Reduced thymic output, cell cycle abnormalities, and increased apoptosis of T lymphocytes in patients with cartilage-hair hypoplasia

Abstract

Background: Cartilage-hair hypoplasia (CHH) is characterized by metaphyseal dysplasia, bone marrow failure, increased risk of malignancies, and a variable degree of immunodeficiency. CHH is caused by mutations in the RNA component of the mitochondrial RNA processing (RMRP) endoribonuclease gene, which is involved in ribosomal assembly, telomere function, and cell cycle control.

Objectives: We aimed to define thymic output and characterize immune function in a cohort of patients with molecularly defined CHH with and without associated clinical immunodeficiency.

Methods: We studied the distribution of B and T lymphocytes (including recent thymic emigrants), in vitro lymphocyte proliferation, cell cycle, and apoptosis in 18 patients with CHH compared with controls.

Results: Patients with CHH have a markedly reduced number of recent thymic emigrants, and their peripheral T cells show defects in cell cycle control and display increased apoptosis, resulting in poor proliferation on activation.

Conclusion: These data confirm that RMRP mutations result in significant defects of cell-mediated immunity and provide a link between the cellular phenotype and the immunodeficiency in CHH.

FIG 1

Lymphocyte subpopulations. In each panel, the shaded areas correspond to the value range of healthy control children from the various age groups.

FIG 2

RTEs. The absolute (A) and relative (B) number of RTEs (CD4⁺CD45RA⁺CD31⁺) in patients with CHH was plotted against the value range of healthy pediatric controls for various age groups.

FIG 3

T-cell proliferation in response to PHA. Values are expressed as SI. The dashed line at SI = 60 represents the lower range of PHA response in healthy individuals.

FIG 4

T-cell proliferation in response to agonistic anti-CD3 antibody. Left, Representative examples of proliferation of control-derived or patient-derived PBMCs in response to anti-CD3 (top) or anti-CD3 + IL-2 (bottom), as measured by CFSE dilution of CD3⁺ cells. Boxes identify different rounds of proliferation, and the proportion of cells within each box is annotated. Gating was done on viable cells. Right, Proportion of PBMCs undergoing 0 to 1 or >2 rounds of cell division in response to anti-CD3 (top) or anti-CD3 + IL-2 (bottom). Individual symbols identify single subjects. In each diagram, the horizontal bar represents the mean value. *P < .05.

FIG 5

Cell cycle analysis. Left, Representative examples of cell cycle analysis in control-derived or patient-derived PBMCs on in vitro stimulation with anti-CD3 (top) or anti-CD3 + IL-2 (bottom). Boxes identify cells in G0/G1 (bromodeoxyuridine [BrdU]⁻7-AAD⁻), S (BrdU⁺ 7-AAD^−/+), and G2/M (BrdU⁺ 7-AAD⁺) phases of the cell cycle. The proportion of cells within each of these is annotated. Right, Proportion of PBMCs in G0-G1, S, and G2/M phases on in vitro culture with anti-CD3 (top) or anti-CD3 + IL-2 (bottom). Individual symbols identify single subjects. In each diagram, the horizontal bar represents the mean value. *P < .05.

FIG 6

Analysis of apoptosis and cell death. Left, Representative example of fluorescence-activated cell sorting plots after staining of PBMCs for AnnV and 7-AAD in healthy controls and patients with CHH on in vitro stimulation with anti-CD3 (top) or anti-CD3 + IL-2 (bottom). Right, Proportion of early apoptotic (AnnV⁺7-AAD⁻) and late apoptotic/necrotic (AnnV⁺7-AAD⁺) PBMCs on in vitro culture with anti-CD3 (top) or anti-CD3 + IL-2 (bottom). Individual symbols identify single subjects. In each diagram, the horizontal bar represents the mean value. *P < .05.

FIG 7

Correction of T-cell proliferation defect after HCT in a patient with CHH. The proportion of T lymphocytes undergoing 0, 1, 2 or >2 rounds of proliferation (as indicated by CFSE dilution) after 72 hours of in vitro culture with anti-CD3 + IL-2 is shown for patient 17 before (black bars) or 11 months after (white bars) HCT from his HLA phenotypically identical father. Tx, Transplantation.