Antibody-mediated allograft rejection is associated with an increase in peripheral differentiated CD28-CD8+ T cells - Analyses of a cohort of 1032 kidney transplant recipients

Abstract

Background: CD28-CD8+ T cells represent a differentiated CD8+ T cell subset that is found to be increased in various conditions associated with chronic antigenic stimulation such as aging, chronic viral infections, autoimmune diseases, cancers, and allotransplantation.

Methods: Using multivariate models, we analyzed a large cohort of 1032 kidney transplant patients in whom 1495 kidney graft biopsies were performed concomitant with a peripheral blood leukocyte phenotyping by flow cytometry. We investigated the association between the level of CD28-CD8+ T cells in the blood and the diagnosis of graft rejection according to the recent Banff classification of renal allograft pathology.

Findings: We found that antibody-mediated rejection (ABMR) was associated with a significant increase in the percentage as well as the absolute number of CD28-CD8+ T cells in the peripheral blood of kidney transplant patients at the time of biopsy. The confounder-adjusted mean difference of log percentage and log absolute value between the ABMR group and the normal/subnormal histology group were 0.29 (p=0.0004) and 0.38 (p=0.0004), respectively. Moreover, we showed that CD28-CD8+ T cells from the patients diagnosed with ABMR responded more rigorously to TCR and FcγRIIIA (CD16) engagement compared to their CD28+ counterparts as evidenced by an increase in the expression of IFNγ, TNFα, and CD107a.

Interpretation: Collectively, our data suggest that differentiated CD28-CD8+ T cells, with increased frequency, number, and function, may participate in the pathobiology of ABMR. Further studies are warranted to clarify the immunological role of this T cell subset in kidney graft rejection.

Funding: Agence nationale de la recherche (France).

Keywords: Antibody; CD28; CD8; Kidney transplantation; Rejection; T lymphocyte.

Figure 1

Histological diagnoses of kidney graft biopsies.

Figure 2

Distribution of the natural logarithm of the percentage (A) and absolute number (B) of CD28-CD8+ T cells according to histological groups. In each boxplot, the central line indicates the median of the data set, the shaded box represents the interquartile range (IQR) which means 25 percentile to 75 percentile or quartile 1 (Q1) to quartile 3 (Q3), and the lower and upper bars indicate Q1 - 1.5 IQR and Q3 + 1.5 IQR, respectively. Biopsy group 1: normal/sub-normal, group 2: IFTA grade 2/3, group 3: ABMR, group 4: TCMR, and group 5: borderline rejection. See also Supplementary Tables 2 and 3 for the unadjusted mean difference in the log percentage and log absolute number, respectively, between biopsy groups.

Figure 3

Confounder-adjusted mean difference, 95% confidence intervals and p-value adjusted by Holm–Bonferroni method for the natural logarithm of the percentage (A) and the absolute number (B) of CD28-CD8+ T cells.

Figure 4

Functional studies of CD8+ T cells in ABMR. Frozen PBMCs (n=24) from patients diagnosed with ABMR (biopsy group 3) was retrieved from our biocollection for functional studies. (A) Flow cytometry staining for CD45RA and CCR7 divides CD8+ T cells into 4 subpopulations: naïve (CD45RA+CCR7+), central memory (CM) (CD45RA-CCR7+), effector memory (EM) (CD45RA-CCR7-), and effector memory expressing CD45RA (TEMRA) (CD45RA+CCR7-). The percentage of these 4 subpopulations in CD28+CD8+ and CD28-CD8+ T cells are shown. (B) and (C) PBMCs were stimulated with plate-bound anti-CD3 mAb, soluble anti-CD28 mAb or IL-15, and plate-bound anti-CD16 mAb as indicated for 4 h. Cells were stained for surface CD8, CD28, and CD107a, followed by intracellular staining for TNFα and IFNγ and analyzed by flow cytometry. The percentage of cells double positive for TNFα and IFNγ (left) and positive for CD107a (right) among CD28+CD8+ (open circles) and CD28-CD8+ (filled circles) T cells are shown. (D) Frozen PBMCs (n=46) were also retrieved from patients with normal/subnormal biopsies (group 1) and stimulated as in (B) and (C). The functional responses of the CD28-CD8+ T cell subpopulation from patients with ABMR and normal/subnormal biopsies were compared (see also Supplementary Figure 3). *: p<0.05; **: p<0.01; ***:p<0.001; ****: p<0.0001.