Rapid T cell repopulation after rabbit anti-thymocyte globulin (rATG) treatment is driven mainly by cytomegalovirus

Abstract

Rabbit anti-thymocyte globulin (rATG) induces a long-lasting lymphocytopenia. CD4(+) T cells remain depleted for up to 2 years, whereas the CD8(+) T cell compartment is refilled rapidly by highly differentiated CD27(-) CD45RA(+) CD57(+) effector-type cells. Because the presence of these highly differentiated CD8(+) T cells has been associated with cytomegalovirus (CMV) infection, we questioned to what extent restoration of CMV T cell immunity contributes to the re-emergence of T cells following rATG treatment. We compared T cell repopulation in six CMV-seropositive patients with CMV reactivation (reactivating CMV(+) ) to that in three CMV(+) patients without reactivation (non-reactivating CMV(+) ), and to that in three CMV-seronegative recipients receiving a kidney from a CMV-seronegative donor (CMV(-/-) ). All patients received rATG because of acute allograft rejection. Total CD4 and CD8 counts, frequency and phenotype of virus-specific CD8(+) T cells were determined. In reactivating CMV(+) patients, total CD8(+) T cells reappeared rapidly, whereas in non-reactivating CMV(+) patients they lagged behind. In CMV(-/-) patients, CD8(+) T cell counts had not yet reached pretransplant levels after 2 years. CMV reactivation was indeed followed by a progressive accumulation of CMV-specific CD8(+) T cells. During lymphocytopenia following rATG treatment, serum interleukin (IL)-7 levels were elevated. Although this was most prominent in the CMV-seronegative patients, it did not result in an advantage in T cell repopulation in these patients. Repopulated CD8(+) T cells showed increased skewing in their Vβ repertoire in both CMV(-/-) and reactivating CMV-seropositive patients. We conclude that rapid T cell repopulation following rATG treatment is driven mainly by CMV.

Fig. 1

Changes in T cell subpopulations after rabbit anti-thymocyte globulin (rATG) treatment. (a) Longitudinal and comparative analysis of the absolute amount of T cells (CD3⁺) and the absolute amount of CD4⁺ and CD8⁺ T cells. (b) Longitudinal and comparative analysis of naive (CD27⁺CD45RA⁺), memory (CD27⁺CD45RA^-), effector (CD27^-) CD4⁺ and CD8⁺ T cells; (c) CD4⁺CD28^- T cells; (d) CD31⁺ naive (CD27⁺CD45RA⁺) CD4⁺ T cells. Analysis was performed before ATG treatment (pre-ATG), 20–50, 100–200, 200–400 and 400–700 days after ATG treatment. Black dots (n = 5 at 400–700 days after rATG treatment, n = 4 at all other time-points) represent the reactivating CMV⁺patients. The grey squares represent the non-reactivating CMV⁺ patients (n = 3 at 100–200 days and 200–400 days after rATG treatment, n = 2 at all other time-points). The open triangles represent the CMV-seronegative patients (n = 3 at pre-ATG and 400–700 days after rATG treatment, n = 2 at all other time-points). Mean absolute numbers are shown; the standard deviation (s.d.) is shown when n > 3.

Fig. 2

Repopulation of cytomegalovirus (CMV)- and Epstein–Barr virus (EBV)-specific CD8⁺ T cells following rabbit anti-thymocyte globulin (rATG) treatment. (a) Longitudinal analysis of CMV-specific cells, demonstrated by tetramer staining for three reactivating CMV⁺ patients (P1, P3 and P5) and one non-reactivating CMV-seropositive or CMV⁺ patient (P7) using four different CMV tetramers (IE 1 B8 QIK, pp65 B7 TPR, pp65 A2 NLV, pp65 B35 IPS). (b) Longitudinal analysis of CMV- and (c) EBV-specific cells. Analysis was performed before ATG treatment (pre-ATG), 20–50, 100–200, 200–400 and 400–700 days after ATG treatment. Mean and standard deviation (s.d.) of absolute numbers of tetramer positive cells are shown. To study CMV-specific cells, a total of six patients were analysed using eight different CMV tetramers. The black bars represent five reactivating CMV⁺ patients (P1–5) and the grey bar represents one non-reactivating CMV⁺ patient (P7) analysed with three different tetramers. To study EBV-specific cells, five patients (four CMV⁺ patients and one CMV-seronegative patient) were analysed using six different EBV tetramers. The black bars represent four reactivating EBV-seropositive patients and the grey bar represents one non-reactivating EBV-seropositive patient. (d) Longitudinal analysis of CMV-specific, tetramer-positive CD8⁺ T cells. Black bars are the effector (CD27^-) cells and grey bars the memory (CD27⁺CD45RA^-) cells as a percentage of the CMV-specific CD8⁺ T cells; n.d.: not determined.

Fig. 3

Changes in serum interleukin (IL)-7 levels and percentages of dividing Ki67⁺ T cells after rabbit anti-thymocyte globulin (rATG) treatment. Longitudinal analysis of (a) serum IL-7 levels of reactivating cytomegalovirus (CMV⁺) patients (black line, n = 5 pre-ATG and n = 4 at all other time-points) and CMV^−/− patients (grey line n = 3 pre-ATG and n = 2 at all other time-points). Analysis was performed before ATG treatment (pre-ATG), 20–50, 100–200 and 200–400 days after the last dose administration of ATG. Longitudinal and comparative analysis of Ki67 expression on naive (CD27⁺CD45RA⁺), memory (CD27⁺CD45RA^-), effector (CD27^-) (b) CD4⁺ and (c) CD8⁺ T cells. The black lines represent the reactivating CMV⁺patients (n = 3 at 20–50, 400–700 days post-ATG treatment, n = 4 at all other time-points), the grey lines represent CMV^−/− patients (n = 3 pre-ATG, n = 2 at all other time-points). Mean absolute numbers and standard deviation (s.d.) are shown. (d) Correlation between the percentage Ki-67⁺ on total CD3⁺ cells and the amount of interleukin (IL)-7 present in the serum (R² = 0·4125, P < 0·0001). This graph represents five reactivating CMV⁺ as well as three CMV^−/− patients.

Fig. 4

Effect of transplantation and rabbit anti-thymocyte globulin (rATG) treatment on Vβ repertoire of CD4 and CD8 T cells. Longitudinal analysis of the CD4⁺ (a/b) and CD8⁺ T cell (c/d) repertoire diversity, studied in four different patients at various time-points before and after ATG treatment. Of each patient, three different representative Vβ families of CD4⁺ and CD8⁺ T cells are shown. Two of the analysed patients are reactivating CMV⁺ (P1 and P6) (a/c) and two are CMV^−/− (P10 and P11) (b/d). The black arrows indicate skewing. (e) Skewing of CD4⁺ T cells, comparing pre-ATG treatment to 1 year post-ATG treatment (post-ATG); (f) CD8⁺ T cells comparing pre-ATG to post-ATG. The grey squares represent two CMV-seronegative patients and the black dots represent two CMV-seropositive patients.