Analysis of the Effects of A Gain of Function Mutation of Rho (

The finding that loss of Rho function did not prevent T cells from making contacts with antigen-presenting cells and the finding that activation of RhoA increased integrin receptor mediated cell adhesion of T cells, raised the question whether T cell specific expression of a constitutively active mutant of RhoA (V14RhoA) had any effects on T cell/APC interaction.

To address this question, P14 TCR transgenic mice that expressed a constitutively active mutant of RhoA (V14RhoA) were used. The data in Figure 7.4A compare the ability of normal control P14 TCR CDS SP T cells and P14 TCR CDS SP T cells from V14RhoA mice in the formation of T cell/APC conjugates over a time course. The data in

Figure 7.4B evaluate conjugate formation in response to increasing concentrations of the

specific LCMV peptide GP (33-41).

The data show that for normal T cells, conjugates were only formed when APCs were pulsed with the specific peptide. Conjugates could be detected within 1 hour of mixing the T cells with the APCs and the percentage of T cells that formed conjugates with APCs increased up to a 5 hour time point. At a 24 hour time point the numbers of conjugates were decreased. Figure 7.4 shows that V14RhoA-expressing T cells did not form conjugates in the absence of the specific antigen but they did make conjugates if the APCs were pulsed with the specific peptide. Activation of RhoA was able to promote integrin-mediated cell adhesion in primary T cell populations and this increased adhesion was expected to promote contacts between the T cell and APC. However, the peptide dose response curves (Figure 7.4B) showed that V14RhoA T cells were less efficient in conjugate formation than normal cells; they were also slower in conjugate formation than normal cells (Figure 7.4A). In an effort to understand why V14RhoA T cells were less efficient in conjugate formation the TCR expression levels were compared in normal and V14RhoA T cells. P14 TCR/V14RhoA T cells showed a subtle reduction in TCR expression levels (Figure 7.5) but this effect was minimal and seems unlikely to be the reason for the decreased conjugate formation, especially since a much larger reduction in TCR levels was observed in P14 TCR/LckC3 T cells and was not associated with any major defects in conjugate formation.

A)

T cell - Antigen Presenting Cell (APC) Conjugate Formation Time course (10 nM peptide : GP33 - 41) 1 6n O P14NLC •P 14V 14R h o A (/) S 1 2- (0 Ui 3 F

8

Figure 7.4. CDS SP T cells from TCR-transgenic mice that express a constitutively active mutant of Rho (P I4 V I4 RhoA) form conjugates with antigen presenting cells (APC) less efficiently than normal T cells . Purified B cells were activated with LPS, pulsed with the specific peptide (100 nM: gp 33-41) over night and were mixed with purified CD8 SP peripheral T cells from P14 TCR transgenic mice: P14 TCR NLC and P14 TCR V14RhoA, respectively. After the indicated time-points the cells were stained for 8220 and Thyl .2 and analysed by flow cytometry for cells that stain positive for both, 8220 and T h y l.2. (A) Time course (10 nM peptide). (B) Different peptide concentrations (5 hrs).

VpS I TCR Expression

— P14TCRV14RhoA [¥1 P14TCRNLC

I 1 11 III

Vp8

Figure 7.5. TCR levels on peripheral blood CD8 SP T cells from normal TCR transgenic mice (P14 TCR NLC) and TCR- transgenic mice that express a gain of function mutation of RhoA (P14 TCR V14RhoA). Peripheral blood cells were stained for CD4, CDS and Vp8 and analysed by flow cytometry. Overlay histogram shows expression of the V(38 chain of the apTCR, gated on CD8 SP T cells.

7.3 Discussion

T cells are activated by antigen presented on the surface of antigen-presenting cells and the formation of stable contacts between T cells and APCs is necessary for T cell activation (Tseng and Dustin, 2002). T cell interactions with APCs are mediated by a number of different adhesion molecules including integrins (Sims and Dustin, 2002). In particular, for the P14 TCR, LFA-1 has been shown to be crucial (Bachmann et al., 1997). Thus when the P14 TCR is expressed on an LFA-1 null background the resultant T cells do not activate efficiently. For integrins to function during the formation of stable conjugates between T cells and APCs it is necessary that they are activated. The current model for how this happens is that initial triggering of the TCR by antigen/MHC results in integrin activation and the active integrins then allow a stronger adhesion of the T cell to the APC, thereby stabilizing the T cell/APC conjugate (Sims and Dustin, 2002). A key finding of the present chapter is that Rho function is not absolutely required for T cell/APC conjugate formation, and that activation of RhoA actually decreases the efficiency of T celFAPC conjugate formation. A conclusion from these data is that Rho is not part of the signal transduction pathways used by the TCR to activate integrins.

