Progenies of fetal thymocytes are the major source of CD4−CD8+ αα intestinal intraepithelial lymphocytes early in ontogeny

Present literature supports the view of an extrathymic origin for the subset of intestinal intraepithelial lymphocytes (IEL) that express the CD4^?CD8⁺ αα phenotype. This subset would include virtually all T cell receptor (TCR) γδ IEL and a portion of TCR αβ IEL. However, these reports do not exclude the possibility that some CD4^?CD8⁺ αα IEL are actually thymically derived. To clarify this issue, we examined the IEL day 3 neonatally thymectomized (NTX) mice. NTX resulted in as much as 80 % reduction in total TCR γδ IEL and in a nearly complete elimination of TCR αβ CD4^?CD8⁺ αα IEL early in ontogeny (3-to 5-week-old mice). The thymus dependency of TCR γδ IEL and TCR αβ CD4^?CD8⁺ IEL was less prominent in older mice (7- to 10-week-old mice), as the total number of these IEL increased in NTX mice, but still remained severalfold less than that in euthymic mice. Furthermore, we demonstrate, by grafting the fetal thymus of CBF1 (H-2^b/d) mice under the kidney capsule of congenitally nude athymic mice of BALB/c background (H-2^d), that a substantial number of TCR γδ IEL and TCR αβ CD4^?CD8⁺ αα IEL can be thymically derived (H-2^b+). In contrast, but consistent with our NTX data, grafting of adult thymi into nude mice generated virtually no TCR γδ IEL and relatively less TCR αβ CD4^?CD8⁺ αα IEL than did the grafting of fetal thymi. These results suggest that the thymus is the major source of TCR γδ and TCR αβ CD4^?CD8⁺ αα IEL early in ontogeny, but that the extrathymic pathway is probably the major source of these IEL later in ontogeny. A reassessment of the theory that most CD4^?CD8⁺ IEL are extrathymically derived is needed.     

Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing γδ T cell receptor in mice

Several cytokines including stem cell factor (SCF) and interleukin (IL)-7 are known to be required for development of γδ T cell receptor (TCR) intestinal intraepithelial lymphocytes (i-IEL) in mice. We show here the effects of IL-15 on the proliferation and maintenance of murine γδ i-IEL in vitro. γδ i-IEL constitutively expressed a high level of IL-15 receptor α mRNA and proliferated in response to IL-15 more vigorously than αβ i-IEL. Vγ/δ repertoire analysis revealed that IL-15, like IL-2, induced polyclonal expansion of γδ i-IEL, whereas γδ i-IEL responding to IL-7 showed a Vγ/δ repertoire skewed towards Vγ1/Vδ4, Vδ5. IL-15 efficiently prevented γδ i-IEL from apoptosis induced by growth factor deprivation. This rescue was accompanied by up-regulation of Bcl-2 expression. These results suggest that IL-15 plays important roles in proliferation and maintenance of γδ i-IEL.     

Host intestinal intraepithelial γδ T lymphocytes present during acute graft-versus-host disease in mice may contribute to the development of enteropathy

We reported that T cell receptor (TcR) γδ intestinal intraepithelial lymphocytes (i-IEL) of host origin increased transiently, then decreased drastically at the early stage of non-irradiated acute graft-versus-host disease (GVHD) in mice. We investigated the role of the TcR γδ i-IEL of host origin in the pathogenesis of the intestinal lesions that occur during acute GVHD. The acute GVHD was induced in mice which had been depleted of TcR γδ by in vivo administration of hamster monoclonal antibody (mAb) against TcR γδ. Although the degree of splenomegaly after the induction of acute GVHD in mice treated with anti-TcR γδ mAb was similar to that in control mice treated with hamster anti-2,4,6-trinitrophenyl mAb, infiltration of donor-derived T cells into the epithelium, and mitosis and apoptosis of crypt cells in the intestinal mucosa were dramatically suppressed in these mice. This suggest that host TcR γδ T cells in i-IEL contribute to the development of enteropathy in acute GVHD in mice.     

Activation of uterine intraepithelial γδ T cell receptor-positive lymphocytes during pregnancy

Intraepithelial lymphocytes (IEL) of the uterus of non-pregnant sheep were analyzed by single- and two-color flow cytometry. Very few lymphocytes carrying classical B and T cell markers (CD5, surface immunoglobulin) were detected in the uterine epithelial cell suspensions and all IEL expressed the CD8 surface marker although with varying intensities. Three distinct subpopulations were identified including a major (46-56%) population of CD8⁺CD45R^?γδ T cell receptor (TcR)-negative cells and approximately equal numbers of CD8⁺CD45R+γδTcR^? and CD8⁺CD45R⁺γδTcR⁺ lymphocytes. The same three subpopulations were also present in the interplacentomal areas of the uterus of ewes at a late stage of pregnancy but there was a dramatic increase (60-70%) in the γδ TcR⁺ subpopulation. In addition, a pronounced increase in both size and granularity was observed in the IEL population of pregnant uteri and this was attributed to the γδ TcR⁺ cells. Light and electron microscopic examination of these γδ TcR⁺ IEL revealed an increase in metabolic activity and the formation of exceptionally large cytoplasmic granules and confirmed their restricted localization within the uterine epithelium close to the trophoblast. These results represent for the first time, a clear example of the activation of γδ TcR⁺ cells which is not associated with an ongoing disease process or infection, γδ TcR⁺ cells have recently been observed in the epithelium of the murine reproductive tract and were characterized by their unique homogeneous receptor structure. The present results indicate that these cells may play an important physiological role during pregnancy.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

T cell receptor-γδ-expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection

We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so-called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCRγδ cells were CD2⁺, suggesting an origin at a post-CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day-15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.     

