Functional Studies of T Regulatory Lymphocytes in Human Schistosomiasis in Western Kenya

Abstract

Immunoregulation is considered a common feature of Schistosoma mansoni infections, and elevated levels of T regulatory (Treg) lymphocytes have been reported during chronic human schistosomiasis. We now report that the removal of Treg (CD4+/CD25^hi/CD127^low lymphocytes) from peripheral blood mononuclear cells (PBMCs) of S. mansoni-infected individuals leads to increased levels of phytohemagglutinin (PHA)-stimulated interferon gamma (IFNγ) production and decreased interleukin-10 (IL-10) responses. Exposure to schistosome antigens did not result in measurable IFNγ by either PBMC or Treg-depleted populations. Interleukin-10 responses to soluble egg antigens (SEA) by PBMC were unchanged by Treg depletion, but the depletion of Treg greatly decreased IL-10 production to soluble worm antigenic preparation (SWAP). Proliferative responses to PHA increased upon Treg removal, but responses to SEA or SWAP did not, unless only initially low responders were evaluated. Addition of anti-IL-10 increased PBMC proliferative responses to either SEA or SWAP, but did not alter responses by Treg-depleted cells. Blockade by anti-transforming growth factor-beta (TGF-β) increased SEA but not SWAP proliferative responses by PBMC, whereas anti-TGF-β increased both SEA- and SWAP-stimulated responses by Treg-depleted cultures. Addition of both anti-IL-10 and anti-TGF-β to PBMC or Treg-depleted populations increased proliferation of both populations to either SEA or SWAP. These studies demonstrate that Treg appear to produce much of the antigen-stimulated IL-10, but other cell types or subsets of Treg may produce much of the TGF-β. The elevated levels of Treg seen in chronic schistosomiasis appear functional and involve IL-10 and TGF-β in antigen-specific immunoregulation perhaps leading to regulation of immunopathology and/or possibly decreased immunoprotective responses.

Figure 1.

Scatter plot of correlation between the percentages of lymphocytes in peripheral blood mononuclear cells (PBMC) from individuals with schistosomiasis stained for CD4/CD25^hi vs. those in the same PBMC population stained with CD4/CD25^hi/CD127^low using Spearman’s correlation test.

Figure 2.

(A) Paired analysis of the percentage of lymphocytes in peripheral blood mononuclear cells (PBMC) from individuals with schistosomiasis stained for CD4/CD25hi before and after processing to remove CD25^hi–positive cells (T regulatory [Treg]-depleted). (B) Paired analysis of the percentage of lymphocytes in PBMC from individuals with schistosomiasis stained for CD4/CD25^hi/CD127^low before and after processing to remove CD25hi cells (Treg-depleted). The mean difference between the percentages in the PBMC populations and the Treg-depleted populations were analyzed by Wilcoxon matched-pairs signed rank test and P values are indicated above each set of pairs.

Figure 3.

(A) Paired analysis of the interferon gamma (IFN-γ) responses of peripheral blood mononuclear cells (PBMC) from individuals with schistosomiasis prior to and after T regulatory (Treg)-depletion in response to phytohemagglutinin (PHA). (B) Paired analysis of the interleukin-10 responses of PBMC from individuals with schistosomiasis prior to and after Treg-depletion in response to PHA. Statistical analyses were by the Wilcoxon matched-pairs signed rank test and P values are indicated above each set of pairs.

Figure 4.

Paired analysis of the interleukin-10 (IL-10) responses of peripheral blood mononuclear cells (PBMC) from individuals with schistosomiasis prior to and after T regulatory (Treg)-depletion in response to (A) soluble egg antigens (SEA) and (B) soluble worm antigenic preparation (SWAP). Mean SEA-stimulated IL-10 responses were not altered significantly by Treg-depletion (A). Soluble worm antigenic preparation-stimulated production of IL-10 (B) was significantly decreased by Treg-depletion (P = 0.0078). Statistical analyses were by the Wilcoxon matched-pairs signed rank test.

Figure 5.