Previous studies have shown that when the TCR cannot activate integrins or T cells which fail to express LFA-1, activate and proliferate very poorly in antigen dependent systems (Bachmann et al., 1997). T cells lacking the adapter FYB/ADAPl which is critical for integrin activation activate poorly in response to TCR triggering (Griffiths et al., 2001; Peterson et al., 2001). Similarly, loss of function of the GTPase Rap-1, another key activator of integrins in T cells, causes T cells to activate poorly in response to antigen/MHC (Katagiri et al., 2002).

Adhesion studies with purified matrix proteins have shown that Rho function is necessary for Rac-1 and Rapl A integrin-mediated cell adhesion (Chapter 4). The failure to find an involvement for Rho in the ability of T cells to form conjugates with APCs was thus surprising, because studies have suggested both Rac-1 and Rapl A are required for T cell conjugate formation with antigen-pulsed APCs (Ardouin et al., 2003; Katagiri et al., 2002; Krawczyk et al., 2002). Hence, loss of Rapl function by expression of a dominant negative N17Rapl or by overexpression of the Rapl GTPase activating protein SPA-1

totally abrogates T cell/APC conjugate formation (Katagiri et al., 2002). The Rac-1 guanine nucleotide exchange factor Vav-1 has also been shown to be required for a normal T cell/APC conjugate formation (Ardouin et al., 2003; Krawczyk et al., 2002). The formation of the T cell/APC synapse is followed by polarization of the cell, as exemplified by the movement of the microtubule-organizing centre (MTOC) within the T cell to face the APC. A greatly reduced fraction of Vav-1 deficient thymocytes showed polarization with the MTOC facing the contact site. Thus, Vav-1 is required to transduce TCR signals, leading to the reorientation of the MTOC to face the APC, suggesting that it may participate in signalling pathways that control the dynamics of the microtubule cytoskeleton. This activity may be due to Vav’s GEF activity, as a number of reports have implicated Rho-family GTPases in the regulation of microtubules (Palazzo et al., 2001; Vicente-Manzanares et al., 2002). In this context MTOC reorientation in LckC3 T cells was not looked at specifically in the studies presented in this Chapter. The only way to resolve this point would be to do detailed analysis of T cell/APC conjugates using confocal microscopy.

Expression of active RhoA or active Rapl A results in integrin activation in T cells. Studies in pro-B cells have described that expression of the active Rapl mutant V12Rapl can cause LFA-l/lCAM-1 dependent cell aggregation (Katagiri et al., 2000). In this chapter it is shown that expression of constitutively active Rho did not cause T cells to spontaneously aggregate with APCs rather it decreased the efficiency of T cell/APC conjugate formation (Figure 7.4). The explanation for this decrease is not known. It was noted that activated V14RhoA T-Lymphoblasts are very round and do not polarize normally (Figure 3.14 Chapter 3), this could impair their ability to make contacts with APCs.

Interestingly, despite the fact that V14RhoA T cells appear to be less efficient in making conjugates with APCs, these cells are hyper-responsive in the context of TCR- induced responses in vivo and in vitro (Corre et al., 2001). For example, in the context of defined TCRs, V14RhoA causes an increase in positive selection. In vitro, V 14RhoA T cells are also hyper-proliferative to CD3 antibodies. The apparent discrepancy between decreased conjugate formation but increased TCR responsiveness could be explained if active RhoA could substitute for some of the costimulatory signals normally provided by APCs. It is normal for T cells to dissociate from APCs once they are activated, but the molecular basis for this dissociation is not clear. One possibility is that active RhoA mimics the signals that activated T cells generate to initiate the dissociation process.

Final Summary of Results

The results presented in this study are as follows:

1. Expression of activated RhoA stimulates (3, and p2 integrin-mediated adhesion of

primary thymocytes and peripheral T cells.

2. Expression of activated RhoA did not induce any discernible morphology changes in primary thymocytes whereas T lymphoblasts expressing active RhoA are large granular cells.

3. Rho function is necessary for efficient pj integrin-mediated adhesion of thymocytes.

4. Investigation of potential signalling networks in integrin-mediated thymocyte adhesion revealed an existing cross-talk between Rho and the GTPases Rac-1 and Rapl A. It was shown that in thymocytes activation of Rho is necessary for enhanced p, integrin-mediated adhesion induced by activated Rapl A and further for Pi integrin-mediated adhesion induced by activated Rac-1.

5. The level of Rho activity is critical for T cell migration. Rho function is necessary for chemokine-induced migration in vitro and the results further imply that Rho function is also required for peripheral emigration of mature SP thymocytes in vivo.

6. Activation of RhoA did not potentiate the ability of T cells to form antigen specific conjugates with APCs nor was Rho function absolutely required for this process. The regulatory actions of RhoA on integrins thus have a selective role on T cells: they do not facilitate and stabilize contacts between the T cell and cells presenting antigen/MHC complexes (APCs), rather they are important for lymphocyte motility.

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