Intracellular cytokines in blood T cells in lung transplant patients--a more relevant indicator of immunosuppression than drug levels

Abstract

Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increase in T-cell pro-inflammatory cytokine expression. Systemic levels of immunosuppressive drugs used to reduce pro-inflammatory cytokine expression are closely monitored to their 'therapeutic range'. However, it is currently unknown if levels of these drugs correlate with pro-inflammatory cytokine expression in peripheral blood T cells. To investigate the immunomodulatory effects of currently used immunosuppressive regimes on peripheral blood T-cell cytokine production, whole blood from stable lung transplant patients and control volunteers were stimulated in vitro and cytokine production by CD8+ and CD4+ T-cell subsets determined using multiparameter flow cytometry. T-cell IL-2 and TNFalpha production was significantly reduced from lung transplant patients compared to controls. CD4+ T-cell production of IFNgamma was also significantly reduced from lung transplant patients but production of IFNgamma by CD8+ T cells remained unchanged. There was an excellent correlation between the percentage of CD8+ T cells and the percentage of CD8+ T cells producing IFNgamma from transplant patients. T-cell IL-4 and CD8+ T-cell production of TGFbeta was significantly increased from lung transplant patients. We now provide evidence that current immunosuppression protocols have limited effect on peripheral blood IFNgamma production by CD8+ T-cells but do up-regulate T-cell anti-inflammatory cytokines. Drugs that effectively reduce IFNgamma production by CD8+ T cells may improve current protocols for reducing graft rejection in these patients. Intracellular cytokine analysis using flow cytometry may be a more appropriate indicator of immunosuppression than drug levels in these patients. This technique may prove useful in optimizing therapy for individual patients.

Fig. 1

Box plot graphs showing the production of pro-inflammatory cytokines by CD4+ (□) and CD8+ () T-cells from lung transplant (T) and control (C) subjects following in vitro stimulation (mean ± SD and range). The percentage of CD4+ and CD8+ T cells producing IL-2 and TNF-α was significantly reduced from lung transplant patients compared to control. The percentage of CD4+ T cells producing IFN-γ was also significantly reduced from lung tranplant patients but not the percentage of CD8+ T cells producing IFN-γ. Note the marked inhibition of inflammaatory T-cell cytokines in CD4+ cells compared with CD8+ cells in transplant patients compared to control group.

Fig. 2

Representative dot plots showing the effective immunosuppression therapy on IFN-γ production by CD4+ and CD8+ T cells from 2 lung transplant patients and control. T cells were identified by CD3 PC5 versus side scatter characteristics. Patient A shows immunosuppression of IFN-γ in CD8–(CD4+) T cells but not in CD8+ T cells. Patient B shows immunosuppression of IFN-γ in both T cells subsets. Note the reduced percentage of CD4 T cells and increased CD8 T cells in transplant patients compared to control.