COMPONENTES BIOACTIVOS DE LOS HONGOS.

Since the aberrant expression of maturation markers TCRβ, CD5 and CD69 suggested an impaired positive selection of DP thymocytes into SP cells in the absence of RhoH, we analyzed positive selection of DP thymocytes into the CD4 lineage using an OT-II TCR transgenic model. We intercrossed RhoH-deficient mice backcrossed to C57BL6 with OT-II transgenic mice, which express an ovalbumin specific TCR restricted to MHC I-Ab as present in the RhoH-null mice and examined thymocyte subsets in these mice.

Control mice expressing OT-II TCR were expected to show an increase in CD4SP and decrease in DP and CD8SP thymocytes. Within the DN compartment, the DN4 population was supposed to be elevated and the DN3 population reduced.

In the OT-II TCR transgenic mice, the loci for the transgenic OT-II TCRβ and TCRα chain are already pre-rearranged, with the consequence that the rearrangement of the endogenous TCRβ and TCRα loci is largely suppressed and the OT-II TCRα and TCRβ chains are preferably expressed. OT-II TCRα and TCRβ pair and form together with CD3 the OT-II TCR, which is the predominant TCR expressed at the DN and later stages. The transgenic mice, however, are not on the RAG-/- background and express the recombination activating gene (RAG) enzyme, which is involved in the rearrangement of the TCR genes. Therefore, rearrangement of the loci for the endogenous TCRβ and TCRα chain can still occur, resulting in the endogenous pre-TCR and TCR expression at the DN and DP stage, respectively, although to a very low degree.

The assembly of and signaling through the OT-II TCR at the DN3 stage gives a positive signal for proliferation and survival, promoting further differentiation and resulting in the enlargement of the DN4 subset. At the DP stage of the thymocyte development, the low- affinity interaction between the OT-II-TCR on DP thymocytes and the MHC class II-self- antigen-complex on the thymic epithelial cells triggers TCR signaling that allows the development of CD4SP cells. CD8SP cells expressing OT-II TCR cannot be generated since OT-II TCR is MHC class II restricted and requires CD4 co-expression on a T cell for binding the antigen presenting cell.

FACS analysis of control mice expressing OT-II TCR confirmed our expectations. Control mice expressing OT-II TCR revealed a skewing to the CD4+ T cell subset and reduction of DP thymocytes (Figure 37A and Figure 26A). Very little CD8SP could be detected in mice expressing OT-II TCR (Figure 37A). Furthermore, the percentage of DN3 cells was decreased, while the DN4 population was increased (Figure 37B and Figure 26B). The ovalbumin TCR was expressed at the DN stage, as detected by Vα2 antibody recognizing the

TCRα chain: nearly 60% of DN cells in control mice expressed OT-II TCR (Figure 38A). Approximately 80% of DP thymocytes and nearly 100% of CD4SP cells carried OT-II-TCR (Figure 38A). In spleen, the skewing towards CD4 lineage was also detected and almost 100% of CD4+ splenic cells expressed OT-II-TCR (Figure 39).

In accordance to TCR signaling, the expression of CD5 increased in the transition from DN to DP and to CD4SP cells (Figure 38B), as well as the expression of the CD69 maturation marker rose on CD4SP cells in the control OT-II transgenic mice (Figure 38C).

Figure 37. Defective positive selection in the absence of RhoH. (A) Thymocytes of 4-7 week old OT-II mice

transgenic for an ovalbumin specific TCR, either expressing or not expressing RhoH were analyzed for the expression of CD4 and CD8 by FACS (upper panel). Quantification of absolute numbers of thymocyte subpopulations (lower panel; *: p< 0.05; **: p<0.01; ***: p<0.001; n=3/4). (B) Thymocytes of 4-7 week old OT-II mice transgenic for an ovalbumin specific TCR, either expressing or not expressing RhoH were gated for lineage negative (B220, CD4, CD8, NK1.1, Mac1, Gr-1, Ter119) cells and analyzed for the expression of CD25 and CD44. (DN1 (CD25-CD44+); DN2 (CD25+CD44+); DN3 (CD25+CD44-); DN4 (CD25-CD44-); upper panel). Quantification of absolute numbers of thymocyte subpopulations (lower panel; *: p< 0.05; n=3/4).

In contrast, RhoH-deficiency severely impaired generation of CD4SP cells in mice expressing the ovalbumin TCR, indicating a defect in positive selection (Figure 37A and Figure 26A). Moreover, no increase in DN4 population was observed, suggesting a defective β-selection, which controls the DN3 to DN4 transition (Figure 37B and Figure 26B). Consequently, no skewing towards CD4+ T cells in spleen was detected in RhoH-null mice expressing OT-II TCR (Figure 39).

The following data comprising the expression of TCRVα2, CD5 and CD69 on RhoH- deficient thymocytes confirm the assumption of impaired TCR signaling and defective positive selection. Among the DN cells, which upregulate Vα2 expression during β-selection,

RhoH-null mice showed a reduced amount of Vα2+ cells compared to controls (Figure 38A). Nearly all DP and CD4SP thymocytes expressed the ovalbumin specific TCR in mutant mice, although the expression level of Vα2 on DP cells was lower than in control OT-II transgenic mice and within the RhoH-deficient CD4SP cells and CD4+ splenocytes a population expressing lower Vα2 levels could be detected (Figure 38A and Figure 39).

Also in the presence of the ovalbumin TCR, the percentage of CD5low cells was higher on DN, DP and CD4SP thymocytes in RhoH-null mice compared to control transgenic mice (Figure 38B), and the relative amount of CD69high cells was lower on DP and CD4SP cells (Figure 38C).

Figure 38. Aberrant expression of maturation markers on RhoH-deficient thymocytes in mice expressing transgenic OT-II TCR. (A) Decreased

amount of Vα2+ DN and CD4SP thymocytes in 4-7 week old OT-II mice transgenic for an ovalbumin specific TCR in the absence of RhoH (*: p< 0.05; **: p<0.01; n=4/4). (B) Increased amount of CD5low thymocytes in 4-7 week old OT-II mice transgenic for an ovalbumin specific TCR in the absence of RhoH (**: p<0.01; ***: p<0.001; n=4/4). (C) Decreased amount of CD69high thymocytes in 4-7 week old OT-II mice transgenic for an ovalbumin specific TCR in the absence of RhoH (*: p< 0.05; **: p<0.01; n=4/4).

Figure 39. Reduced numbers of CD4+ splenocytes in mice expressing OT-II TCR in the absence of RhoH.

(A) Splenocytes of 4-7 week old OT-II mice transgenic for an ovalbumin specific TCR, either expressing or not expressing RhoH were analyzed for the expression of CD4 and CD8 by FACS. (B) Decreased amount of Vα2+ CD4+ splenocytes in 4-7 week old OT-II mice transgenic for an ovalbumin specific TCR in the absence of RhoH (*: p< 0.05; **: p<0.01; n=4/4).