Abstract

Abstract 2286

It is known that major surgery and allogeneic blood transfusion mediate immunosuppression by interfering on cytokine secretion, lymphocyte count and cytotoxic response. However, which cytokines and lymphocyte subpopulations participate in such immunosuppressive state remains poorly understood. Regulatory T cells (Tregs) are suppressive CD4(+) cells with a central role in immunosuppression of trauma victims, cancer patients, and transplant recipients. Several markers have been identified with regulatory properties, such as FOXP3, CTLA-4, GITR and the recently described CD69. Recently, allogeneic blood transfusion has been shown capable of inducing regulatory CD4+FOXP3+ T cells in vitro.

Purpose:

In this study, we aimed to investigate regulatory CD4+ T cells induction and cytokine profile in transfused and non-transfused surgical patients.

Patients and Methods:

Thirty-five patients undergoing elective hip replacement were recruited for this study and prospectively evaluated. Blood samples were obtained before surgery (D0) and on days 1 (D1) and 4 (D4) after surgery. Quantification of regulatory T lymphocytes was done by flow cytometry using CD4, CD25, FoxP3, CTLA-4, GITR and CD69, while cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-α, IFN-γ and TGF-β) levels were determined by multiplexed immunoassay system.

Results:

From the 35 patients recruited for this study, 18 (51.4%) received allogeneic non-leukodepleted red cells transfusion during or after surgery. There were no significant differences in clinical or epidemiological characteristics between transfused and non-transfused groups, except for the duration of surgery, being longer in transfused patients (p<0.001). Results of total lymphocyte count and subsets of CD4+ T cells in the 35 patients are summarized in the table:

D0D1D4p value
Total lymphocyte count (×109/mL)* 1,907 (1579-2472) 1,450 (1205-1864) 1,266 (1092-1817) D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
CD4+ (%)** 24.5±9.8 33.3±11.7 38±11.7 D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
D1 vs. D4; p<0.001 
CD4+CD25-CD69+ (%)* 0.55 (0.27-0.77) 0.6 (0.3-1.23) 1.13 (0.56-2.52) D0 vs. D4; p<0.001 
D1 vs. D4; p<0.001 
CD4+GITR+ (%)** 1.64±1.64 2.92±1.94 4.38±3.00 D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
D1 vs. D4; p<0.001 
CD4+CTLA-4+ (%)* 0.53 (0.46-0.97) 0.89 (0.72-1.3) 0.89 (0.64-1.33) D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
D0D1D4p value
Total lymphocyte count (×109/mL)* 1,907 (1579-2472) 1,450 (1205-1864) 1,266 (1092-1817) D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
CD4+ (%)** 24.5±9.8 33.3±11.7 38±11.7 D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
D1 vs. D4; p<0.001 
CD4+CD25-CD69+ (%)* 0.55 (0.27-0.77) 0.6 (0.3-1.23) 1.13 (0.56-2.52) D0 vs. D4; p<0.001 
D1 vs. D4; p<0.001 
CD4+GITR+ (%)** 1.64±1.64 2.92±1.94 4.38±3.00 D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
D1 vs. D4; p<0.001 
CD4+CTLA-4+ (%)* 0.53 (0.46-0.97) 0.89 (0.72-1.3) 0.89 (0.64-1.33) D0 vs. D1; p<0.001 
D0 vs. D4; p<0.001 
*

Results reported as median (P25-P75);

**

Results reported as mean±SD

The total lymphocyte count was significantly decreased only in transfused patients. No significant difference was seen in the percentage of CD4+CD25highFoxp3+ T lymphocytes by comparing days D0, D1 and D4. However, a significant increase of CD4+CD25-CD69+ cells was observed by comparing D1 vs. D4 [0.61(0.23–2.69) vs. 2.27 (0.64–4.45); p=0.001] in transfused patients, but not in the group of non-transfused patients [0.36(0.31–0.81) vs. 1.02(0.42–1.52); D1 vs. D4. Similarly, we also detected a significant increase in the number of CD4+GITR+ T cells in the group of transfused patients [D1 vs. D4; 3.12±2.01 vs. 5.19±3.33; p=0.007). Total hip replacement also led to substantial increase of the following cytokines: IL-6 (p<0.001, D0 vs. D1 and p<0.001, D0 vs. D4), IL-8 (p<0.001, D0 vs. D1 and p=0.017, D0 vs. D4), IL-10 (p<0.001, D0 vs. D1). However, no differences regarding cytokines levels were seen comparing groups of transfused and non-transfused patients.

Conclusions:

This study demonstrated that although allogeneic non-leukodepleted red cells transfusion is associated with decrease of total lymphocyte count following major surgery, there was a significant increase of total CD4+ T lymphocytes with no difference observed in transfused and non-transfused patients. However, a significant increase of CD4+CD25-CD69+ and CD4+GITR+ T lymphocytes was observed in the transfused group on D4, possibly reflecting the late effect of transfusion on induction of these subsets of CD4+ regulatory cells. The increase of pro-inflammatory IL-6, IL-8 and anti-inflammatory IL-10 levels indirectly highlights the imbalance on immune response after surgical trauma. Further studies, with a larger cohort and functional assays, investigating these CD4+ T cells subpopulations following major surgery and the impact of allogeneic non-leukodepleted red cells transfusion on these cells should be addressed.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.