Altered Tcell development function and selection in SKG mice

The ZAP-70W163C mutation and resultant altered signal transduction in T-cells then affects thymic development and differentiation of T-cells, in particular their positive and negative selection, responsiveness of mature T-cells to self and non-self antigens, and the generation and function of CD25+CD4+ natural regulatory T (TR) cells [22]

Compared with normal BALB/c mice, TCRhigh mature thymocytes decrease in number and ratio whereas TCRlow immature thymocytes increase in the SKG thymus, although the levels of TCR expression on mature thymocytes and peripheral T-cells are comparable between SKG and BALB/c. The number of peripheral CD4+ and CD8+ T-cells also decreases in SKG mice with a relative increase of B-cells.

Functionally, SKG T-cells are hyporesponsive to in vitro polyclonal T-cell stimulation, for example, with anti-CD3 monoclonal antibody (mAb), whereas they respond well to phorbol myristate acetate and ionomycin stimulation, indicating altered transduction of TCR-proximal signals in SKG T-cells. When the skg gene is homozygously introduced to DO11.10 TCR-transgenic (Tg) mice, in which the majority of T-cells express Tg TCRs specific for an ovalbumin (OVA) peptide, T-cells from such mice (designated DO.SKG mice) require ten times higher peptide concentrations to exhibit an equivalent magnitude of proliferation as DO11.10 T-cells. Furthermore, SKG thymocytes are less sensitive than BALB/c thymocytes to apoptosis induced by in vivo and in vitro TCR stimulation, in contrast to equivalent susceptibility to dexamethasone-induced apoptosis.

The ZAP-70W163C mutation notably affects thymic positive and negative selection of T-cells. In DO.SKG (i.e., DOskg/skg) mice, the number of CD4+CD8- mature thymocytes decreases to ~20% of normal DO11.10 mice. Furthermore, only ~30% of CD4+CD8- thymocytes and CD4+ peripheral T-cells are Tg-TCRhigh in contrast to ~80% in DO11.10 mice (Fig. 2a); the rest of CD4+CD8- thymocytes/T-cells (i.e., Tg-TCRlow thymocytes/T-cells) in DOskg/skg mice appear to express endogenous TCR a-chains associated with transgenic P-chains, as the majority of their T-cells express transgenic P-chains at equivalent levels to DO 11.10 mice. In skg-heterozygous DOskg/+ mice, the reduction of the percentage of Tg-TCRhigh T-cells and increase in Tg-TCRlow T-cells in the thymus and the periphery are intermediate between DOskg/skg and DO11.10 mice (Fig. 2a). Similar findings were also made with HY-TCR Tg mice that express transgenic TCRs specific for male-specific HY antigens on an H-2b background (Fig. 2b). Female HY-TCR Tg mice positively select HY-TCR+CD8+CD4- thymocytes, whereas female HY-TCR Tg mice with the homozygous skg gene (designated HY-TCR.SKG Tg mice) on an H-2b background hardly show positive selection of HY-TCR+CD8+CD4- thymocytes and T-cells. On the other hand, in contrast to substantial deletion of HY-TCR+CD8+CD4- thymocytes in male HY-TCR Tg mice, male HY-TCR.SKG Tg mice show efficient positive selection of HY-TCR+CD8+CD4- thymocytes. Notably, both DO.SKG and HY-TCR.SKG mice develop arthritogenic T-cells expressing endogenous TCR a-chains paired with transgenic P-chains, and consequently autoimmune arthritis at high incidences, due to positive selection of self-reactive T-cells that would not be positively selected in normal TCR Tg mice.

Figure 2

Altered thymic T-cell selection in SKG mice.

A. Staining of thymocytes or lymph node cells from a 4-month-old DO11.10, DOskg/skg, or DOskg/+ mouse with indicated mAbs. KJ1.26 is a mAb specific for the transgenic TCR. Dot-plot figures are scaled logarithmic. Ordinates of histograms denote cell number (arbitrary units). B. Staining of thymocytes or lymph node cells from a 4-month-old female or male HY-TCR or HY-TCR.SKG transgenic mouse with indicated mAbs. C. Selection shift of the T-cell repertoire and thymic production of arthritogenic T-cells.

DO11.10

DOskg/+

Thymocytes 2.0% 1.5x108

9.0x107

Doskg/skg

CD4+8-Thymocytes i

10.1%1.0x108

KJ1.26

LN cells CD4+T cells

L"

KJ1.26

KJ1.26

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