Final Progress Report

Proposal No. IBD-0029
Principal Investigator:  Adrian G. Cummins, M.D., Ph.D.
Applicant Organization:  The Queen Elizabeth Hospital (Woodville South, Australia)
Project Title:  Deficiency of immunoregulatory NK T cells in inflammatory bowel disease
Award Period:  November 1, 2002 - March 31, 2005

Regulatory (invariant, i) NK T-cells have the surface phenotype of Vα24Ja18 and Vβ11 chains of the CD3 T-cell receptor and are characterized as responding rapidly with interleukin (IL)-4 production to the α-galactosylceramide model antigen that is presented by CD1d HLA molecules.  These iNK T cells have been shown to be deficient in several autoimmune conditions.  Our hypothesis was to investigate any deficiency of iNK T-cells in inflammatory bowel disease (IBD).  The project aims were to investigate any deficiency in blood, any mucosal deficiency, any functional cytokine deficiency and any functional cytotoxic deficiency.  We did not pursue the fourth aim on assessment of cytotoxicity.

We recruited 104 subjects with Crohn’s disease, 91 subjects with ulcerative colitis and 153 normal subjects for blood collection for flow cytometry.  iNK T-cells were identified as Vα24+ Vβ11+ α-galactosylceramide/CD1d tetramer+ or Vα24+ 6B11+ CD3+ cells.  A smaller number in each of the subject categories had ileocolonic biopsies.  We identified that there was a systemic deficiency of NK cells, NK T-cells, Vα24+ T-cells and iNK T-cells in Crohn’s disease.  In particular iNK T-cells were reduced in Crohn’s disease to 0.3% of levels in normal subjects.  We found that NK cells and iNK T-cells were deficient in ulcerative colitis.  Vα24+ T-cells were not deficient in ulcerative colitis.  However, iNK T-cells in ulcerative colitis were reduced to 7% of levels in normal subjects.  In ileocolonic biopsies, intestinal Vα24/GAPDH mRNA expression by real time PCR was reduced 7% in Crohn’s disease and to 9% in ulcerative colitis.  Cell mucosal counts of Vα24+ T-cells by immunofluorescence was reduced to 23% in Crohn’s disease.  Residual systemic iNK T cells had deficient IL-4, IL-10, and IL-13 production after 4 h of anti-CD3 antibody or PMA/ionomycin stimulation in both Crohn’s disease and ulcerative colitis.  We conclude that there is a deficiency of regulatory iNK T-cells both systemically in blood and mucosally in the intestines in both Crohn’s disease and in ulcerative colitis.

Lay summary:

This study attempted to answer why the bowel becomes inflamed in inflammatory bowel disease (Crohn’s disease and ulcerative colitis). We proposed that the immune system lacks a normal suppressor cell called the invariant NK T-cell (iNK T-cell.  The function of this cell might be to suppress inappropriate overactivity of the immune system to the myriad of food and particularly bacterial antigens in the intestine.  We counted these cells in the blood of 153 healthy subjects, 104 subjects with Crohn’s disease and 91 subjects with ulcerative colitis.  The level of iNK T-cells was markedly reduced to 0.3% in Crohn’s disease and 7% in ulcerative colitis compared to healthy subjects.  A subgroup of these subjects had colonoscopy and biopsy of the mucosal lining of the intestine.  A marker of these cells is called Vα24+ and can be detected under the microscope using an antibody label or using mRNA from the expressed gene.  Both these measures showed deficiency of Vα24+ cells in the intestine in Crohn’s disease and in ulcerative colitis.  Finally, we investigated the function of these iNK T-cells after stimulation to see whether they produce chemicals called cytokines that directly inhibit other immune cells and prevent them becoming activated.  iNK T-cells from healthy subjects rapidly produced interleukin-4 cytokine, whereas equivalent cells from subjects with Crohn’s disease or those with ulcerative colitis failed to produce interleukin-4 cytokine.  We conclude that there is a deficiency of iNK T-cells both in the blood and also in the intestine in inflammatory bowel disease.  This deficiency could in part explain why the bowel becomes inflamed in susceptible people in inflammatory bowel disease.