Materials and methods

Results

CLONAL T CELL RESPONSES TOWARDS ANAEROBICBACTEROIDES ANDBIFIDOBACTERIUM

We have previously shown that CD4 T cells specifically recognise different enterobacteria and that enterobacteria specific T cell clones are increased in intestinal IBD lesions.15 Using new T cell clones we now determined the T cell response to major constituents of the anaerobic intestinal flora in humans—that is,Bacteriodes andBifidobacterium—and assessed whether T cell cross reactivity between anaerobes and enterobacteria occurs. T cell clones were isolated from peripheral blood (n = 26) and lamina propria T cell populations (n = 70) from two patients with IBD (both ulcerative colitis) as described above, and subsequently restimulated with sonicated preparations from enterobacteria, H pylori, B thetaiotaomicron, andB bifidum. Confirming previous data,16 T cell clones responding to enterobacteria were obtained (fig 1). In addition, [³H]thymidine incorporation identified a total of seven out of 96 T cell clones from peripheral blood and intestine with reactivity towardsB thetaiotaomicron and B bifidum. Of these seven T cell clones, two were reactive towardsB thetaiotaomicron only (RP3 and BB−22), and T cell clone RB+16 was cross reactive between B thetaiotaomicron and B bifidum. Cross reactivity, however, was not restricted to anaerobic bacteria as T cell clones reactive with B bifidum andY enterocolitica (RB−9 and BB+6) orB bifidum, B thetaiotaomicron, and Y enterocolitica (RB+2 and BB−17) were also identified. All bacteria specific T cell clones were CD4+TCRαβ+ as determined by single-cell immunocyte assay.

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Figure 1

Clonal T cell responses towards Bacteroides and Bifidobacterium. T cell clones were isolated, using phytohaemagglutinin (PHA), from two patients with ulcerative colitis (patient R: peripheral blood n = 14, non-involved colon n = 12, involved colon n = 18; patient B: peripheral blood n = 12, non-involved colon n = 17, involved colon n = 23) and restimulated with the indicated antigens for 42 hours. Mean proliferation of T cell clones from peripheral blood (RP3), non-involved colon (RB−9 and BB−17), and involved colon (RB+2, RB+16, BB+6, and BB+22) stimulated by Bacteriodes thetaiotaomicron (ATCC 12290) or Bifidobacterium bifidum (ATCC 35914) is shown as measured by [³H]thymidine incorporation in triplicate cultures. Clone BP6 is representative of other T cell clones reactive with enterobacteria only. All T cell clones were CD4+TCRαβ+ by single-cell immunocyte assay.

CLONAL T CELL RESPONSES TO CRUDE SONICATES FROM AEROBIC INTESTINAL FLORA

We have previously shown that crude sonicate preparations from indigenous intestinal flora stimulate proliferation in mononuclear cells from peripheral blood and intestinal lamina propria of a foreign subject but not those of the same subject.16 To determine T cell reactivity against these sonicates on a clonal level, we now isolated T cell clones from peripheral blood and lamina propria from two controls (n = 77) and three patients with IBD (n = 133) by cloning with phytohaemagglutinin. These clones were then restimulated with crude sonicates of bacteria from autologous intestine (BsA) or heterologous intestine (BsH). To assess T cell clone cross reactivity, T cell clones were also restimulated with sonicate preparations from different enterobacteria and H pylori, previously shown to selectively stimulate bacteria specific T cell clones.15 As shown in fig 2 and table 1, [³H]thymidine incorporation identified a total of five out of 77 T cell clones from controls and 21 out of 133 T cell clones from patients with IBD with reactivity towards BsH. In controls, three of five BsH reactive T cell clones were stimulated by BsH only and two of five T cell clones (WB−5 and WB−15) cross reacted with eitherE coli or S typhimurium. In patients with IBD, 15 out of 21 T cell clones were stimulated by BsH only, two out of 21 T cell clones (DB−8 and SP24) showed weak cross reactivity between BsH andH pylori or BsH, H pylori, and E coli, and four out of 21 T cell clones (DP15, DP18, DB−5 and FB+2) were cross reactive with BsA and BsH. The fraction of T cell clones recognising autologous bacteria was 0/77 in controls and 8/133 in patients with IBD. Four of eight T cell clones (DP15, DP18, DB−5, and FB+2) were cross reactive with BsA and BsH, and four of eight T cell clones (DB+5, DB+8, FP15, and FB+11) reacted to BsA only.

