Onset of Clinical Tolerance to Milk Protein is Associated with Increased CD25+ CD27+ Casein Specific T Cells

Abstract

RATIONALE: Co-expression of CD25 and CD27 has been reported to correspond to regulatory T cell function. We hypothesized that milk-allergic patients may have increased milk-specific T regulatory function as they develop clinical tolerance.METHODS: PBMC were labeled with CFSE and cultured for seven days with casein as well as medium and anti-CD3, -CD28 controls. Live (propidium iodine negative) proliferating (CFSElow) CD4 T cells (CD3+CD4+) were assessed for expression of CD25 and CD27 by flow cytometry. Patients were characterized by standard testing, history and challenge criteria as non-allergic (n=9), allergic (n=10), tolerating heated milk (n=18) and outgrown (n=8).RESULTS: As a percentage of total live CD3+ CD4+ cells, patients recently passing challenge to heated dietary milk protein had the highest percentage of casein-specific CD25+ CD27+ T cells (21.3 [13.6 - 29.1]; mean [95% confidence intervals]), which were significantly higher than in milk allergic patients (7.3 [2.2 - 12.3], p<0.05), or non-allergic controls (5.0 [-2.9 - 13], p<0.05). Patients with complete resolution of milk allergy had a level of CD25+ CD27+ T cells (9.9 [1.4 - 18.3]) that was intermediate those of allergic and tolerating heated milk patients. There were significant differences in the percent CD25+ CD27+ T cells following polyclonal stimulation with anti-CD3, -CD28 antibodies.CONCLUSIONS: Tolerance to dietary milk protein is associated with an expansion of putative casein-specific T regulatory cells. RATIONALE: Co-expression of CD25 and CD27 has been reported to correspond to regulatory T cell function. We hypothesized that milk-allergic patients may have increased milk-specific T regulatory function as they develop clinical tolerance. METHODS: PBMC were labeled with CFSE and cultured for seven days with casein as well as medium and anti-CD3, -CD28 controls. Live (propidium iodine negative) proliferating (CFSElow) CD4 T cells (CD3+CD4+) were assessed for expression of CD25 and CD27 by flow cytometry. Patients were characterized by standard testing, history and challenge criteria as non-allergic (n=9), allergic (n=10), tolerating heated milk (n=18) and outgrown (n=8). RESULTS: As a percentage of total live CD3+ CD4+ cells, patients recently passing challenge to heated dietary milk protein had the highest percentage of casein-specific CD25+ CD27+ T cells (21.3 [13.6 - 29.1]; mean [95% confidence intervals]), which were significantly higher than in milk allergic patients (7.3 [2.2 - 12.3], p<0.05), or non-allergic controls (5.0 [-2.9 - 13], p<0.05). Patients with complete resolution of milk allergy had a level of CD25+ CD27+ T cells (9.9 [1.4 - 18.3]) that was intermediate those of allergic and tolerating heated milk patients. There were significant differences in the percent CD25+ CD27+ T cells following polyclonal stimulation with anti-CD3, -CD28 antibodies. CONCLUSIONS: Tolerance to dietary milk protein is associated with an expansion of putative casein-specific T regulatory cells.