γ by mixed CD4 and CD8 T cells in the absence of CD4⁻8⁻ T cells. Columns and bars depict mean ± sd enhancement of IFN-γ generation attained by adding 1 or 2 cytokines to CD4⁻8⁻ T-cell-free CD4 + CD8 T cells from 3 healthy donors before stimulation by anti-CD3 + anti-CD28 antibodies for 3 d. The 100% control value represents level of IFN-γ production by same number of CD4 + CD8 T cells mixed with 3% CD4⁻8⁻ T cells before stimulation. ⁺P < 0.05, *P < 0.025, and **P < 0.01; t test.

Figure 5.

Prevention of CD4⁻8⁻ T-cell regulation of mixed CD4 + CD8 T-cell generation of IFN-γ by antagonism of cytokines. Columns and bars depict mean ± sd change induced by adding anti-cytokine/cytokine receptor antibodies and/or the MIF receptor antagonist Iso-1 (I) to CD4 + CD8 T cells mixed with 3% CD4⁻8⁻ T cells from 3 healthy donors before stimulation by anti-CD3 + anti-CD28 antibodies (no additions control=100%). Antibodies were to Serpin E1 (S; 2 μg/ml), RANTES (R; 10 μg/ml), and MIF + MIF receptor (M; 30 and 6 μg/ml, respectively); Iso-1 (I) level was 40 μM. Bars in each pair depict data from d 2 and 3 in that order. Statistical analyses and symbols as in Fig. 2, except that brackets over Iso-1 data in left frame show significantly greater inhibition at both days when S is added to Iso-1.