Thymus influences the development of extrathymically derived intestinal intraepithelial lymphocytes

Overwhelming evidence suggests that the majority of murine small intestinal intraepithelial lymphocytes (IEL) are extrathymically derived. These IEL include those with T cell receptor (TCR) γδ and someTCR αβ (CD8αα and Thy-1^?). In contrast, congenitally athymic nude mice have low numbers of γδ TCR IEL as well as very few αβ TCR IEL, far less than that would be expected if one assumes that γδ TCR IEL and αβ TCR (CD8αα and Thy-1^?) IEL in euthymic mice are extrathymically derived. To examine this discrepancy, we followed extrathymic IEL differentiation in IEL of day 3-thymectomized (NTX) mice as another athymic mouse model and found that γδ TCRIEL and extrathymically derived αβ TCR IEL in NTX mice are markedly reduced, almost to the level of nude mice. We further show that it is probably the absence of a thymic stroma that is responsible for the lower amounts of extrathymically derived IEL in nude mice, as the low amounts can be corrected to euthymic levels by syngeneic fetal thymus grafting but not by direct injection of F₁ thymocytes. Lastly, unlike TCR/CD3⁺ extrathymically derived IEL, we noted a large proportion of extrathymic CD3^?CD8^? and CD3^?CD8⁺ IEL; they were threefold more frequent in nude and NTX than in euthymic mice. This suggests that the thymus influences extrathymically derived IEL in its development from CD3^? to CD3⁺ at the small intestinal epithelium.     

Migration patterns of thymus-derived γδ T cells during chicken development

Cell transfer experiments in congenic chick strains, one of which expresses the ov antigen marker, indicate that intestinal γ δ T cells are derived from γ δ ⁺ thymocytes in embryos and newly hatched birds, and this early intestinal colonization occurs in two discrete waves. Here, we extend these studies to show that splenic colonization by γ δ T cells occurs in essentially the same way. Following the engraftment of ov⁺ thymic lobes in thymectomized ov^? recipients, γ δ T cells migrate both to the spleen and intestine. By 1 week after hatching, a third generation of thymus-derived γ δ T cells begins to migrate to both peripheral lymphoid organs, and this thymus-dependent seeding process is sustained over the first weeks of life. The survival time for splenic γ δ migrants is significantly less than for the intestinal migrants. Tissue section analysis indicates that γ δ T cells enter the intestinal epithelium at all villus levels. A shift in the γ δ intraepithelial lymphocyte distribution toward the villus tip in thymectomized birds suggests the comigration of enterocytes and γ δ intraepithelial lymphocytes. However, survival kinetics of the donor γ δ population and a relatively high division rate of intestinal γ δ T cells indicate that founder thymic migrants produce relatively long-lived clones of intestinal γ δ T cells.     

T cell receptor heterogeneity in γδ T cell clones from intestinal biopsies of patients with celiac disease

In celiac disease large numbers of γδ T lymphocytes infiltrate the intestinal epithelia. We have isolated intestinal γδ T cell clones from patients with celiac disease and have analyzed their T cell receptor repertoire. T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, αβ and γδ T cell receptor, by Southern blot with γ and δ specific probes and by polymerase chain reaction using Vδ-specific oligonucleotides. Intestinal γδ cells from patients with celiac disease differed from those of controls with normal jejunal histology in that Vδ1⁺ cells and Vδ Vδ2 cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of γδ T cell receptor.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

Lymphocytes bearing the γδ T cell receptor in acute Brucella melitensis infection

A phenotypical analysis carried out by indirect immunofluorescence and two-color cytofluorometry showed that the number of lymphocytes bearing the γδ T cell receptor (TcR) heterodimer was dramatically increased in the blood of six children with Brucella melitensis infection. Most in vivo expanded γδ T cells reacted with a monoclonal antibody which identifies Vδ2 gene products and a significant proportion expressed CD25 and HLA-DR activation antigens. In addition, whereas only a few γδ T lymphocytes were CD8⁺, nearly all were CD4^?. Highly enriched populations of both αβ and γδ T cells were obtained by negative immunoselection from three subjects with brucellosis sampled during convalescence. Despite the different form of their TcR, the proliferation of these two major T cell subsets in response to a mitogenic anti-CD3 monoclonal reagent (OKT3) was optimal. In contrast, αβ, but not γδ, T lymphocytes proliferated vigorously in response to the antigenic stimulus elicited by heat-killed Brucella. Further studies are, therefore, needed to determine whether the selective expansion of the γδ T cell subpopulation observed during the clinical course of the infection is driven by antigenic determinant(s) borne by the pathogen in vivo or is due to host-derived stimuli, such as autologous heat-shock proteins expressed on the surface of the infected cells.     