Mean ± SEM proliferative responses of peripheral blood mononuclear cells from individuals with schistosomiasis in response to phytohemagglutinin (PHA) (Day 3 of culture), soluble egg antigen (SEA) or soluble worm antigenic preparation (SWAP) (both Day 5 of culture) as measured by optical density (O.D.) based on BrdU incorporation, minus the incorporation in culture medium alone (PHA, Day 3; SEA and SWAP, Day 5). N values indicate the number of individuals contributing to each mean.

Figure 6.

Peripheral blood mononuclear cells (PBMCs) and their parallel T regulatory-depleted populations were cultured in the presence of phytohemagglutinin (PHA), schistosome soluble egg antigen (SEA) or schistosome soluble worm antigenic preparation (SWAP) and their level of proliferation determined by incorporation of BrdU/labeled anti-BrdU as optical density values (O.D.) as Experimental (E; PHA, SEA or SWAP) or Control (C; media alone). Panels are as follows: A, PHA; B, SEA; C, SWAP. Panels D and E are replotted and reanalyzed from Panels B and C, respectively, showing the responses of very low (E–C O.D. values < 0.150) responders to SEA or SWAP. E–C O.D. values for each pair are plotted and were analyzed by Wilcoxon matched-pairs signed rank test. P values for differences in the mean E–C values are given above each pair in each panel.

Figure 7.

(A and B) Peripheral blood mononuclear cells and their parallel T regulatory (Treg)-depleted populations were cultured in the presence of schistosome soluble egg antigen (SEA) plus isotype control monoclonal antibody (mAb) (IsoControl) compared with culture in the presence of SEA plus mAb against interleukin-10 (anti-IL-10). (C and D) Peripheral blood mononuclear cell and their parallel Treg-depleted populations were cultured in the presence of schistosome soluble worm antigenic preparation (SWAP) plus IsoControl compared with culture in the presence of SWAP plus anti-IL-10. The levels of proliferation in the cultures were determined by incorporation of BrdU/labeled anti-BrdU expressed as optical density values (O.D.) as Experimental (E) or Control (C). E–C O.D. values for each pair are plotted and were analyzed by Wilcoxon matched-pairs signed rank test. P values for differences in the mean E–C values are given above each pair in each panel.

Figure 8.

(A and B) Peripheral blood mononuclear cells and their parallel T regulatory (Treg)-depleted populations were cultured in the presence of schistosome soluble egg antigen (SEA) plus isotype control monoclonal antibody (mAb) (IsoControl) compared with culture in the presence of SEA plus mAb against TGF-β (anti-TGF-β). (C and D) Peripheral blood mononuclear cell and their parallel Treg-depleted populations were cultured in the presence of schistosome soluble worm antigenic preparation (SWAP) plus IsoControl compared with culture in the presence of SWAP plus anti-TGF-β. The levels of proliferation in the cultures were determined by incorporation of BrdU/labeled anti-BrdU expressed as optical density values (O.D.) as Experimental (E) or Control (C). E–C O.D. values for each pair are plotted and were analyzed by Wilcoxon matched-pairs signed rank test. P values for differences in the mean E–C values are given above each pair in each panel.

Figure 9.

(A and B) Peripheral blood mononuclear cells and their parallel T regulatory (Treg)-depleted populations were cultured in the presence of schistosome soluble egg antigen (SEA) plus two isotype control monoclonal antibodies (IsoControl) compared with culture in the presence of SEA plus monoclonal antibodies against both interleukin-10 (IL-10) and TGF-β. (C and D) Peripheral blood mononuclear cell and their parallel Treg-depleted populations were cultured in the presence of schistosome soluble worm antigenic preparation (SWAP) plus two IsoControls compared with culture in the presence of SWAP plus both anti-IL-10 and anti-TGF-β. The levels of proliferation in the cultures were determined by incorporation of BrdU/labeled anti-BrdU expressed as optical density values (O.D.) as Experimental (E) or Control (C). E–C O.D. values for each pair are plotted and were analyzed by Wilcoxon matched-pairs signed rank test. P values for differences in the mean E–C values are given above each pair in each panel.