*Purpose: Alemtuzumab induction leads to profound T cell depletion followed by a prolonged period of repopulation. Repopulation is thought to be influenced both by thymic output and homeostatic expansion of mature peripheral cells, though the relative contribution of each has not been well studied.

*Methods: We have longitudinally investigated the kinetics of repopulating T cells in 40 patients (median 47 years, range 20-69) following alemtuzumab induction over 36 months posttransplantation. Maintenance immunosuppression was belatacept-based. In vitro, we studied purified CD57⁺ and CD57^– cells with or without IL-7 or autologous mature dendritic cells (mDCs).

*Results: Substantial homeostatic T cell proliferation was seen in all patients and was characterized by increased intracellular Ki67 expression (p<0.05) that retuned to baseline over a period on 6-18 months. This was not observed in a control cohort of non-depleted patients. Following repopulation, alemtuzumab-treated patients were enriched significantly for naïve T cells (T_N, CCR7⁺, CD45RA⁺, CD57^–PD1^–, CD28⁺) when compared with baseline (p<0.05). Use of a generalized estimating equation (GEE) linear model revealed a strong negative linear association between frequencies of reconstituting CD4⁺ and CD8⁺ T_N cells and advancing patient age (Bonferroni-adjusted p<0.0036) that was evident within 18 months post-depletion. An direct relationship between age and persistence of CD4⁺ and CD8⁺ effector memory T cells (T_EM) including CD57⁺ CD57⁺ memory subtypes was also shown (p<0.0036). To assess the role of thymic output in T cell expansion, we analyzed 28/40 patients for repopulating CD31⁺CD4⁺ cells within 12 months posttransplantation, and found significant increase of frequency for CD4⁺CD31⁺ cells when compared with baseline (p<0.002 at 6 months, p<0.001 at 12 months). Patients under 30 years old showed significantly more CD4⁺CD31⁺ cells (p<0.0009), while the frequency of CD4⁺CD31⁺ cells in patients over 55 years old was similar to baseline (p<0.6), and was significantly lower than young patients (p<0.0087) before and after depletional induction. CD4⁺CD57^– cells proliferated in the presence of IL-7 (78.7±15.4%) or mDCs (71.3±28.1%). In contrast, CD4⁺CD57⁺ cells did not proliferate in the presence of IL-7 (8.2±5.3%), but autologous mDCs induced CD57⁺ cell proliferation (37.6±5.6%).

*Conclusions: In summary, our data establish insight into the importance of age-related thymic output and hoemostatic expansion of T cells after alemtuzumab induction. We also find a subordinate role for autologous mDCs for expansion of residual memory T cells.

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