Age-related effects on thymic output and homeostatic T cell expansion following depletional induction in renal transplant recipients

Abstract

Thymic output and homeostatic mature cell proliferation both influence T cell repopulation following depletional induction, though the relative contribution of each and their association with recipient age have not been well studied. We investigated the repopulating T cell kinetics in kidney transplant recipients who underwent alemtuzumab induction followed by belatacept/rapamycin-based immunosuppression over 36-month posttransplantation. We focused specifically on the correlation between repopulating T cell subsets and the age of patients. Substantial homeostatic Ki67-expressing T cell proliferation was seen posttransplantation. A repertoire enriched for naïve T (TNaïve ) cells emerged posttransplantation. Analysis by generalized estimating equation linear models revealed a strong negative linear association between reconstituting TNaïve cells and advancing age. A relationship between age and persistence of effector memory cells was shown. We assessed thymic output and found an increase in the frequency of recent thymic emigrants (RTEs, CD4+ CD31+ ) at 12-month posttransplantation. Patients under 30 years of age showed significantly higher levels of CD4+ CD31+ cells than patients over 55 years of age pre- and posttransplantation. IL-7 and autologous mature dendritic cells (mDCs) induced CD57- cell proliferation. In contrast, mDCs, but not IL-7, induced CD57+ cell proliferation. This study establishes the relationship between age and thymic output during T cell homeostatic repopulation after alemtuzumab induction. Trial Registration: ClinicalTrials.gov - NCT00565773.

Keywords: basic (laboratory) research/science; clinical research/practice; immunobiology; immunosuppressant - fusion proteins and monoclonal antibodies: alemtuzumab; immunosuppressant - fusion proteins and monoclonal antibodies: belatacept; immunosuppression/immune modulation; immunosuppressive regimens - induction; kidney transplantation/nephrology; lymphocyte biology: differentiation/maturation; lymphocyte biology: proliferation.

Conflict of interest statement

Disclosure: The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1.. Increasing homeostatic proliferation post-alemtuzumab induction over the course of renal allograft transplantation.

CD3+ T cells are segregated by CD4+ (left) and CD8+ (right) cells. T cell homeostatic proliferation is measured by intracellular staining for Ki67 expression. Patients demonstrate significant increasing homeostatic proliferation within 18–24 months posttransplantation. The box borders indicate the 75th and 25th percentiles, and the line within the box indicates the median. The upper and lower whiskers represent the 90th and 10th percentiles. The dots represent outliers. (* p≤0.01, ** p≤0.001, *** p≤0.0001)

Figure 2.. Increased recent thymic emigrants during CD4+ cell reconstitution at 12 months post-alemtuzumab induction (n=28).

Shown are the percentage (a,b) and absolute numbers (c), of recent thymic emigrants, defined as CD31+ cells gating on CD3+CD4+ cells. Patients show a significant increase in the frequency of CD4+CD31+ cells following alemtuzumab induction, and the recent thymic emigrants are phenotypically CD45RA+CCR7+ naïve cells (a). In contrast, CD4+CD31- cells, phenotypically defined as CD45RA- CCR7- memory cells, decrease significantly post-alemtuzumab induction (b). Repopulating CD4+CD31+ naïve cells were still significantly below baseline levels (p=0.0493). In contrast, the repopulation of effector memory cells in CD31+ or CD31- populations was dramatically suppressed posttransplantation (c). The box borders indicate the 75th and 25th percentiles, and the line within the box indicates the median. The upper and lower whiskers represent the 90th and 10th percentiles. The dots represent outliers.

Figure 2.. Increased recent thymic emigrants during CD4+ cell reconstitution at 12 months post-alemtuzumab induction (n=28).

Shown are the percentage (a,b) and absolute numbers (c), of recent thymic emigrants, defined as CD31+ cells gating on CD3+CD4+ cells. Patients show a significant increase in the frequency of CD4+CD31+ cells following alemtuzumab induction, and the recent thymic emigrants are phenotypically CD45RA+CCR7+ naïve cells (a). In contrast, CD4+CD31- cells, phenotypically defined as CD45RA- CCR7- memory cells, decrease significantly post-alemtuzumab induction (b). Repopulating CD4+CD31+ naïve cells were still significantly below baseline levels (p=0.0493). In contrast, the repopulation of effector memory cells in CD31+ or CD31- populations was dramatically suppressed posttransplantation (c). The box borders indicate the 75th and 25th percentiles, and the line within the box indicates the median. The upper and lower whiskers represent the 90th and 10th percentiles. The dots represent outliers.

Figure 3.. Functional form of age in relation to the frequency of repopulating naïve T cells.

(a) The association between a patient’s age and reconstituting CD4+ (left) and CD8+ naïve cells (right). Age is coded using restricted cubic splines with three knots at the 5th, 50th, and 95th percentiles of age. The Y-axis represents the difference in the naive cell frequency between individuals of any age and individuals 47 years of age. The dashed lines indicate 95% confidence intervals. The knots are presented by dots. The frequencies of naïve cells are significantly different between the ages, and the association between naïve cell frequency and age is linear (p=0.0034 for CD4+ and p=0.0001 for CD8+). (b) The dynamics of repopulating naïve B cells post-alemtuzumab induction (left). The frequency of naïve cells changes significantly over time posttransplantation. The box borders indicate the 75th and 25th percentiles, and the line within the box indicates the median. The association between age and the frequency of naïve B cells (right). No association between age and the frequency of naïve cells was found during repopulation posttransplantation (p=0.506). The dashed lines indicate 95% confidence intervals. The dots represent the knots of restricted cubic splines at the 5th, 50th, and 95th percentiles of age. (**** p≤0.00001)

Figure 4.. Dynamics and phenotype of repopulating CD31+CD4+ cells post-alemtuzumab induction in three age groups.