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Figure 2

Clonal T cell responses to aerobic intestinal flora. T cell clones were isolated with phytohaemagglutinin (PHA) from two controls (patient A, bile acid induced diarrhoea: peripheral blood n = 23, colon n = 15; patient W, irritable bowel syndrome: peripheral blood n = 20, colon n = 19) and restimulated with the indicated antigens for 42 hours. Aerobic intestinal flora was obtained from intestinal biopsy specimens touched to blood agar plates and grown under aerobic conditions. Sonicates from outgrowing bacteria were used as BsA (sonicates and mononuclear cells from the same person) or BsH (sonicates and mononuclear cells from different people). Mean proliferation of T cell clones from peripheral blood (WP2 and WP18) and colon (AB−3, WB−5, and WB−15) stimulated by BsA or BsH is shown as measured by [³H]thymidine incorporation of triplicate cultures. All T cell clones were CD4+TCRαβ+ by single-cell immunocyte assay. Incubation of antigen presenting cells with the indicated stimuli without T cell clones resulted in <500 cpm.

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Table 1

Clonal T cell responses towards aerobic intestinal flora

Discussion

The intestinal flora is increasingly incriminated as a source of antigens inducing or perpetuating intestinal inflammation in IBD. Therefore bacteria specific or antigen specific immunomodulation of antibacterial immune responses may represent an option for IBD treatment. The feasibility of this approach, however, will depend on the complexity of the cellular immune response to intestinal bacteria, the nature of the relevant antigens, and whether some bacterial species are more relevant to the inflammatory process than others.24

To address these questions, we determined the complexity of the CD4+TCRαβ+ immune response to antigens from the intestinal flora and show that (a) both anaerobic intestinal bacteria and enterobacteria express discrete and shared antigens, (b) T cell clones cross react with anaerobic intestinal bacteria and enterobacteria, (c) T cell clones recognising foreign intestinal flora can cross react with enterobacteria, (d) T cell clones recognising indigenous aerobic intestinal flora of the same subject can be isolated in patients with IBD. These data from peripheral blood and lamina propria T cells therefore support the concept that the CD4+TCRαβ+ T cell response to antigens from the resident intestinal flora in controls and patients with IBD is specific but also comprises the potential for broad cross reactivity between different bacterial species.

In IBD, and here especially in Crohn’s disease, abnormalities in the composition of the intestinal flora have been shown for a variety of aerobic and anaerobic bacteria.37-42 Providing a link to the pathological immune response associated with Crohn’s disease inflammation and tissue injury, these descriptive studies have been complemented by investigations of the humoral or cellular antibacterial immune response. Thus increased T cell reactivity towardsE coli and other microbial recall antigens14 and an increase in enterobacteria specific T cells15 in intestinal lesions of patients with IBD have implicated T cell responses to enterobacterial antigens in IBD. With regard to anaerobic bacteria, luminal concentrations ofPeptostreptococci, Eubacteria, andCoprococcus 40 and serum antibodies to these bacteria43 were found to be selectively increased in patients with Crohn’s disease. Furthermore, purified peptidoglycan−polysaccharide polymers fromPeptostreptococcus,Eubacteria, Salmonella faecum, and E coli were shown to induce chronic inflammation in susceptible rat strains.5

As we had previously determined the response of human CD4+TCRαβ+ T cell clones to different enterobacteria and H pylori, we now used Bacteroidesspecies, Bifidobacterium species, and direct isolates from indigenous intestinal flora to investigate the potential complexity of the immune response to different components of the intestinal flora. Importantly, the bacterial antigens used in this study differ from each other in various ways. Firstly,Bacteroides andBifidobacterium are predominant anaerobic isolates from normal faecal—that is, luminal colonic bacteria—whereas direct isolates from indigenous intestinal flora were generated using a technique favouring the growth of aerobic intestinal bacteria from the bowel wall. The bacterial flora at the bowel wall differs considerably from the faecal flora, and in the distal ileum and colon contains a much higher percentage of aerobic bacteria.7 As it is not known whether luminal bacteria or bacteria at the bowel wall are more relevant to the mucosal immune response in IBD, it may be important to study the two groups of bacteria separately. Secondly,B thetaiotomicron had been isolated from purulent material from an incision during appendicectomy, whereasB bifidum had been isolated from human faeces. Thirdly, Bacteroides andBifidobacterium were established isolates from foreign donors, whereas direct isolates from indigenous intestinal flora allowed us to test the immune response of a subject towards antigens from his/her own flora.

