Reduced proportions of natural killer T cells are present in the relatives of lupus patients and are associated with autoimmunity

Abstract

Introduction: Systemic lupus erythematosus is a genetically complex disease. Currently, the precise allelic polymorphisms associated with this condition remain largely unidentified. In part this reflects the fact that multiple genes, each having a relatively minor effect, act in concert to produce disease. Given this complexity, analysis of subclinical phenotypes may aid in the identification of susceptibility alleles. Here, we used flow cytometry to investigate whether some of the immune abnormalities that are seen in the peripheral blood lymphocyte population of lupus patients are seen in their first-degree relatives.

Methods: Peripheral blood mononuclear cells were isolated from the subjects, stained with fluorochrome-conjugated monoclonal antibodies to identify various cellular subsets, and analyzed by flow cytometry.

Results: We found reduced proportions of natural killer (NK)T cells among 367 first-degree relatives of lupus patients as compared with 102 control individuals. There were also slightly increased proportions of memory B and T cells, suggesting increased chronic low-grade activation of the immune system in first-degree relatives. However, only the deficiency of NKT cells was associated with a positive anti-nuclear antibody test and clinical autoimmune disease in family members. There was a significant association between mean parental, sibling, and proband values for the proportion of NKT cells, suggesting that this is a heritable trait.

Conclusions: The findings suggest that analysis of cellular phenotypes may enhance the ability to detect subclinical lupus and that genetically determined altered immunoregulation by NKT cells predisposes first-degree relatives of lupus patients to the development of autoimmunity.

Figure 1

Flow cytometry profiles showing gates used to identify various lymphocyte populations. Peripheral blood mononuclear cells from representative control individuals and lupus patients were stained with combinations of conjugated mAbs, fixed, and analyzed by flow cytometry, gating on the lymphoid population as determined by forward and side staining characteristics. (a) Cells were stained with a combination of anti-CD20, anti-CD38, and anti-CD27 mAbs to distinguish peripheral blood B-cell subsets. Shown are dot plots, gated on CD20⁺ cells, with four regions defined by the levels of staining with anti-CD27 and anti-CD38, as determined by staining with a relevant isotype control. Using this combination of stains, B cells can be divided into naïve transitional (CD27^-CD38⁺⁺) naïve mature (CD27^-CD38^-/+), memory (CD27⁺CD38^-/+), and pre-germinal center (CD27⁺CD38⁺⁺) populations. (b) Cells were stained with anti-CD3 in combination with anti-Vα24 and anti-Vβ11 mAbs. Shown are dot plots gated on the CD3⁺ population. The top right quadrant represents the Vα24⁺Vβ11⁺ invariant NKT cell population that has been proposed to play a regulatory role in autoimmunity. (c) Cells were stained with anti-CD4 or anti-CD8 (shown) in combination with anti-CD45RA and anti-CD45RO to identify naïve (CD45RA⁺CD45RO^-; bottom right) and memory (CD45RA^-CD45RO⁺; top left) cell subsets. mAb, monoclonal antibody; NK, natural killer; SLE, systemic lupus erythematosus.

Figure 2

Scatter plots for cell populations that demonstrated significant differences between first-degree relatives and control individuals. Peripheral blood mononuclear cells were stained with various combinations of conjugated mAbs, fixed, and analyzed by flow cytometry (as outlined in the Materials and methods section and shown in Figure 1). (a) Shown are plots for the proportion of activated naïve B cells (CD20⁺CD27^- cells that were CD86⁺), the proportion of B cells (CD20⁺) that had a mature naïve phenotype (CD27^-CD38^-/+), the proportion of NKT cells (CD3⁺ cells that were Vα24⁺Vβ11⁺), and the proportion of memory CD4⁺ T cells (CD45RA^-CD45RO⁺). Results shown are for 144 (143 for NKT cells) lupus probands, 356 family (parents and siblings) members (355 for NKT cells), 287 parents (286 for NKT cells), 69 siblings, and 102 control individuals. (b) The proportion of NKT cells in controls, probands, and family members, stratified for the presence or absence of positive ANA status. Significant differences (*P < 0.05, **P < 0.005, and ***P < 0.0005) were determined using the Wilcoxon test. In panel a differences are as compared with control individuals, and in panel b comparisons are between indicated populations. mAb, monoclonal antibody; NK, natural killer.