Antigen receptor V-segment usage in mucosal T cells

Abstract

In the accepted model of lymphocyte intestinal homing, naïve T cells recirculate via organized lymphoid tissues, whilst induced effector/memory cells home to the intestinal mucosa. In order to assess the T-cell-receptor repertoire in the intestine and gut-associated lymphoid tissue (GALT), spectratyping was performed on the proximal and the distal intestine, spleen and mesenteric lymph node tissue from six PVG rats. The products were analysed with an automated sequencer and statistical analyses were performed with hierarchical cluster analysis. This demonstrated the presence of a restricted T-cell repertoire in the small intestine compared with that in the mesenteric lymph nodes and the spleen. It also demonstrated marked differences in repertoire between individual, fully inbred rats maintained under apparently identical conditions in the same cage and fed identical diets. In addition, this work demonstrated marked differences between repertoires in the proximal and the distal intestine. Such marked differences are likely to reflect the end result of increasing divergence over time produced by relatively subtle effects of environment and antigenic load. Equally, marked differences in repertoire between small intestinal segments within individual rats indicate selective recruitment or retention of specific clones, presumably antigen-driven.

Figure 1

Agarose-gel electophoresis of polymerase chain reaction (PCR) products from proximal intestine amplified using primers in the constant region (lanes 2 and 3) and using an example Vβ primer (Vβ5, lanes 4 and 5). Templates used in lanes 3 and 5 were not subjected to reverse transcription, as a negative control. C, C-region primer; C con, reverse C-region primer.

Figure 2

(a) Normalizing of spectratype peak data. Peak height data were extracted from ‘raw’ spectratypes, as shown on the left. This data was then normalized, so that for each Vβ family, for each tissue sample, the proportion that each peak contributed to the total spectratype was calculated. When this data was plotted, as on the graphs on the right, as each peak is a proportion of the total, the height of peaks also relates to spread. Thus, when a peak height is 1, this represents a highly restricted repertoire, with a single CDR3 length. (b) Plots of normalized data, from all six animals, with the proportion of the spectratype contributed to on the y-axis and each of the 22 Vβ families on the x-axis. Red, proximal intestine; green, distal intestine; blue, mesenteric lymph node; grey, spleen.

Figure 3

Hierarchical cluster analysis of normalized peak height data for animals numbered 1–6. Based upon the Vβ repertoire, most of the organized lymphoid tissue (mesenteric lymph nodes and spleen) cluster together. In contrast, proximal intestinal tissues do not cluster with the organized tissues, with marked differences observed between the animals, indicating markedly different repertoires between individuals. Some distal intestinal samples clustered strongly with the unselected repertoires of the organized lymphoid tissues, whereas others showed highly selected repertoires, as in the proximal intestine. s.i., small intestine.