Table 1.
Demographic and clinical characteristics of TB patients, latent infection individuals and healthy controls.
Figure 1.
Representative flow cytometric plots showing gating strategy and expression of CD244/2B4.
(A) Viable lymphocytes in PBMCs were gated based on characteristic forward and side scatter profiles. (B) The gate depicting CD4/CD244 expression was defined by both control isotype staining and CD244 expression on CD3−CD4− cells that showed clearly two cell subsets with positive and negative staining of CD244. (C and D) CD4+ T cells were defined as CD3+CD4+ cells (C) and CD244/2B4 expression on total CD4+ T cells was shown (D). (E and F) Antigen-responsive CD4+ T cells were identified by appearance of activation marker CD69 in the CD3+CD4+ cell population (E) and CD244/2B4 expression on M. tuberculosis antigen-responsive CD4+ T cells was shown (F). Isotype: isotype antibody control; LI: latent TB infection; TB: active TB patients.
Figure 2.
The expression of CD244/2B4 on CD4+ T cells in active pulmonary TB patients, latent infection individuals and healthy controls.
(A) The frequencies of CD244/2B4-expressing total CD4+ T cells were similar among active TB patients, latent infection individuals and healthy controls. (B) Mean fluorescent intensity (MFI) of CD244/2B4 expression on total CD4+ T cells was significantly higher in active TB patients than in latent infection individuals and healthy controls. (C) The frequencies of CD244/2B4-expressing antigen-responsive CD4+ T cells increased significantly in retreatment active TB patients as compared with latent infection individuals and new active TB patients. (D) MFI of CD244/2B4 expression on antigen-responsive CD4+ T cells was significantly higher in retreatment active TB patients as compared with latent infection individuals and new active TB patients. New TB patients had significantly elevated expression of CD244/2B4 as compared with latent infection individuals. (E and F) When only M. tuberculosis culture-positive TB cases were selected for analysis, retreatment TB patients also had higher frequencies of CD244/2B4-expressing antigen-responsive CD4+ T cells (F) and elevated expression of CD244/2B4 as compared with latent infection individuals and new TB patients (G). (G and H) TB patients with more than 3 months of treatment, which contained all of the retreatment cases, had higher frequencies of CD244/2B4-expressing antigen-responsive CD4+ T cells (G) and elevated expression of CD244/2B4 as compared with those who were treated less than 1 month or between 1 to 3 months (H). HC: healthy controls; new: new TB patients; retreat: retreatment TB patients; ≤1 m: less than 1 month of treatment; 1–3 m: between 1 to 3 month of treatment; ≥3 m: more than 3 month of treatment. Mann-Whitney test was used for statistical analysis between groups. *: p<0.05; **: p<0.01; ***: p<0.001.
Figure 3.
Influence of CD244/2B4 expression on IFN-γ production.
(A and B) Representative flow cytometric plots showing gating strategy of CD244/2B4-dull (AW) –middle (AU) and -bright (AV) CD4+ T cells, and Intracellular IFN-γ production in CD244/2B4-dull (AZ), -middle (AY) and -bright (AX) CD4+ T cells. (C) CD244/2B4-bright CD4+ T cells contained significantly lower ratio of IFN-γ-producing CD4+ cells, compared with CD244/2B4-dull CD4+ T cells. (D) CD244/2B4-bright CD4+ T cells had greatly reduced expression of IFN-γ, compared with CD244/2B4-dull and –middle CD4+ T cells. (E and F) T-SPOT.TB-negative TB patients had higher frequencies of CD244/2B4-expressing cells than T-SPOT.TB-positive TB patients. ELI+: ELISPOT-positive TB patients; ELI-: ELISPOT-negative TB patients. Mann-Whitney test was used for statistical analysis between groups. *: p<0.05; ***: p<0.001.
Figure 4.
Activation of CD244/2B4 signaling resulted in significantly decreased IFN-γ production.
(A) Representative flow cytometric plots showing IFN-γ production in CD3+CD4+ T cells incubated with isotype control antibody (ISO) or with anti-CD244/2B4 antibody clone C1.7 (CK). (B to D) Activation of CD244/2B4 signaling by cross-linking led to significantly lower frequencies of IFN-γ-producing CD4+ T cells (B), decreased expression of IFN-γ as determined by flow cytometry (C), and reduced numbers of spot forming units in ELISPOT assay (D). Paired t-test was used for statistical analysis. *: p<0.05; **: p<0.01.
Figure 5.
Rescue of IFN-γ production following CD244/2B4 receptor blocking.
(A) Representative flow cytometric plots showing IFN-γ production in stimulated CD3+CD4+ T cells when incubated with isotype control antibody (ISO) or blocking anti-CD244/2B4 antibody clone eBioPP35 (BL). (B to D) Blocking of CD244/2B4 signaling resulted in significantly higher frequencies of IFN-γ-producing CD4+ T cells (B), increased level of IFN-γ production in CD3+CD4+ T cells as determined by flow cytometry (C), and elevated numbers of spot forming units in ELISPOT assay (D), as compared with those incubated with isotype control antibody. Paired t-test was used for statistical analysis. **: p<0.01.