T cells from patients with Candida sepsis display a suppressive immunophenotype

Abstract

Background: Despite appropriate therapy, Candida bloodstream infections are associated with a mortality rate of approximately 40%. In animal models, impaired immunity due to T cell exhaustion has been implicated in fungal sepsis mortality. The purpose of this study was to determine potential mechanisms of fungal-induced immunosuppression via immunophenotyping of circulating T lymphocytes from patients with microbiologically documented Candida bloodstream infections.

Methods: Patients with blood cultures positive for any Candida species were studied. Non-septic critically ill patients with no evidence of bacterial or fungal infection were controls. T cells were analyzed via flow cytometry for cellular activation and for expression of positive and negative co-stimulatory molecules. Both the percentages of cells expressing particular immunophenotypic markers as well as the geometric mean fluorescence intensity (GMFI), a measure of expression of the number of receptors or ligands per cell, were quantitated.

Results: Twenty-seven patients with Candida bloodstream infections and 16 control patients were studied. Compared to control patients, CD8 T cells from patients with Candidemia had evidence of cellular activation as indicated by increased CD69 expression while CD4 T cells had decreased expression of the major positive co-stimulatory molecule CD28. CD4 and CD8 T cells from patients with Candidemia expressed markers typical of T cell exhaustion as indicated by either increased percentages of or increased MFI for programmed cell death 1 (PD-1) or its ligand (PD-L1).

Conclusions: Circulating immune effector cells from patients with Candidemia display an immunophenotype consistent with immunosuppression as evidenced by T cell exhaustion and concomitant downregulation of positive co-stimulatory molecules. These findings may help explain why patients with fungal sepsis have a high mortality despite appropriate antifungal therapy. Development of immunoadjuvants that reverse T cell exhaustion and boost host immunity may offer one way to improve outcome in this highly lethal disorder.

Fig. 1

Co-inhibitory molecule expression on CD4 and CD8 T cells. PD-1, PD-L1, and BTLA are co-inhibitory molecules. The percentage of PD-L1 expression on CD8 T cells and the GMFIs of CD4 and CD8 T cells were significantly increased in patients with CBSI compared to CINS (p ≤0.01). Several patients with CBSI had higher percentage of PD-L1 expression on CD4 T cells than those with CINS, although there was no statistical significance between the groups (p = 0.161). Horizontal lines indicate mean or median values. Asterisks indicate significant differences between CINS and CBSI (^* p ≤0.05 and ^** p ≤0.01). BTLA B and T lymphocyte attenuator, CBSI Candida bloodstream infection, CINS critically ill non-sepsis, GMFI geometric mean fluorescence intensity, PD-1 programmed cell death 1, PD-L1 programmed cell death ligand 1

Fig. 2

PD-L1 expression on CD8 T cells in CINS and CBSI. Representative flow cytometric findings in patients with CINS and CBSI are shown. The percentage of PD-L1-positive CD8 T cells was higher in CBSI than in CINS. CBSI Candida bloodstream infection, CINS critically ill non-sepsis, PD-1 programmed cell death 1, PD-L1 programmed cell death ligand 1

Fig. 3

Co-stimulatory molecule expression and markers related to cell activation on CD4 and CD8 T cells. CD28 is a co-stimulatory molecule that is necessary for optimal T cell activation. CD69 is marker of cellular activation. IL-7R activation leads to T cell activation and proliferation. CD28 expression on CD4 T cells was significantly decreased in patients with CBSI, compared to those with CINS. In CD8 T cells, both the percentage and GMFI for CD69 were increased in CBSI compared to CINS. On the other hand, the percentage and GMFI of the IL-7R tended to be lower in CD4 T cells from CBSI versus CINS patients. However, this difference did not reach statistical significance, p = 0.07 and p = 0.08 respectively. Horizontal lines indicate mean or median values. Asterisks indicate significant differences between CINS and CBSI (^* p ≤0.05 and ^** p ≤0.01). CBSI Candida bloodstream infection, CINS critically ill non-sepsis, GMFI geometric mean fluorescence intensity, IL-7R interleukin-7 receptor

Fig. 4

Interleukin 7 receptor expression on CD4 and CD8 T cells - differences between CINS and CBSI. Representative flow cytometric findings in patients with CINS and CBSI are shown. The percentages of IL-7R-positive CD4 and CD8 T cells were lower in CBSI than in CINS. CBSI Candida bloodstream infection, CINS critically ill non-sepsis, IL-7R interleukin- 7 receptor

Fig. 5

CD marker expression on CD4 and CD8 T cells related to senescence and inhibitory or stimulatory effects. CD57 is a marker that is used to identify senescent, poorly functional T cells. TIM-3 is a co-inhibitory molecule that impairs T cell function. 2B4 (CD244) is a dual-functional receptor that may have either inhibitory or stimulatory effects on T cells depending upon other stimuli and environmental settings. Horizontal lines indicate mean or median values. Asterisks indicate significant differences between CINS and CBSI (^* p ≤0.05 and ^** p ≤0.01). CBSI Candida bloodstream infection, CINS critically ill non-sepsis, TIM-3T cell immunoglobulin domain and mucin domain 3