Long-term effects of alemtuzumab on regulatory and memory T-cell subsets in kidney transplantation

Abstract

Background: Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes.

Methods: We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied.

Results: We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells.

Conclusions: Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

Figure 1. Identification and quantitation of peripheral blood T cell subsets

Proportion (%) and absolute numbers of CD3⁺ (A), CD3⁺CD4⁺ (B), and CD3⁺CD8⁺ (C) T cells in each study group. (D) Gating strategy for identifying T_N (CD45RO⁻CD62L⁺), T_CM (CD45RO⁺CD62L⁺), T_EM (CD45RO⁺CD62L⁻), and T_EMRA (CD45RO⁻CD62L⁻) CD4 and CD8 T cells. (E–F) Proportion (%) of CD4⁺ T_N, T_EM, and CD8⁺ T_N, T_EMRA cells for each study group. Each symbol represents a single subject, while the horizontal line reprsents the mean value. *p<0.05; ** p<0.01; *** p<0.005.

Figure 2. Identification and quantitation of peripheral blood CD4 T_REG and T_EFF cells

(A) Gating strategy and representative flow cytometry of T_REG (CD3⁺CD4⁺CD25^highFOXP3⁺CD127^low) and T_EFF (CD3⁺CD4⁺CD25^highFOXP3⁻CD127^high) in the CD3⁺CD4⁺CD25^high T cell gate in each of the study groups. (B) Proportion (%) of T_REG and of T_EFF within the CD3⁺CD4⁺CD25^high T cell gate. (C) CD4 T_EM/T_REG and CD8 T_EMRA/T_REG ratios in each of the study groups. Each symbol represents a single individual, and the horizontal line represents the mean value. *p<0.05; ** p<0.01; *** p<0.005.

Figure 3. T cell proliferation and IFNγ and peforin/granzyme B expression in one-way CFSE-MLR

Analyses were performed after excluding PKH-26⁺ stimulator cells and gating on CD3⁺CD8⁻ (CD4⁺) and CD3⁺CD8⁺ T cells at the end of the 5-day MLR. (A) Proliferation of responder CD4⁺ and CD8⁺ T cells, presented as proportion (%) of cells that diluted CFSE, after stimulation with allogeneic donor (R+D) or third party PBMC (R+3^rd). (B) IFNγ production by proliferated CD4⁺ and CD8⁺ T cells 4 hrs after re-stimulation with donor or third party CD3-depleted allogeneic PBMC. (C) Perforin/granzyme B expression by proliferated responder CD4⁺ and CD8⁺ T cells. HC (n=14), quiescent (n=12), reactive (n=4), and ACR (n=2). *p<0.05; ** p<0.01; ***p<0.005.

Figure 4. Identification of T cell subsets in renal allograft tissue of a patient undergoing ACR

(A) H&E staining shows irregularly shaped tubules and mononuclear cell infiltrates (tubulitis) (yellow arrows). (B and C) Multiplex quantum dot staining of CD45RO (green), CD62L (red), CD8 (cyan), and CD3 (yellow) in the same tissue section as A. Panel B depicts merged image, red arrows indicate infiltrating cells which are mostly CD8⁺CD3⁺CD45RO⁺CD62L⁻ T cells (T_E or T_EM). Panel C depicts cropped images from Panel B. White arrow heads indicate tubules. Purple arrows identify CD3⁺CD8⁻CD45RO⁻CD62L⁻ (T_EMRA) while white arrows identify CD3⁺CD8⁺CD45RO⁺CD62L⁻ (T_EM) infiltrating T cells.