Intracellular cytokine analysis of peripheral blood T-lymphocytes for noninvasive diagnosis of acute rejection after lung transplantation

Yui Watanabe¹, Yoshinori Okada¹, Hisashi Oishi¹, Tatsuaki Watanabe¹, Shunsuke Eba¹, Yasushi Hoshikawa¹, Takashi Kondo¹.

¹Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.

Purpose: Acute allograft rejection (AR) after lung transplantation remains a serious problem. Currently, the diagnosis of AR is primarily obtained by histological examination of TBLB specimens, and thus noninvasive diagnostic approach is desirable. AR is associated with an increased expression of intra-graft pro-inflammatory cytokines. We hypothesized that intracellular pro-inflammatory cytokine expression in peripheral blood T-lymphocytes (PBTL) may also be elevated in lung transplant recipients undergoing AR. In the present study, we examined intracellular cytokine expression in PBTL by flow cytometry in a rat model of AR.

Methods and Materials: Left single lung transplantations were performed between a highly histoincompatible combination of inbred rats (BN to LEW). Syngeneic transplants (LEW to LEW) served as the control. On day 3, 5 and 7 posttransplantation, PBTL were divided and stimulated by PMA and Ionomycin. Cell surface antigens (CD4, 8) and intracellular cytokines (IL-2, 4, 10, IFN-γ, TNF-α) were stained, and cells were analyzed on a flow cytometer. Harvested allografts were histologically examined and degree of AR was graded (A0 to A4) according to the ISHLT grading system.

Results: In general, the stage of AR of allografts was A2 on day 3, A3 on day5, and A4 on day 7. The proportions of IL-2, 10, IFN-γ, and TNF-α positive PBTL increased in the allograft model as the stage of AR progressed. Of noteworthy, the proportion of TNF-α + cells showed a significant increase compared with the control (day 3: 29.6% vs. 11.6%, p<0.05, day 5: 49.3% vs. 21.4%, p<0.01, day 7: 43.9% vs. 22.9%, p<0.05). Additionally, the proportion of CD4 + and TNF-α+ cells showed a marked elevation compared with the control (day 3: 37.3% vs. 16.3%, p<0.01, day 5: 63.3% vs. 24.4%, p<0.001).

Conclusions: Analysis of Intracellular TNF-α expression in PBTL may be useful for noninvasive diagnosis of AR after lung transplantation. Additional study will be necessary to more precisely assess the clinical utility of this monitoring system.