Reversion mutations in patients with leukocyte adhesion deficiency type-1 (LAD-1)

Abstract

Leukocyte adhesion deficiency type-1 (LAD-1) is an autosomal recessive immunodeficiency caused by mutations in the beta2 integrin, CD18, that impair CD11/CD18 heterodimer surface expression and/or function. Absence of functional CD11/CD18 integrins on leukocytes, particularly neutrophils, leads to their incapacity to adhere to the endothelium and migrate to sites of infection. We studied 3 LAD-1 patients with markedly diminished neutrophil CD18 expression, each of whom had a small population of lymphocytes with normal CD18 expression (CD18(+)). These CD18(+) lymphocytes were predominantly cytotoxic T cells, with a memory/effector phenotype. Microsatellite analyses proved patient origin of these cells. Sequencing of T-cell subsets showed that in each patient one CD18 allele had undergone further mutation. Interestingly, all 3 patients were young adults with inflammatory bowel disease. Somatic reversions of inherited mutations in primary T-cell immunodeficiencies are typically associated with milder clinical phenotypes. We hypothesize that these somatic revertant CD18(+) cytotoxic T lymphocytes (CTLs) may have altered immune regulation. The discovery of 3 cases of reversion mutations in LAD-1 at one center suggests that this may be a relatively common event in this rare disease.

Figure 1

Peripheral blood phenotyping. (A) Flow cytometric detection of CD18 expression of peripheral blood neutrophils and lymphocytes in 3 LAD-1 patients and a healthy control. CD18 expression is shown as histograms. For PMNs, anti-CD18 antibody binding is expressed as relative fluorescence intensity (RFI), defined as geometric mean channel (GMC) fluorescence of CD18 FITC divided by GMC fluorescence of isotype control. For lymphocytes, percentage of lymphocytes staining positive for CD18 is shown. All 3 patients have a lymphocyte population distinctly positive for CD18 compared with the rest of the lymphocyes. The solid peaks represent CD18, while dashed lines represent the isotype controls. (B) Four-color flow cytometry analysis of PBMCs from 3 patients with LAD-1. Backgating and multigating techniques were used to assess coexpression of multiple cell surface markers on CD18⁺ lymphocytes. CD18 is expressed on the x-axis of all dot plots, while the indicated surface antibody is shown on the y-axis. CD18 was coexpressed with CD3 and CD8 but not with CD4 or CD20. CD18⁺ T cells did not stain with CD27 or CD28 (data not shown). TCR staining revealed more than 95% TCRαβ⁺ in the CD3⁺/CD18⁺ population. Patients 1 and 2 had small populations of CD18⁺ cells carrying NK (CD3⁻/CD56⁺/16⁺) cell surface markers (1% and 5% of all CD18⁺ cells, respectively).

Figure 2

Mutation analysis. (A) Mutations in ITGB2 patients with LAD-1. The 4 mutations in these 3 patients all affect the highly conserved I/A domain of the protein. The numbering of the mutated residues is calculated from the beginning of the signal sequence. (B) Comparison of sequences. CD3⁺/CD18⁺ and CD3⁺/CD18⁻ cells from 3 LAD-1 patients were sorted and genomic DNA was extracted. Mutation sites and surrounding sequence were PCR amplified and subcloned. Clones were sequenced and compared for each patient. All of the 20 CD18⁺ clones screened for patient 3 had wt intronic sequence. The mutation A>G at nt 1052 was maintained (not shown).

Figure 3

Flow cytometric evaluation of 24 TCRVβ subfamilies in revertant and mutant populations. TCRVβ expression of CD18⁺ or CD18⁻ CD8⁺ T lymphocytes for each patient was evaluated by flow cytometry, analyzed using Cellquest software and summarized using bar graphs. Gating is based on CD8⁺/CD18⁺ or CD8⁺/CD18⁻ lymphocytes. CD18⁺ revertant T cells are represented by black bars; CD18⁻ T cells are represented by gray bars. TCRVβ subset was considered not expressed if it was detected in less than 0.1% of the gated population. For all 3 patients, revertant cells expressed fewer TCR Vβ subsets compared with CD18⁻ T cells. Skewing of TCVβ subfamilies is most prominent in patient 3.

Figure 4

Transfection of wild-type and mutant CD11/CD18 dimers into COS-7 cells. Wild-type CD18 cDNA or the mutants G284S, G284R, Y131S, and Y131F were transfected into COS-7 cells together with the wild-type cDNA for CD11a, CD11b, or CD11c. CD11/CD18 expression was monitored by flow cytometry using the monoclonal antibody 1B4. The background histograms (broken lines) were obtained using an irrelevant isotype-matched antibody. Expression profiles of CD11/CD18 transfectants are shown as solid lines. The subtracted profiles (using Cellquest software) are shown in solid black. The percentage of gated positive cells (%GP), mean fluorescence intensity (MFI), and expression index (EI), derived from %GP × MFI are also shown.

Figure 5

Adhesion of LFA-1 transfectants. Adhesion of LFA-1 (CD11a/CD18) transfectants to ICAM-1 (A), ICAM-2 (B), and ICAM-3 (C). Adhesion was done in the absence of activating agents, in the presence of 1.5 mM EGTA and 5 mM MgCl₂ (Mg/EGTA), or with the activating monoclonal antibody KIM185 (2.5 μg/mL) or with both Mg/EGTA and KIM185 (for ICAM-2 and ICAM-3 only). CD18-independent adhesion (background) was determined by inhibition with monoclonal antibody 1B4 (10 μg/mL) and has been subtracted.

Figure 6

Adhesion of transfectants. Adhesion of Mac-1/(CD11b/CD18) (A) and p150,95/(CD11c/CD18) (B) transfectants to iC3b. Adhesion was done in the absence of activating agents, in the presence of 0.5 mM MnCl₂, or with the activating monoclonal antibody KIM185 (2.5 μg/mL).