Altered frequency and phenotype of CD4+ forkhead box protein 3+ T cells and its association with autoantibody production in human immunodeficiency virus-infected paediatric patients

Abstract

The association between immune dysfunction and the development of autoimmune pathology in patients with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is not clear. The frequency and phenotype of regulatory T cells, as well as the presence of autoantibodies, were evaluated in a paediatric cohort of HIV-infected patients without clinical evidence of autoimmune disease. Lower absolute counts but higher percentages of total CD4(+) forkhead box protein 3 (FoxP3)(+) T cells were recorded in children with severe immunosuppression than in those without evidence of immunosuppression. The frequencies of classical CD4(+) CD25(+) FoxP3(+) regulatory T cells were not altered, whereas CD4(+) FoxP3(+) CD25(-) T cells were found increased significantly in patients with severe immunosuppression. Like classical regulatory T cells, CD4(+) FoxP3(+) CD25(-) T cells display higher cytotoxic T-lymphocyte antigen 4 (CTLA-4) but lower CD127 expression compared with CD4(+) FoxP3(-) CD25(+) T cells. An improvement in CD4(+) T cell counts, along with a decrease in viral load, was associated with a decrease in CD4(+) FoxP3(+) CD25(-) T cells. The majority of the patients with severe immunosuppression were positive for at least one out of seven autoantibodies tested and displayed hypergammaglobulinaemia. Conversely, HIV-infected children without evidence of immunosuppression had lower levels of autoantibodies and total immunoglobulins. A decline in CD4(+) FoxP3(+) T cell numbers or a variation in their phenotype may induce a raise in antigen exposure with polyclonal B cell activation, probably contributing to the generation of autoantibodies in the absence of clinical autoimmune disease.

Fig. 1

The frequency and phenotype of CD4⁺forkhead box P3 (FoxP3)⁺ T cells are altered in human immunodeficiency virus (HIV)-paediatric patients. Peripheral blood mononuclear cells (PBMC) were collected and stained with anti-CD4, anti-CD25 and FoxP3 monoclonal antibodies. CD4⁺ T cells were gated from a side-scatter (SSC) versus CD4 dot-plot and the expression of FoxP3 and CD25 was analysed with CellQuest Pro software. Blood cell count was recorded in all subjects. (a) Absolute numbers (shown as cells per millilitre of blood) of total CD4⁺FoxP3⁺ T cells. The horizontal lines denote the median values for each clinical group. (b) FoxP3⁺ T cell levels (expressed as percentages of CD4⁺ T cells) in the different clinical groups. Boxes represent values between the 25th and 75th percentiles and medians; bars indicate 10th and 90th percentiles. (c) Frequency of regulatory CD4⁺ T cell subsets according to CD25 expression. CD4⁺CD25^–FoxP3⁺ T cells in group A were significantly higher compared with those in group B and uninfected controls.

Fig. 2

Phenotypic analysis of forkhead box P3 (FoxP3)-expressing CD4⁺ T cells. Peripheral blood mononuclear cells (PBMC) were collected and stained with anti-CD4, anti-CD25 and anti-FoxP3 in combination with anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) (n = 14), CD69 (n = 14), CD127 (n = 6), interferon (IFN)-γ (n = 4) or human leucocyte antigen D-related (HLA-DR) (n = 6). Lymphocytes were identified by forward- and side-scatter properties. From this population, a CD4 versus FoxP3 dot-plot was set. Subsequently, CD4⁺FoxP3⁺ or CD4⁺FoxP3^- T cell populations were gated and analysed for CD25 versus CTLA-4, CD127, IFN-γ, CD69 or HLA-DR. (a) Representative histogram plot from a single human immunodeficiency virus (HIV)-infected child showing CTLA-4 (upper panel) and CD69 (bottom panel) expression on CD4⁺FoxP3^–CD25⁺, CD4⁺FoxP3⁺CD25⁺ and CD4⁺FoxP3⁺CD25^- T cells. Proportions of CD4⁺FoxP3^–CD25⁺, CD4⁺FoxP3⁺CD25⁺ and CD4⁺FoxP3⁺CD25^- T cells co-expressing CTLA-4 (b), CD127 (c), IFN-γ (d), CD69 (e) or HLA-DR (f) were measured in HIV-infected children exhibiting immunosuppression (closed circle) or without signed of immunosuppression (open circle).

Fig. 3

Relationship among total regulatory T cells (T_reg), anti-cardiolipin (ACA), rheumatoid factor (RF) and CD4 count. Correlation analysis was performed by Spearman's rank method. (a) ACA versus percentage of CD4⁺ T cells. (b) RF versus CD4⁺ T cell counts (cells/ml). (c) RF versus total CD4⁺FoxP3⁺ T cell counts (cells/ml).

Fig. 4

Monitoring of CD4⁺ forkhead box P3 (FoxP3)⁺CD25⁺ T cells and CD4⁺ T cell counts in human immunodeficiency virus (HIV)-infected children. The frequency of CD4⁺FoxP3⁺CD25⁺ and CD4⁺ T cells were determined at two different time-points during an average of 12-month follow-up period. (a) Correlation analysis between changes in the levels of CD4⁺ T cells (Δ% CD4) and changes in regulatory T cell (T_reg) phenotype (Δratio %CD4⁺FoxP3⁺CD25⁺/%CD4⁺FoxP3⁺CD25^–). (b) Representative dot-plot of CD25 and FoxP3 expression in CD4⁺ T cells during follow-up (t₀ and t₁₂ patient 3 in Table 2). CD4⁺ T cells were gated from a side-scatter (SSC) versus CD4 dot-plot and the expression of CD25 and FoxP3 was calculated. The figures in the upper left quadrants show the percentages of CD4⁺FoxP3⁺CD25^- T cells, while the percentages of CD4⁺FoxP3⁺CD25⁺ T cells are shown in the upper right quadrants. (c) Summary of immune/virological status of the same patient showed in (b).