Coupled regulation of interleukin-12 receptor beta-1 of CD8+ central memory and CCR7-negative memory T cells in an early alloimmunity in liver transplant recipients

Abstract

This study investigated how CD8(+) T cell subsets respond to allo- and infectious immunity after living donor liver transplantation (LDLT). Early alloimmunity: 56 recipients were classified into three types according to the post-transplant course; type I demonstrated uneventful post-transplant course, type II developed severe sepsis leading to multiple organ dysfunction syndrome or retransplantation and type III with acute rejection. In 23 type I recipients, the interleukin (IL)-12 receptor beta-1 (R beta 1)(+) cells of central memory T cells (Il-12R beta 1(+) T(CM)) were increased above the pretransplant level. In 16 type II recipients, IL-12R beta 1(+) T(CM) was decreased markedly below the pretransplant level on postoperative day (POD) 5. In 17 type III recipients, IL-12R beta 1(+) T(CM) was decreased for a more prolonged period until POD 10. Along with down-regulation of IL-12R beta 1(+) T(CM), the IL-12R beta 1(+) cells of CCR7-negative subsets (CNS) as well as perforin, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha decreased gradually, resulting in the down-regulation of effectors and cytotoxicity. The down-regulation of IL-12R beta 1(+) T(CM) was suggested to be due to the recruitment of alloantigen-primed T cells into the graft, and then their entry into the secondary lymphoid organ, resulting in graft destruction. Infectious immunity: immunocompetent memory T cells with the capacity to enhance effectors and cytotoxicity were generated in response to post-transplant infection along with both up-regulation of the IL-12R beta 1(+) T(CM) and an increase in the CNS showing the highest level of IL-12R beta 1(+) cells. In conclusion, this work demonstrated that the IL-12R beta 1(+) cells of T(CM) and CNS are regulated in a tightly coupled manner and that expression levels of IL-12R beta 1(+) T(CM) play a crucial role in controlling allo- and infectious immunity.

Fig. 1

Flow cytometric assay of all CD8⁺ T cell subsets. For flow cytometry, CD8⁺ T cells were classified into three subsets on the basis of triple-staining using allophycocyanin (APC)-conjugated anti-CD8 (Coulter Immunotech, Marseille, France), fluorescein isothiocyanate (FITC)-conjugated anti-CD45RO (Coulter Immunotech) and RD1-conjugated anti-CD45RA (Coulter Clone 2H4-RD1; Beckman Coulter, Miami, FL, USA) for CD8⁺CD45RO^- cells, CD8⁺CD45RO⁺ cells and CD8⁺CD45RO⁺⁺ cells in gated lymphocytes. This figure shows the classification of three subsets from a representative recipient. In 10 recipients before living donor liver transplantation (LDLT), the average rate of the CD45RA phenotype was 99·98% in CD8⁺CD45RO^- cells, 84·29% in CD8⁺CD45RO⁺ cells and 3·34% in CD8⁺CD45RO⁺⁺ cells. The CD8⁺CD45RO⁺ subset was designated as double-positive cells (DP). The expression of interleukin (IL)-12Rβ1⁺ cells, perforin, interferon (IFN)-γ and tumour necrosis factor (TNF)-α was measured similarly in these three subsets.

Fig. 2

(a) Changes in CCR7(+)- and CCR7(−) subsets as well as interleukin (IL)-12Rβ1⁺ T_CM after living donor liver transplantation (LDLT) in three types, respectively, representing the three types (type I, nine recipients; type II, 12 including eight severe sepsis combined multiple organ dysfunction syndrome (SS/MODS) and four re-transplantation (Re-Tx); and type III, 12 recipients). (b) Changes in the IL-2 expression of CD4⁺ T_CM and T_EM after tacrolimus administration related to tacrolimus (Tac) trough level.

