Rosemary K. Lees

A Role for CD147 in Thymic Development

We have previously identified a mAb that binds to a molecule expressed preferentially on the surface of cycling thymocytes. In this study the molecule recognized by this mAb has been identified in the mouse as CD147 (basigin) by expression cloning. We show that CD147 expression correlates with cycling of immature thymocytes even in the absence of TCRβ selection and that ligation of this molecule on immature fetal thymocytes inhibits their further development into mature T cells.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM–ITAM signaling is likely to play an important role in the developmental program of NKT cells.

TCR-α CDR3 Loop Audition Regulates Positive Selection

How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-β-transgenic mice in which MHC shapes TCR-α use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of αβ pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the α CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.

Selective absence of CD8+ TCRα β+ intestinal epithelial cells in transgenic mice expressing β2‐microglobulin‐associated ligands exclusively on thymic cortical epithelium

Whereas interactions between the TCRα β and self MHC:peptide complexes are clearly required for positive selection of mature CD4+ and CD8+ T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14‐β2‐microglobulin (K14‐β2m)‐transgenic mice expressing β2m‐associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8+ T cellhomeostasis. Our data indicate that CD8+ T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8+ T cells in the intestinal epithelium is severely affected by the absence of β2m‐associated ligands. Indeed TCRα β+ IEL subsets expressing CD8α β or CD8α α are both dramatically reduced in K14‐β2m mice, suggesting that the development, survival or expansion of CD8+ IEL depends upon interaction of the TCR with MHC class I:peptide or other β2m‐associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8+ T cell homeostasis by β2m‐associated ligands in the intestine as compared to peripheral lymphoid organs.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Does negative selection involve accumulation of self-reactive thymocytes in thymic rosettes?

Thymic rosettes, the natural associations between thymocytes and either macrophages or dendritic cells, were isolated from the thymus by collagenase digestion and unit-gravity elutriation. Rosettes from mouse strains where either the V beta 6-bearing thymocytes are deleted because of reactivity with products of the Mlsa allele of the minor lymphocyte stimulating locus, or where V beta 17a-bearing thymocytes are deleted because of reactivity with IE class II MHC molecules, were compared with rosettes from appropriate control strains to test if a selective association with stromal cells preceded deletion. Rosettes from an Mlsa-bearing strain were able to stimulate an Mlsa-reactive T-hybridoma, but much of this stimulatory activity was attributable to the few B cells associated with the rosette preparations; the stromal components of the rosettes appeared to be poor presenters of Mlsa gene products. There was no enrichment of thymocytes bearing high or low levels of V beta 6 TcR in the rosettes from the Mlsa-bearing strain, which might have reflected the poor presentation by the stromal cells. However, nor was there detectable selective association of thymocytes bearing C beta 17a in the rosettes from an IE-positive mouse strain. This argues against binding and immobilisation on stromal cells as part of the deletion process, but not against the stromal cells delivering a rapid signal during a transient association, leading later to deletion.

Multicellular Tumor Spheroids of Human Colon Carcinoma Origin

The relationship between destruction and concomitant host cell infiltration of human tumor xenografts has been quantitatively investigated by using the multicellular tumor spheroid model. Multicellular tumor spheroids of HT-29 human colon carcinoma cells were grown in vitro and subsequently implanted in the peritoneal cavity of BALB/c mice. At various times thereafter, spheroids were recovered and dissociated and their viability was quantitatively assessed by using a clonogenic assay. Little damage to spheroids was observed during the initial 4 days after implantation, but essentially complete destruction (greater than 99% reduction in clonogenic tumor cells) occurred between days 4 and 7. In parallel studies, host cell infiltration was assessed by light and electron microscopy both in situ on sections and on dissociated suspensions of spheroid cells. The data demonstrate the value of utilizing a model system in which both functional and morphological techniques can be combined in a quantitative assessment of the relationship between host cell infiltration and graft destruction in situ.