Analysis of T-lymphocyte subpopulations in inflammatory bowel diseases by three-color flow cytometry

In order to better define changes in the relative proportion of peripheral blood T-lymphocyte subpopulations in patients with inflammatory diseases of the bowel, we performed simultaneous three-color fluorescence-activated cytometric (FACS) analysis using fluorophore-conjugated monoclonal antibodies with specificity for CD4, CD8, Leu 8, and CD45RA on 22 normal control subjects, 28 patients with Crohn's disease (CD), 15 patients with ulcerative colitis (UC), and 11 patients with intestinal inflammation secondary to etiologies other than inflammatory bowel disease (NIBD). This staining combination allowed enumeration of distinct T-cell subpopulations as follows: virgin CD4⁺, recall antigen helper T cells, nonspecific B-cell helper T cell, virgin CD8⁺, cytotoxic effector and suppressor effector and recall antigen cytotoxic T cells based on a synthesis of published functional analyses. No differences in the proportion of CD4⁺ or CD8⁺ cells or in the CD4⁺/CD8⁺ ratios were evident when UC and NIBD patients were compared to normal subjects. A significant reduction in the proportion of CD4⁺ cells and an increase in CD8⁺ cells was observed, however, in the CD group. When two-color analysis was performed, several significant differences in the proportions of circulating lymphocytes were seen. Specifically, these included significant increases in the number of CD4⁺, Leu 8^– (P<0.01) cells in all disease groups and an increase in CD4⁺, CD45RA⁺ cells in the NIBD group. Conversely, significant decreases in the proportions of CD8⁺, Leu 8⁺ (P<0.01) cells were evident in the Crohn's disease group. Three-color FACS analysis revealed significant differences in the relative proportions of the defined T-cell subpopulations enumerated in the various groups as compared with the normal controls. These included a decrease in the proportions of Leu 8⁺, CD45RA⁺, (virgin) CD8⁺ T cells (P<0.05) and Leu 8^–, CD45RA⁺, (putative recall antigen helper) CD4⁺ T cells (P<0.01) in all patient groups as compared with normal controls. Conversely, an increase in the proportions of Leu 8^–, CD45RA⁺, (putative suppressor effector) CD8⁺ T cells (P<0.01), and Leu 8^–, CD45RA⁺, (function unknown) CD4⁺ T cells (P<0.05) was seen in all patient groups as compared with normal controls. CD patients but not UC or NIBD patients demonstrated a significant increase in the proportion of Leu 8^–, CD45RA⁺, (putative cytotoxic effector) CD8⁺ T cells (P<0.01). An increase in the ratio of Leu 8^–, CD45RA⁺, CD8⁺ (suppressor effector)/Leu 8⁺, CD45RA^–, CD8⁺ (putative cytotoxic effector) T cells was observed in all of the patient groups, but was most accentuated in those with Crohn's disease. Significant decreases in the ratios of Leu 8^–, CD45RA^–, CD4⁺ (putative nonspecific B cell helper)/Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper) T cells and Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper)/Leu 8^–, CD45RA⁺, CD4⁺ (function unknown) T cells were observed in all of the disease groups studied as compared with normal controls. These results suggest that the proportions of certain peripheral blood T-cell subpopulations are significantly altered in gastrointestinal inflammatory states. Further analysis of these T-lymphocyte subpopulations in the blood and tissues might provide valuable insights into immunological aberrations in inflammatory bowel diseases and might be of value in distinguishing among inflammatory diseases of the intestine.