Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation

Abstract

The association between lymphopenia and autoimmunity is recognized, but the underlying mechanisms are poorly understood and have not been studied systematically in humans. People with multiple sclerosis treated with the lymphocyte-depleting monoclonal antibody alemtuzumab offer a unique opportunity to study this phenomenon; one in three people develops clinical autoimmunity, and one in three people develops asymptomatic autoantibodies after treatment. Here, we show that T-cell recovery after alemtuzumab is driven by homeostatic proliferation, leading to the generation of chronically activated (CD28(-)CD57(+)), highly proliferative (Ki67(+)), oligoclonal, memory-like CD4 and CD8 T cells (CCR7(-)CD45RA(-) or CCR7(-)CD45RA(+)) capable of producing proinflammatory cytokines. Individuals who develop autoimmunity after treatment are no more lymphopenic than their nonautoimmune counterparts, but they show reduced thymopoiesis and generate a more restricted T-cell repertoire. Taken together, these findings demonstrate that homeostatic proliferation drives lymphopenia-associated autoimmunity in humans.

Keywords: T lymphocytes; immunotherapy; reconstitution.

Fig. 1.

CD4 and CD8 reconstitution slows with repeated cycles and is dominated by effector memory populations. (A) Mean CD4 and CD8 counts ± SEM up to 18 mo after each cycle. Dotted horizontal lines represent the lower limits of normal, and dashed lines represent the fitted models. There were too few data points for the model to be reliably fitted after cycle 4. (B) Mean CD4 and CD8 subsets ± SEM from 29 healthy controls (HCs) and 87 RRMS patients pre- and postalemtuzumab. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

T cells after alemtuzumab show evidence of chronic activation and have cytotoxic potential. (A) Mean percentage of the parent population expressing a given maker ± SEM: a cross-sectional analysis of 86 patients pre- and postalemtuzumab vs. 29 HCs. HLADR, human leukocyte antigen-DR. (B) Mean percentage of cytokine positive cells ± SEM from 11 patients postalemtuzumab. ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

Mature aTregs are expanded after alemtuzumab, and long-term functional regulation is restored. (A) Percentage of CD4 cells with a CD4⁺CD25^hi Treg phenotype. (B) Percentage of CD4 with an aTreg (CD45RA⁻FoxP3hi) vs. rTreg (CD45RA⁺FoxP3lo) vs. non-Treg (CD45RA⁻FoxP3lo) phenotype. (C) Suppressive capacity of CD4⁺CD25hi Tregs from 29 patients 3–4 y postalemtuzumab compared with 12 healthy controls. All graphs show mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Thymic function is reduced after alemtuzumab. (A) Percentage and absolute number of CD4 cells with recent thymic emigrants (RTEs) phenotype (CD4⁺CD45RA⁺CCR7⁺CD31⁺). (B) Sj/βTREC ratio in a cross-sectional analysis of 12 healthy controls vs. 12 RRMS patients pre- and postalemtuzumab. All graphs show mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

T-cell clonality is reduced after alemtuzumab. (A) CDR3 spectratyping of 22 RRMS patients pre- and postalemtuzumab; mean numbers of undetectable BV families and mean complexity scores for detectable families are shown ± SEM. (B) CD4 and CD8 cells from 10 patients <12 mo of treatment and 5 patients >2 y postalemtuzumab. (C) TCR sequencing of CD4 and CD8 cells from one patient 3 mo after alemtuzumab and a second patient 12 mo posttreatment. V-J histograms are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6.

T-cell homeostatic proliferation drives autoimmunity after alemtuzumab. (A) Clonal frequency in 5 patients without autoimmunity vs. 13 patients with autoimmunity (10 thyroid and 2 autoantibodies) at 6 mo and clonal frequency in 7 nonautoimmune vs. 11 autoimmune patients (9 thyroid and 2 autoantibodies) at 12 mo postalemtuzumab. (B) CDR3 spectratyping of nine autoimmune patients (six thyroid and three autoantibodies) vs. nine nonautoimmune patients 12 mo postalemtuzumab. Mean undetectable BV subfamilies and mean complexity scores for detectable subfamilies are shown ± SEM. (C) Mean absolute and mean Sj/βTREC ratios relative to baseline ± SEM in 10 patients without autoimmunity vs. 16 patients with autoimmunity (14 thyroid and 2 autoantibodies)12 mo after alemtuzumab. *P < 0.05; **P < 0.01.