Development of γδ T cells in the adult murine thymus

The development of T cells belonging to the γδ lineage is not well understood. We have analyzed the cells in the adult murine thymus which express the γδ TcR on the surface in order to learn more about this process. Our data demonstrate a number of clear subpopulations of γδ expressing cells in the thymus based on the expression of Thy-1 and HSA (heat-stable antigen). Only one of these subpopulations, the one expressing both Thy-1 and HSA, contains dividing cells or has a significant rate of turnover. Together with the fact that emigrant γδ cells are HSA⁺Thy-1⁺, this suggests that this thymic subpopulation is the sole, or major, source of exported cells. However, the turnover of cells from this population is 5 × 10⁴ - 10 × 10⁴ cells per day, while previous estimates of the rate of export of γδ cells are in the order of 10⁴ cells per day. Furthermore the Vγ profile of recent γδ⁺ emigrants differs from that of the thymic HSA⁺Thy-1⁺ cells. This raises the possibility that only a selected subpopulation of the thymic γδ⁺HSA⁺Thy-l⁺ population is exported, and that some γδ cells may die in situ in the thymus. The function of the other γδ thymic subpopulations, which are turning over very slowly or not at all, (i.e. the HAS^?Thy-l^? and HAS^?Thy-l⁺ subpopulations) remains unclear.     

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

CD3−CD8+ intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL

Present evidence suggests that a majority of murine CD3⁺ intraepithelial intestinal lymphocytes (IEL) are extrathymically derived T cells and that these extrathymically derived IEL phenotypically express the CD8 homodimer (CD8αα). Recently, CD3^? IEL have been reported to express the recombination activating gene (RAG-1), suggesting that precursors to extrathymically derived CD3⁺CD8⁺αα IEL exist on the intestinal epithelium. To study in detail whether these CD3^? IEL can develop into CD3⁺CD8⁺αα IEL, we analyzed the CD3^? IEL subset and found that it can be separated into two subsets, namely CD3^?CD8^? and CD3^?CD8⁺ IEL. We show that (1) CD3^?CD8^? IEL are mostly small, non-granular and phenotypically Pgp-1⁺ IL-2R⁺ B220^?, while CD3^?CD8⁺ IEL are mostly large, granular and phenotypically Pgp-1^? IL-2R⁺ B220⁺, (2) CD3^?-CD8⁺ IEL express the RAG-1 gene, and (3) CD3^?CD8^?, CD3^?CD8⁺ and CD3⁺CD8⁺αα IEL, respectively, appear sequentially in normal ontogeny and in bone marrow-reconstituted thymectomized radiation chimeras. In the latter, virtually all CD3⁺CD8⁺αα IEL expressed the γδ T cell receptor (TCR), but not the αβ TCR. From this and what is presently known about T cell development, we propose that CD3^?CD8⁺ IEL are an intermediate in extrathymic IEL development and that the development of extrathymically derived IEL occurs at the intestinal epithelium from CD3^?CD8^? to CD3^?CD8⁺ to CD3⁺(γδ TCR)CD8⁺αα.     

Development of T cell receptor αβ-bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentration

Organ culture (OC) of murine fetal thymuses on a membrane filter floating on the medium has been used as an important strategy in studies of the mechanism of T cell development. On the other hand, the submersion organ culture (S-OC) system, in which fetal thymus lobes are submerged in the culture medium, is not popularly used because the growth of T cells is much lower than that in OC at the air-medium border (AMB-OC). In the present work, we tried to culture the fetal thymuses in the S-OC system at 5% CO₂ and various concentrations of O₂. We found that in the environment containing 5% O₂, γδ cells were selectively generated, though the cell recovery was less than one-tenth of that seen in AMB-OC. Generation of γδ T cells was hardly affected by increasing the O₂ concentration. In marked contrast, the development of αβ T cells was highly dependent upon the O₂ concentration. In the S-OC at more than 60% O₂, differentiation and growth of T cells, as determined in terms of recovered cell number, CD4 vs. CD8 profile and predominance of αβ lineage, were comparable to those observed in AMB-OC. It was further shown that clonal deletion of Vβ6⁺ T cells occurred in high O₂ S-OC of CBA/J (Mls-1^a) but not C3H/HeJ (Mls-1^b) mice, the extent of the deletion being low but comparable with that seen in AMB-OC. These results indicated that the fetal thymus microenvironment was potentially capable of supporting the development of both γδ and αβ T cells, and that skewing to one of these lineages was determined by an additional factor(s) such as the concentration of O₂ dissolved in the medium.