(a) Patients ≤ 30 years of age show higher frequencies of CD31+ cells than patients ≥ 55 years of age. In the ≤ 30 year and 31–54 year age groups, a significant increase in the frequency of CD4+CD31+ cells over time is observed when compared with patients ≥ 55 years of age posttransplantation (left). CD4+CD31+ cells prior to depletional induction contain large fractions of TNaïve cells in the ≤ 30 and 31–54 year age groups but not in the ≥ 55 year age group (right). The frequency of TNaïve cells remained unchanged following the repopulation of the CD4+CD31+ subset following depletional induction. (b) The absolute CD4+CD31+ cells and naïve cells (CD45RA+CCR7+) in CD4+CD31+ population repopulated in patients ≤ 30 but not ≥ 55 years of age. The circle represents each patient. (* p≤0.01, ** p≤0.001, *** p≤0.0001)

Figure 5.. Dynamics of repopulating naïve and effector memory cells post-alemtuzumab induction in three age groups.

(a) The effect of age on the absolute counts and frequencies of CD4+ and CD8+ naïve cells is significant, and the effect of age differs at different time points posttransplantation. (b) The effect of age on the absolute CD4+ TEM cell counts is not significant. The effect of age on the frequency of CD4+ TEM cells is significant, and the effect of age differs at different time points. The effect of age on the frequency of CD8+ TEM cells is significant, and the effect of age differs at different time points posttransplantation. The data are expressed as mean ± SD (standard deviation).

Figure 5.. Dynamics of repopulating naïve and effector memory cells post-alemtuzumab induction in three age groups.

(a) The effect of age on the absolute counts and frequencies of CD4+ and CD8+ naïve cells is significant, and the effect of age differs at different time points posttransplantation. (b) The effect of age on the absolute CD4+ TEM cell counts is not significant. The effect of age on the frequency of CD4+ TEM cells is significant, and the effect of age differs at different time points. The effect of age on the frequency of CD8+ TEM cells is significant, and the effect of age differs at different time points posttransplantation. The data are expressed as mean ± SD (standard deviation).

Figure 6.. Dynamics of repopulating CD57- and CD57+cells following depletional induction in three age groups.

(a) The frequency of repopulating CD57- cells is significantly associated with age (p≤0.0004), and younger age shows more CD57- cells at all time points. Similarly, the effect of age on the absolute count of the CD4+CD57- subset is also significant (p≤0.0104). In contrast, the association between age and the absolute counts of the CD4+CD57+ subset is not significant due to prolonged suppression of cell repopulation within first 36 months posttransplantation. However, the effects of age on the frequency of CD4+CD57+ cells is significant (p≤0.0002), and older age shows a higher frequency at all time points when compared with the other two age groups. (b) The absolute count of the CD8+CD57- subset is significantly associated with advancing age (p≤0.0033), and younger age shows higher absolute counts of the CD8+CD57- subset. The effects of age on the frequency of CD8+CD57- cells is significant (p≤0.0004), and younger age shows a higher CD8+CD57- cell frequency. However, the interaction between age and time is not significant. The absolute counts of repopulating CD8+CD57+ cells is not significantly correlated with age and time. The effect of age on the frequency of repopulating CD8+CD57+ cells is significant (p≤0.0001), and older age shows a higher CD57+ cell frequency pre- and posttransplantation. However, the interaction between age and time is not significant. The data are expressed as mean ± SD (standard deviation).

Figure 6.. Dynamics of repopulating CD57- and CD57+cells following depletional induction in three age groups.

(a) The frequency of repopulating CD57- cells is significantly associated with age (p≤0.0004), and younger age shows more CD57- cells at all time points. Similarly, the effect of age on the absolute count of the CD4+CD57- subset is also significant (p≤0.0104). In contrast, the association between age and the absolute counts of the CD4+CD57+ subset is not significant due to prolonged suppression of cell repopulation within first 36 months posttransplantation. However, the effects of age on the frequency of CD4+CD57+ cells is significant (p≤0.0002), and older age shows a higher frequency at all time points when compared with the other two age groups. (b) The absolute count of the CD8+CD57- subset is significantly associated with advancing age (p≤0.0033), and younger age shows higher absolute counts of the CD8+CD57- subset. The effects of age on the frequency of CD8+CD57- cells is significant (p≤0.0004), and younger age shows a higher CD8+CD57- cell frequency. However, the interaction between age and time is not significant. The absolute counts of repopulating CD8+CD57+ cells is not significantly correlated with age and time. The effect of age on the frequency of repopulating CD8+CD57+ cells is significant (p≤0.0001), and older age shows a higher CD57+ cell frequency pre- and posttransplantation. However, the interaction between age and time is not significant. The data are expressed as mean ± SD (standard deviation).

Figure 7.. Proliferation of purified CD57+ and CD57- cells in response to autologous mature dendritic cells.

(a) CD57+ and CD57- cells are sorted from normal human PBMCs after labeling with anti-CD57 FITC antibody. Cells are labeled with proliferation dye VPD-450 and incubated with IL-7 or autologous dendritic cells (mDCs). Unstimulated cells are used as negative controls. The proliferation of cells is analyzed by flow cytometry. Unlike unstimulated cells, CD57- cells demonstrate proliferation in the presence of Il-7 or mDCs, respectively. In contrast, CD57+ cells demonstrate barely detectable proliferation in the presence of IL-7. However, autologous mDCs effectively induce CD57+ cell proliferation. (* p≤0.01, ** p≤0.001). (b) Proliferation of purified CD57- cells following coincubation with autologous mDCs is inhibited by belatacept (100 μg/mL). In contrast, limited inhibitory effects of belatacept on CD57+ cell proliferation are observed. The data are expressed as mean ± SD (standard deviation).