Immune reconstitution following rabbit antithymocyte globulin

Abstract

Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T-cell subsets and subsequent T-cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T-cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ naïve and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27- CD4+ effector memory or CD45RA+CD31-, CD45RO+CD27+ and CD45RO+CD27- CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution. We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells. We found that in adults, administration of rATG-induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome.

© 2010 The Authors Journal compilation © 2010 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1. Pattern of CD4+ T cell reconstitution in adult transplant recipients following lymphocyte depletion with rATG

(A) Absolute CD4+ T cell number over time following rATG [correction made after publication 20 October 2010: CD4+ added after Absolute]. (B) the percentage CD4+ RTE (CD27+CD31+CD45RA+), näive peripheral (CD27+CD31 –CD45RA+), memory effector (CD45RO+CD27–) and central memory (CD45RO+CD27–) T cells in adult CD27 renal transplant recipients that received rATG (n = 40) was determined over the first 6 months post-transplantation = by flow cytometry. Data is expressed as median with IQR. [Correction added after publication 20 October 2010: Figure 2 became Figure 1; Figure 3 became Figure 2 and cm3 was changed to mm3 in Figure 1A]

Figure 2. Pattern of CD8+ T cell reconstitution in adult transplant recipients following lymphocyte depletion with rATG

(A) The absolute number of CD8+ T cells over time following rATG. (B) the percentage CD8+ RTE (CD27+CD31+CD45RA+), näive peripheral (CD27+CD31 –CD45RA+), (CD45RO+ CD27–) effector memory and central memory (CD45RO+ CD27–) cells in adult renal transplant recipients that received rATG (n = 40) was determined over the first 6 months post-transplantation = by flow cytometry. Data is expressed as median with IQR. [Correction added after publication 20 October 2010: Figure 3 became Figure 2; Figure 1 became Figure 3 and cm3 was changed to mm3 in Figure 2A]

Figure 3. Percentage recent thymic emigrants at baseline correlates with age

Percentage (A) CD4+ ((n = 66) and (B) CD8 = 63) CD45RA+CD31+ recent thymic emigrant correlation with age. CD4+ and CD8+ RTE T cells correlated negatively with age. [Correction added after publication 20 October 2010: Figure 1 became Figure 3; Figure 2 became Figure 1]

Figure 4. T-cell phenotypes over time in adult renal transplant recipients that did not receive depletional induction therapy

(A) The percentage CD4+ and (B) CD8+ RTE, na peripheral, EM and CM T cells in adult näive renal transplant recipients (n = 34) that did not received rATG. Data are expressed as median with IQR.

Figure 5. Pattern of immune reconstitution in pediatric transplant recipients following lymphocyte depletion with rATG

(A) The percentage CD4+ and (B) CD8+ RTE (CD27+CD31+CD45RA+), näive peripheral (CD27+CD31–CD45RA+), EM (CD45RO and (CD45RO+CD27+CD27–) T cells in pediatric renal transplant-recipients (n = 17) that received rATG. Data are expressed as median with IQR.

Figure 6. CD4+ and CD8+ Treg increase following administration of rATG in vivo

(A) CD4+ and (B) CD8+ FoxP3 T cells were quantitated by flow cytometry in adult renal transplant patients and compared between patients (n = 28) that did and did not receive=rATG (n = 23). Data are expressed as median with IQR.

Figure 7. CD4+ CD45RA- FoxP3 high Treg are increased post-transplant in patients that received rATG

PBMCs from patients treated with and without rATG were stained for CD4+, CD45RA+ and FoxP3. Data are expressed as median with IQR.

Figure 8. CD4+ Treg phenotypes over time

(A) CD45RA+CD31 –FoxP3+ näive thymic emigrant, (B) CD45RA+CD31 –FoxP3+ näive peripheral/, (C) CD45RA– CD45RO+FoxP3 memory Treg were quantitated by flow cytometry in adult transplant recipients that had (n = 25) and had not (n = 20) received rATG over 6 months posttransplant. Data are expressed as median with IQR.

Figure 9. CD8+ Treg phenotypes over time

(A) CD45RA+CD31 –FoxP3+ näive thymic emigrant, (B) CD45RA+CD31 –FoxP3+ näive peripheral, and (C) CD45RA+CD45RO –FoxP3+ memory Treg were quantified by flow cytometry in adult transplant recipients that had (n = 25) and had not (n = 20) received rATG over 6 months post-transplant. Data are expressed as median with IQR.

Figure 10. Increased regulatory T cells in pediatric transplant recipients compared to healthy pediatric controls

(A) The percentage CD4+ and (B) CD8+ (FoxP3) regulatory T cells was determined in pediatric renal transplant (Peds Tx) (n = 17) patients at 6 months and compared to healthy pediatric controls (Peds HC) (data presented as median with IQR).