Identical T cell clones are located within the mouse gut epithelium and lamina propia and circulate in the thoracic duct lymph.

Murine gut intraepithelial (IEL) T cell receptor (TCR)-alpha/beta lymphocytes bearing CD8alpha/13 or CD8alpha/alpha coreceptors have been shown previously to express different oligoclonal TCR beta chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8alpha/beta+ IELs being fully thymus dependent. CD8alpha/beta+, but not CD8alpha/alpha+, T lymphocytes are also present in the lamina propria. Here, we show that CD8alpha/beta+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-beta clonotypes in the same mouse, as is also the case for CD4alpha T cells. Furthermore, identical T cell clones were detected among CD8alpha/beta IELs and CD8alpha/beta+ blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire.

Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal.

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN-gamma and TNF; these activities are increased by in vitro exposure to IL-12. Mice injected with IL-12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus-dependent as well as natural killer-T cell IEL) and the release of IFN-gamma. The critical role of IFN-gamma may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN-gamma into mutant mice lacking IEL showed that IFN-gamma can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL-12-induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL-12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN-gamma and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.

Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages.

Gut thymo-dependent (CD8 alpha + beta + or CD4+) or -independent (CD8 alpha + beta -) intraepithelial lymphocytes (IEL) mediate cytotoxicity following T cell receptor (TCR)-CD3 signaling, but only TCR gamma delta + and alpha beta + thymo-independent IEL show cytotoxicity of natural killer (NK) and antibody-dependent cell-mediated cytotoxicity types. Moreover, TCR alpha beta + and gamma delta + thymo-independent IEL express NK receptors, and may therefore be referred to as NK-TIEL. NK-TIEL cytotoxicity is mediated through perforin, Fas, or both pathways. In contrast to that of other NK cells, this cytotoxicity is not negatively regulated by signals delivered through the recognition of major histocompatibility complex class I molecules. Thus, gut IEL include T cell subsets with unique specificities and functions, ontogenically distinct from other T cell lineages, which may increase the antigenic repertoire diversity of the immune system participating in the defense of the epithelial barrier.

Different use of T cell receptor transducing modules in two populations of gut intraepithelial lymphocytes are related to distinct pathways of T cell differentiation.

Most gut intraepithelial cells (IEL) of the mouse are T cells that bear CD8 molecules, present either as alpha-beta chain heterodimers (CD8 beta+) or as alpha chain homodimers (CD8 beta-). All CD8 beta+ IEL bear alpha/beta T cell receptors (TCR); CD8 beta- IEL bear either alpha/beta or gamma/delta TCR and are considered to be a thymus-independent (TI) population, probably arising locally from a small fraction of CD3- IEL containing the recombinant activating gene RAG proteins. Here we report that TI CD8 beta- IEL, whether bearing alpha/beta or gamma/delta TCR, contain, in normal mice, mRNAs for both zeta and Fc epsilon RI gamma chains. These chains are present in their CD3-TCR complexes as homo- or heterodimers. In contrast, only zeta chain mRNA and homodimers are found in gut CD8 alpha/beta+ IEL and in peripheral T lymphocytes. Intestinal CD3- precursor cells contain only gamma chain, and CD3- IL-2R+ thymocyte precursors only zeta chain mRNAs. Only very primitive thymocyte precursors contain detectable gamma chain mRNA, and it thus appears that Fc epsilon RI gamma chain use is switched off at a very early stage during thymocyte differentiation. Thus, T cell differentiation in the gut epithelium differs from that occurring in the thymus, from which CD8 beta+ IEL appear to derive. Use of different TCR transducing modules and CD8 accessory molecules between the TI and the thymus-derived T cell populations provides an explanation for their difference in reactivity to antigenic stimulations and thus in selection of repertoires.

Different expression of the recombination activity gene RAG-1 in various populations of thymocytes, peripheral T cells and gut thymus-independent intraepithelial lymphocytes suggests two pathways of T cell receptor rearrangement.

