CD4⁺ CD25⁺ T-Cell Production in Healthy Humans and in Patients with Thymic Hypoplasia

Abstract

Regulatory T cells are found primarily in the CD4⁺ CD25⁺ fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4⁺ CD25⁺ T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4⁺ CD25⁺ T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome.

Regulatory T cells constitute an important contributor to the state of tolerance (6). Little is known about their function and production in humans, and nothing is known about their development in humans (2, 5). Murine studies first defined these cells as a component of CD4⁺ CD25⁺ (CD4 CD25) T cells. Several important features of these murine cells have been described. They exit the thymus from day 4 of life onwards, and they appear to require a T-cell receptor with high affinity (1, 4). They are antigen specific in their activation but are non-antigen specific as effectors. As effectors, they are naturally nonproliferative, and their function is generally detected by their ability to inhibit proliferation of other cells or other effector functions, such as cytolytic activity. Although their exact mechanism of action is controversial, there is agreement that cell contact is required.

These cells have been demonstrated to be responsible for the prevention of organ-specific autoimmunity in several murine models. Mice which have undergone a thymectomy between days 1 and 3 of life have a delay in the appearance of CD25⁺ T cells. These mice develop organ-specific autoimmune disease; the specific disease is dependent on the strain of mice used (1). Infusion of CD25⁺ T cells can abolish the organ-specific autoimmune disease. In thymectomy models, the ratio of CD25⁺ to CD25⁻ T cells is normal. The CD25⁺ T cells appear late and in lower numbers but retain their normal proportion in the T-cell compartment. This has suggested that adequate quantitative production of regulatory T cells early in life may be a critical part of the development of tolerance. Human cells with regulatory properties express very high levels of CD25 and have suppressive effects on effector functions similar to that seen in mice (2). They are present in adults at levels similar to those seen in mice, i.e., approximately 6 to 10% of CD4 T cells.

The studies described above documented the important role of the thymus in the production of CD4 CD25 T cells and the role of CD4 CD25 T cells in the prevention of autoimmunity. In this study, we examined a population of children who have thymic hypoplasia as a result of a heterozygous deletion of chromosome 22q11.2. This deletion results in a phenotype which has also been called DiGeorge syndrome, velocardiofacial syndrome, or conotruncal anomaly face syndrome. Approximately 80% of patients with the deletion will have an immunodeficiency as a consequence of the thymic hypoplasia (7). The immunodeficiency is typically a pure defect in T-cell production, and patients have on average 50 to 70% of the normal numbers of T cells (3). Autoimmune disease develops in approximately 10% of patients, although the pathophysiologic mechanism for this is not understood. This study examined the developmental appearance of CD4 CD25 T cells in the peripheral blood of 33 children and adults with chromosome 22q11.2 deletion and in 33 healthy age-matched controls in an effort to determine whether thymic hypoplasia could be associated with diminished numbers of this important T-cell subset, as is seen in mice with neonatal thymectomies.

Unselected patients with hemizygous deletions of chromosome 22q11.2 as detected by the Oncor/Vysis N25 probe were included as “patients” in this analysis. Control samples were selected from clinics performing well-child care or allergy evaluations (children with negative RAST tests). Institutional review board approval was obtained for this study, and consent was obtained from each participating patient or parent of the patient. Three-color flow cytometry was performed to define CD3, CD4, and CD25.

Only CD25^hi CD4 T cells were included in the CD25-positive gate, in accordance with what is known regarding the expression of CD25 on human regulatory T cells (2). The fraction of CD25 T cells within the CD3 population and the frequency of CD25 T cells within the CD4 population did not vary with age and showed no difference between controls and patients. In both patients and controls, the CD25 fraction of CD4 T cells was typically 6 to 15%. In both patients and controls, the CD25 fraction of CD3 T cells was typically 2 to 10%. Differences were readily apparent between patients and controls when the CD4 CD25 proportion of total lymphocytes was examined and the CD4 CD25 absolute number was determined (Fig. 1). There is a distinct age-related decline in the CD4 CD25 fraction and number of cells from birth through approximately 36 months of age in the controls. The differences between the CD4 CD25 T-cell numbers in children less than 3 years of age and those over 9 years of age are significant, with P being 0.001. In contrast, the fraction and number of CD4 CD25 T cells were similar throughout all ages examined in the chromosome 22q11.2 deletion population. As a result, the fraction and the absolute number are markedly lower in the patients than controls in infancy. The mean CD4 CD25 T-cell count in patients under 3 years of age was 7 cells/mm³, while it was 75 cells/mm³ in the controls (P = 0.001). The mean CD4 CD25 T-cell count in patients 3 to 9 years of age was 3 cells/mm³, while it was 14 cells/mm³ in controls (P = 0.005). The mean CD4 CD25 T-cell count in patients over the age of 9 years was 3 cells/mm³, while it was 10 cells/mm³ in controls (P = 0.002). Six patients had autoimmune disease. The mean CD4 CD25 T-cell count in these six patients was no different than the mean in the non-autoimmune disease patients.

FIG. 1.

(Top) Fractions of CD4 CD25 T cells within the total lymphocyte gate; (bottom) absolute numbers of CD4 CD25 T cells. Gray diamonds represent patients with autoimmune disease. m, months.

This is the first study to examine peripheral blood CD4 CD25 T cells in human children. We have shown that newborns generally have high numbers that decline rapidly over the first 2 to 3 years of life. This is dependent on T-cell production, because the fraction of CD4 CD25 cells within the T-cell subset remains relatively constant throughout childhood. Patients with chromosome 22q11.2 deletion with developmental thymic hypoplasia have markedly fewer CD4 CD25 T cells in infancy.

The significance of these findings is twofold. Patients with chromosome 22q11.2 deletion syndrome have a relatively pure quantitative defect in T-cell production. Thus, this study suggests that regulation of CD4 CD25 T-cell production early in life in humans is directly related to thymic capacity. CD4 CD25 T cells are produced in similar proportions with other T cells postnatally. Secondly, this study suggests that one contributor to the increased prevalence of autoimmune disease could be diminished CD4 CD25 T cells. This is comparable to what was seen in the murine neonatal thymectomy model. The CD4 CD25 T cells appear late, and the absolute numbers remain diminished. In this murine model, approximately 20% of the mice developed autoimmune gastritis by 1.5 to 3 months of age after neonatal thymectomy (1). Extrapolating from the murine model, our finding of markedly diminished CD4 CD25 T-cell numbers early in life suggests that this phenomenon could play a role in the predisposition to autoimmune disease in patients with chromosome 22q11.2 deletion syndrome. Other factors are required to define the type of autoimmune disease and to further determine susceptibility.