Angelika Rudolphi

Regional Specialization of Intraepithelial T Cells in the Murine Small and Large Intestine

We investigated intraepithelial T cells from the small intestine, SI (jejunum, ileum) and the large intestine, LI (colon) of euthymic (BALB/c, H–2d; C. B–17 +/+, H–2d; C57BL/6, H–2b) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. From individual euthymic and athymic mice, 7 × 106 intraepithelial lymphocytes (IEL) per mouse were isolated from the SI. Ten–fold lower numbers of IEL were obtained from the LI epithelium (4 × 105 IEL per mouse). Thymus–dependent and ‐independent T cells represented > 80% of SI–IEL but the fraction of T cells was reduced from 20% to 40% in LI–IEL. In euthymic mice, αβ T cells predominated in SI–IEL and in particular in LI–IEL populations, while SI–IEL and LI‐IEL populations of athymic mice contained predominantly αβ T cells. The intraepithelial T cell subset distribution was different in SI versus LI: mainly CD8+ T cells were present in the SI, but a large CD4+ T cell subset was present in the LI.‘Double positive’ CD4+ CD8α+ T cells were present mainly in the SI epithelium but were rare in the LI epithelium. In euthymic as well as athymic mice, T cells expressing the homodimeric CD8αβ isoform predominated in the SI epithelium, while T cells expressing the heterodimeric CD8αβ isoform predominated in the LI epithelium. LI–derived TCRα+ IEL displayed the CD2+ CD28+ LPAM–1/2− M290+ phenotype, and a fraction of them expressed the L–selectin LECAM–1. In contrast, a large fraction of TCRα+ SI‐IEL was CD2− CD28− LPAM–1/2− M290+ and LECAM–1−. RAG–1/2 expression was detectable by RT–PCR in IEL from the SI but not the LI. Striking differences in phenotype were thus apparent between thymus–dependent and thymus–independent T cells in the epithelial layer of the jejunum/ileum and the colon of the mouse.

CD4+ T lymphocytes injected into severe combined immunodeficient (SCID) mice lead to an inflammatory and lethal bowel disease

Transfer of 2 × 105 congenic or semiallogenic purified TCRαβ+ CD4+ T cells to SCID mice leads to an infiltration of the recipient gut lamina propria and epithelium with a donor‐derived CD4+ T cell subset which induces a lethal inflammatory bowel disease (IBD) in the recipients. In contrast, IBD was not observed in SCID mice transplanted with unfractionated splenic cells. The earliest detectable pathological changes after CD4+ T cell transfer were proliferation and hypertrophy of the entire colonic epithelial layer, including increased mitotic activity, increased expression of epithelial nuclear proliferation antigen, and elongation of the crypts. Later on, massive mononuclear cell infiltration, hypertrophy of all layers of the colon and occasional epithelial ulcerations were observed. At this stage, accumulations of IgA, IgM and small numbers of IgG1‐, IgG2‐and IgG3‐secreting plasma cells were present in the lamina propria of both the small and large intestine. We conclude that low numbers of intraveneously transferred CD4+ T cells induce IBD in SCID mice. In the late stages of CD4+ T cell‐induced IBD, the colonic lamina propria becomes infiltrated with macrophages, neutrophils and plasma cells secreting IgA, IgM, and to a lesser degree IgG antibodies which might play an accessory role in the pathogenesis of IBD.

Adoptive Transfer of Low Numbers of CD4+ T Cells into SCID Mice chronically treated with Soluble IL‐4 Receptor does not prevent Engraftment of IL‐4‐Producing T Cells