The presence of transcripts of the recombination activating gene RAG-1 was studied by in situ hybridization on selected populations of murine thymocytes, peripheral lymphocytes and gut intraepithelial lymphocytes (IEL), obtained by cell sorting. RAG-1 mRNA was found in a majority of "double-positive" (DP) thymocytes, but was absent in "single-positive" thymocytes and peripheral T lymphocytes. The only other T lineages in which about 10%-20% of the cells contained RAG-1 mRNA, and in smaller amounts, were "double-negative" (DN), T cell receptor (TcR) gamma delta- cortical thymocytes and gut CD3- IEL. These observations suggest that (a) the high expression of RAG-1 transcripts in DP thymocytes is related to the process of expansion-selection of these cells, probably accompanied by repeated TcR rearrangements, and that (b) in contrast, CD3- IEL from the gut (which are thymus independent) as well as some DN thymocytes undergo limited TcR rearrangement giving rise locally to TcR+ T cells without prior extensive process of local expansion-selection. A small percentage of peripheral B cells also contained RAG-1 mRNA, raising the possibility that this protein may also be involved in immunoglobulin class switching.

Cytotoxic differentiation of mouse gut thymodependent and independent intraepithelial T lymphocytes is induced locally. Correlation between functional assays, presence of perforin and granzyme transcripts, and cytoplasmic granules.

Mouse gut intraepithelial lymphocytes (IEL), whether thymodependent (CD4+ or CD8 alpha/beta +; TCR-alpha/beta +) or thymoindependent (CD8 alpha/alpha +; TCR-alpha/beta + or -gamma/delta +), all display cytotoxic activity in a "redirected lysis assay" using anti-CD3 or anti-TCR beta or delta chains secreting hybridomas as targets; this is also observed with IEL of germ-free mice, indicating that this activity, which is absent in peripheral T lymphocytes, does not require stimulation by bacterial antigens. Perforin and granzyme transcripts are detectable in unselected gut IEL, in contrast to normal T lymphocytes of peripheral lymphoid organs. Cytological labeling (with [3H]DFP) of IEL smears reveals labeled granules (i.e., containing serine-esterases, presumably granzymes) in all subsets of gut IEL. This indicates that the gut micro-environment has an inductive role on the cytotoxic differentiation of lymphocytes of various origins when they reach the gut wall to become IEL.

Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation.

Mouse gut intraepithelial lymphocytes (IEL) consist mainly (90%) of two populations of CD8+ T cells. One bears heterodimeric alpha/beta CD8 chains (Lyt-2+, Lyt-3+), a T cell receptor (TCR) made of alpha/beta chains, and is Thy-1+; it represents the progeny of T blasts elicited in Peyer's patches by antigenic stimulation. The other bears homodimeric alpha/alpha CD8+ chains, contains no beta chain mRNA, and is mostly Thy-1- and TCR-gamma/delta + or -alpha/beta +; it is thymo-independent and does not require antigenic stimulation, as shown by its presence: (a) in nude and scid mice; (b) in irradiated and thymectomized mice repopulated by T-depleted bone marrow cells bearing an identifiable marker; (c) in thymectomized mice treated by injections of monoclonal anti-CD8 antibody, which lead to total depletion of peripheral CD8+ T lymphocytes; and (d) in germ-free mice and in suckling mice. In young nude mice, alpha/alpha CD8 chains, CD3-TCR complexes, and TCR mRNAs (first gamma/delta) are found on IEL, while they are not detectable on or in peripheral or circulating lymphocytes or bone marrow cells. IEL, in contrast to mature T cells, contain mRNA for the RAG protein, which is required for the rearrangement of TCR and Ig genes. We propose that the gut epithelium (an endoderm derivative, as the thymic epithelium) has an inductive property, attracting progenitors of bone marrow origin, and triggering their TCR rearrangement and alpha/alpha CD8 chains expression, thus giving rise to a T cell population that appears to belong to the same lineage as gamma/delta thymocytes and to recognize an antigenic repertoire different from that of alpha/beta CD8+ IEL.