Using these antigens for stimulation of T cell clones, our results provide evidence that not only enterobacteria and H pylori 15 ,44 ,45 but also members of the anaerobic intestinal flora express dominant discrete protein antigens recognised by specific T cells. Cross reactive T cell clones in our study show that anaerobic bacterial species share antigens with one another andY enterocolitica. Thus, complementing T cell specificity,15 ,44 ,45 cross-reactivity, as shown between different enterobacteria,15 different intestinal anaerobic species as well as enterobacteria and intestinal anaerobic species, is a distinct feature of CD4+TCRαβ+ T cell responses to intestinal bacteria. These findings suggest that CD4+TCRαβ+ T cell mediated immune responses to different bacteria within the intestinal flora, relating to either protection or pathogenicity, are regulated by a complex network of T cell specificities. The mechanisms regulating these immune responses are not understood but certainly contrast with the non-specific antibacterial immune response of TCRγδ+ T cells.46 In this context, it may be interesting that antigenic cross reactivity between autochthonousBacteroides species and intestinal tissue of neonatal mice has been suggested as a mechanism for causing selective unresponsiveness to Bacteroides species in adult mice.47 B vulgatus in contrast has recently been identified as a critical bacterium mediating chronic colitis, gastritis, and arthritis in HLA-B27/human β2-microglobulin transgenic rats.24 In addition, the same bacterium was shown to play an essential role in the pathogenesis of carrageenan induced colitis in guinea pigs.25 The reason for the increased capacity of B vulgatus to induce intestinal inflammation in these animal models is not known and may include immunological and non-immunological causes.48 ,49 In contrast,Bifidobacterium, for which there was cross reactivity with B thetaiotamicron in this study, has been associated with qualities that are beneficial to the host. Thus it has been suggested that cell association with enterocytes and epithelial invasion by pathogenic enterobacteria are inhibited byBifidobacteria.50Furthermore, Bifidobacteria have been shown to excrete antimicrobial substances51 and to reduce the incidence of acute diarrhoea and rotavirus shedding in infants.52

As mentioned above, the intestinal flora is a complex ecosystem. It reaches stable expression shortly after birth and its individual composition is influenced by the genetic characteristics of the host.6 ,40 ,41 Therefore instead of using established or non-indigenous bacterial isolates, it may be more reasonable to investigate the immune response to bacteria isolated from an individual’s own intestinal flora. Using this approach, we have recently shown using mononuclear cell bulk cultures that controls are specifically tolerant to their own flora and that this tolerance is lost in intestinal lesions of patients with IBD.16Investigating the response of clonal T cells, we here show that CD4+TCRαβ+ T cells from peripheral blood and intestinal mucosa specifically recognise antigens from foreign flora and, at least in patients with IBD, also their own intestinal flora. The results showing that clonal T cells cross react with their own and foreign intestinal flora as well as enterobacteria were expected. They reflect the fact that different people share a pool of indigenous bacteria and that our technique for direct isolation of indigenous intestinal bacteria favoured growth of aerobic bacteria from the bowel wall. This was also shown by the fact that enterobacteria were exclusively found in an isolate stimulating BsH specific T cell clone FB−10. Adding a further level of complexity, a more detailed analysis of the bacteria recognised by BsH specific T cell clone FB−10 indicated that the relevant antigens were expressed by different enterobacteria and that antigen expression by these enterobacteria was not constitutive.

T cell clones with specificity for their own intestinal flora in IBD lesions may be the cellular equivalent of the loss of tolerance to BsA observed in mucosal mononuclear bulk cultures. However, BsA reactive T cell clones were also present in peripheral blood mononuclear cells and uninvolved mucosa of these patients, which were tolerant in bulk culture. This indicates that, in these compartments, suppression or anergy rather than deletion may normally be responsible for tolerance to BsA.

T cell cross reactivity between different intestinal bacteria suggests that targeting of single bacterial species may not significantly reduce intestinal inflammation in IBD. This does not, however, exclude the possibility that T cell reactivity in IBD is directed against a limited number of dominant bacterial antigens, especially if there is no strict correlation between expression of stimulating antigens and bacterial genus or species. Further studies using clonal T cells to define in detail the relevant antigens and their expression among different intestinal bacteria will be required to gain a better understanding of antibacterial immunity, IBD pathogenesis, and whether antigen specific immunomodulation of antibacterial immune responses is a viable option for IBD treatment.