Increased frequency of intestinal CD4⁺ T cells reactive with mycobacteria in patients with Crohn's disease

Figures & data

Table I. Patient characteristics.

Patient	Diagnosis	Disease localisation and current activity	Time since diagnosis (years)	Medication (duration)*
IBD18	CD	Small intestine, stenosis	31	Budesonide (7 years)
IBD55	CD	Colon, moderate	20	Budesonide (5 years)
IBD56	CD	Small intestine and colon, stenosis	29	Budesonide (2 years)
IBD57	CD	Small intestine, moderate	8	None
IBD58	CD	Small intestine, moderate	1	Adalimumab (2 months) Azathioprine (1 year)
IBD62	UC	Pancolitis, moderate	3	Prednisolone (2 weeks) 5-ASA (3 years)
IBD63	UC	Proctitis, moderate	13	5-ASA (13 years)
IBD64	UC	Pancolitis, mild	4	Azathioprine (2 years), Prednisolone (4 years) 5-ASA (4 years)
IBD65	UC	Pancolitis, moderate	18	Salazopyrin (1 year)

*Duration of medication at the time of the biopsy is shown

Table II. Frequency of reactive CD4 T cells after direct cloning from biopsies using two different cut-offs.

Patient ID	Diagnosis	Total T- cell clones tested	no reactive (%) SI >10		no reactive (%) SI >5 and Δ CPM >3000
			MAP	E. coli	MAP	E. coli
IBD18	CD	1854	14 (0.76)	2 (0.11)	15 (0.81)	3 (0.16)
IBD55	CD	3972	17 (0.43)	7 (0.18)	15 (0.38)	7 (0.18)
IBD56	CD	3552	6 (0.17)	1 (0.03)	8 (0.23)	1 (0.03)
IBD57	CD	798	13 (1.63)	ND	38 (4.76)	ND
IBD58	CD	1440	3 (0.21)	0 (0.00)	7 (0.49)	1 (0.07)
IBD62	UC	408	0 (0.00)	ND	0 (0.00)	ND
IBD63	UC	1076	0 (0.00)	2 (0.19)	1 (0.09)	3 (0.28)
IBD64	UC	383	3 (0.78)	2 (0.52)	6 (1.57)	3 (0.78)
IBD65	UC	3156	2 (0.06)	0 (0.00)	4 (0.13)	0 (0.00)

Abbreviation: ND = Not determined.

Figure 1. Screening of 3972 clones from one CD patient (IBD55). CD4 T cells were isolated from intestinal biopsies and expanded in vitro. The responses to MAP and E. coli was tested in a ³H thymidine incorporation assay using autologous adherent cells as APC. Each symbol represents one tested well. The line indicates the cut-off of a stimulation index >10 (response in antigen stimulated well/response in unstimulated wells). The response to the two bacterial antigens was compared using a binominal test (p = 0.032).

Figure 2. Cytokine secretion in CD4 T cell clones. Production of IFN-γ, IL-10, IL-17 and IL-4 by CD4 T cell clones (n = 20) from two different patients after stimulation with and anti-CD3/anti CD28 beads for IL-10 and IL-4, and PMA/Ionomycin for IL-17 and IFN-γ. The cells were stimulated in duplicates for 24 h. The cytokine production in the supernatant was measured in the Bio-Plex™ assay.

Figure 3. Expression of surface markers in a Th1 clone (left panel) and a Th17/Th1 clone (right panel). The clones were stained with anti-CD4 and then mixed with unstained PBMC to be used as an internal control. The mix was subsequently stained with antibodies against the surface markers β7 integrin, CCR6 and CD161 and analysed by flow cytometry. The negative population of the PBMC for the respective markers were used as an internal control (open histograms) while gating on the CD4+ T cells gave the expression of the various surface markers on the tested clone (grey histograms). Left panels depicts TCC955.M.1 and the right panel TCC958.B.M.1.