Fig. 3

(a) Flow cytometric assay of the changes in CD8⁺ T cell subsets after living donor liver transplantation (LDLT) in a type I recipient. In flow cytometry, using peripheral blood mononuclear cells (PBMCs), the lymphocytes were stained with monoclonal antibodies to CD45RO and CCR7. The dot plots show double-staining for CD8⁺CCR7/CD45RO on gated lymphocytes which identified six subsets of CD8⁺ T cells: naive (T_N) (CD45RO^-CCR7⁺), central memory (T_CM) (CD45RO⁺CCR7⁺), effector memory (T_EM) (CD45RO⁺CCR7^-), effector T cells (T_E) (CD45RO^-CCR7^-), DP⁺ T cells (T_DP+) (CD45RORACCR7⁺) and DP^- T cells (T_DP−) (CD45RORACCR7^-). Cells in six segments are presented as ratio (%). Subsets, % proportion per CD8⁺ T cells (i) and % difference (ii); WBC: white blood cell; CRP: C-reactive protein; AST: aspartate aminotransferase; B30: bacterial infection on postoperative day (POD) 30 (Pseudomonas aeruginosa, bile); B33: bacterial infection on POD 33 (P. putida and Chryseobacterium, bile); C29: cytomegalovirus on POD 29; and Tac: tacrolinus. In (b), the dot plots show the interleukin (IL)-12Rβ1-expressing cells superimposed on double-staining for IL-12Rβ1 and CCR7 in gated CD8⁺CD45RO^- cells (iii), gated CD8⁺CD45RO⁺ cells (iv) and CD8⁺CD45RO⁺⁺ cells (v). The % proportion (vi) and % difference (vii, viii) of IL-12Rβ1⁺cells; CNS: CCR7-negative subsets; CPS: CCR7-positive subsets.

Fig. 4

(a) Changes on flow cytometry in the % proportion of CD8⁺ T cell subsets after living donor liver transplantation (LDLT) in a type II recipient. In flow cytometry, using peripheral blood mononuclear cells (PBMCs), the lymphocytes were stained with monoclonal antibodies to CD45RO and CCR7, as shown in Fig. 3a. Cells in six segments are presented as ratio (%). Tac: tacrolimus; MODS: multiple organ disorder syndrome; SS/MODS: severe sepsis-combined MODS; B6: bacterial infection on postoperative day (POD) 6 (Staphylococcus haemolyticus and Enterococcus faecium, CV catheter); B26 (Pseudomonas aeruginosa, urine); B33 P. aeruginosa, urine); B61 (S. epidermidi, bile): B82 (P. aeruginosa, urine); B89 (P. aeruginosa, urine); C12, C40 and C50, cytomegalovirus infection on PODs 12, 40 and 50; and Tac: tacrolimus. (b) Changes on flow cytometry in the IL-12Rβ1⁺ cells of CD8⁺ T cell subsets after LDLT. The dot plots show the IL-12Rβ1-expressing cells superimposed on double staining of IL-12Rβ1⁺ and CCR7 in gated CD8⁺CD45RO^- cells (iii), gated CD8⁺CD45RO⁺ cells (iv) and CD8⁺CD45RO⁺⁺ cells (v). The % proportion (vi) and % difference (vii, viii) of IL-12Rβ1⁺ cells; CNS: CCR7-negative subsets; CPS: CCR7-positive subsets.

Fig. 5

Changes on flow cytometry in the % difference of perforin, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in CD8⁺ T cells (T_CD8) as well as interleukin (IL)-12Rβ1⁺ T _CM after living donor liver transplantation (LDLT) in the same recipient as shown in Fig. 4. The dot plots show the perforin-, IFN-γ- and TNF-α-expressing cells superimposed on a double staining of each of three variables and CCR7 in gated CD8⁺CD45RO^- cells (i), gated CD8⁺CD45RO⁺ cells (ii) and CD8⁺CD45RO⁺⁺ cells (iii). Tac: tacrolimus.

Fig. 6

Flow cytometric assay of the changes in CD8⁺ T cell subsets after living donor liver transplantation (LDLT) in a type III recipient. In flow cytometry, using peripheral blood mononuclear cells (PBMCs), the lymphocytes were stained with monoclonal antibodies to CD45RO and CCR7, as shown in Fig. 3a. Cells in six segments are presented as ratio (%). Subsets, % proportion per CD8⁺ T cells (i) and % difference (ii); B4: bacterial infection on postoperative day (POD) 4 (Pseudomonas aeruginosa, catheter); C13: cytomegalovirus on POD 13, C17 and C20; Tac: tacrolinus; and Pre. injury: preservation injury. In (b), the dot plots show the IL-12Rβ1-expressing cells superimposed on double-staining for IL-12Rβ1 and CCR7 in gated CD8⁺CD45RO^- cells (iii), gated CD8⁺CD45RO⁺ cells (iv) and CD8⁺CD45RO⁺⁺ cells (v). The % proportion (vi) and % difference (vii, viii) of IL-12Rβ1⁺ cells; CNS: CCR7-negative subsets; CPS: CCR7-positive subsets.