After intravenous injection of 105 purified, lymph node (LN)‐derived dm2 (H‐2d/Ld) CD4+ T cells into young C.B‐17 scidjscid (severe combined immunodeficiency, SCID) mice (H‐2d/Ld+), the transplanted Ld‐ T cells show a selective pattern of engraftment: they repopulate the spleen, the lamina propria of the small intestine and the mesenteric LN (but not other peripheral LN) of the immunodeficient host. CD4+ cells repopulating different lymphoid organs of the SCID recipient mice produce interleukin‐2 (IL‐2) and interleukin‐4 (IL‐4) in response to polyclonal stimulation in vitro. Some evidence has recently been provided that cytokines (e.g. IL‐4) present at the site of antigen stimulation in vivo decisively influence the pattern of cytokines expressed by T cells activated at these sites. We therefore asked if neutralization of IL‐4 by chronic treatment of SCID mice with high doses of recombinant soluble IL‐4 receptor (sIL‐4R) changes the IL‐4 or IL‐2 expression pattern of CD4+ T cells adoptively transferred into young SCID recipients. Transplanted SCID mice were chronically treated with two different, recombinant murine sIL‐4R proteins. The experimental series further included groups of transplanted SCID mice treated with a recombinant human sIL‐4R protein (which does not bind murine IL‐4), treated with the anti‐murine IL‐4 monoclonal antibody (MoAb) 11B11, or non‐treated. Transplanted SCID mice treated with the recombinant murine sIL‐4R protein preparations displayed detectable sIL‐4R serum levels, which demonstrates that the substitution therapy could maintain neutralizing serum levels of anti‐IL‐4 activity in SCID mice. By contrast, no serum sIL‐4R levels were detectable in the sensitive ELISA readout in transplanted SCID mice which were non‐treated, treated with the MoAb 11B11, or treated with the recombinant human sIL‐4R protein. The efficiency and the pattern of CD4+ T‐cell engraftment, and the lymphokine‐producing phenotype of the engrafted dm2 CD4+ cells, was not affected by the continuous IL‐4‐neutralizing treatment of mice with either the MoAb 11B11 or the soluble IL‐4R preparations. Hence, in contrast to the published evidence of the dramatic effect of IL‐4 on the lymphokine‐producing phenotype of CD4+ T cells stimulated in vitro or in vivo, the chronic suppression in vivo of IL‐4 activity (by either different sIL4‐R protein constructs, or by the anti‐IL‐4 MoAb 11B11) did not lead to preferential engraftment of Th1‐type CD4+ T cells after adoptive transfer of CD4+ T‐cell populations into an immunodeficient recipient.

CD3+ T Cells in Severe Combined Immunodeficiency (SCID) Mice. V. Allogeneic T Cells Engrafted into SCID Mice Do Not Induce Graft‐Versus‐Host Disease in spite of the Absence of Host Veto and Natural Suppressor Cell Activity

Intravenous injection of 106 to 107 non‐fractionated spleen ceils (SC) from C57BL,6 (B6. H‐2b) mice into completely allogeneic, immuno deficient H‐2d severe combined immuno deficiency (scid) mice leads to engraftment of allogencic donor T cells. Mice analysed in the tenth week post‐transfer had engrafted donor‐type CD4+ and CDS+ T cells in the spleens but showed no clinical evidence of graft‐versus‐host disease (GVHD). Transfer of allogeneic T cells engrafted in scid recipients did not induce GVHD upon i.v. injection into secondary scid recipients and lead in most recipients to engraftment of a pureCD4+ T‐cell population. Experiments were carried out to investigate the reason(s) for the lack of GVHD in recipient scid mice, i.e. the presence of allotolerance in the engrafted donor T cells. Scid spleen cells (SC) efficiently stimulated alloreactive responses of B6 T cells: scid SC stimulated H‐2d‐specific cytotoxic responses in a B6 antiscid mixed lymphocyte culture in vitro, and scid SC injected i.v. into B6 mice efficiently primed splenic cytotoxic lymphocyte precursors against H‐2d alloantigens. Moreover when assayed in vitro, no veto activity or natural suppressor activity was detectable in scid SC. These data demonstrate that tolerizing mechanisms currently believed to operate in vivo can not explain the fact that allogeneic T cells injected i.v. into immunodeficient scid mice become tolerized against host‐type alloantigens. Our results are discussed in the tight of clinical experience of allogeneic T‐cell transfer in scid infants.