A Novel Mechanism for CTL Mediated Veto Activity: CTLs Respond to Recognition by Alloreactive T Cell with Activation and Cytotoxic Granule Secretion.
Authors: Milstein Oren , Lask Assaf , Reich-Zeliger Shlomit , Reisner Yair 
Published: April 2020

Abstract
Cytotoxic T lymphocytes (CTLs) are endowed with potent veto activity. As such, they induce death of alloreactive T cells directed against them, in a manner that does not require engagement of their own TCR with MHC-p of the alloreactive cells. Thus, CTLs are currently being evaluated in patients for their potential to induce specific transplantation tolerance. CTL veto activity has been shown to be mediated by engagement of the CTL CD8 molecule with the alloreactive T cell MHC class I α3 domain. We have further shown a role for Fas/FasL in CTL veto activity when assayed at late time points and at low CTL concentrations. In the current study, we apply imaging for the first time to observe CTL-alloreactive CD8+ T cell interactions at the cell-cell level at early time points. For this we employed a model of veto activity in which the alloreative cell is a 2C (H-2b) TCR transgenic CD8 T cell, alloreactive towards H-2d, and the CTL is of an F1 origin (H-2b X H-2d) and thus readily recognizable by the 2C cell, yet void of alloreactivity towards it. Confocal microscopy revealed that F1 CTLs polarize their cytotoxic granules towards the contact area with the 2C cells. This occurred in 71% of contacts and was indicative of granule mediated killing. Accordingly, we found that 46% (±1.2) of 2C cells are killed by F1 CTLs within 5 hours when cultured at a 1:1 ratio. This killing is strictly granule mediated as the intracellular Ca2+ chelator Bapta-AM completely abolishes killing (2% ±2.5). Moreover, this granule mediated killing is reliant on perforin as concanamycin A reduces killing to 15% (±4.2). To directly link granule polarization and secretion with alloreactive cell death, live cell imaging was applied. This revealed that CTL granule polarization occurs rapidly after contact and that alloreactive T cell death follows within a median time of 35 minutes. As F1 CTLs do not recognize the alloreactive cell MHC-p, the signaling event leading to degranulation is not initiated by TCR. The importance of the interaction of the CTL CD8 molecule with the target cell MHC class I α3 domain documented for TCR dependent granule mediated killing, prompted us to evaluate its importance in granule mediated veto activity. To that end, a blocking Ab (Lyt 2.1) which specificaly blocks the CTL CD8 allelic form was added and was found to significantly reduce killing of 2C cells (23% ±7.7, p&lt;0.01) compared to control Ab. (46% ±2.2). Moreover, blocking the CTL CD8 molecule had a detrimental effect on granule polarization. As the primary candidate for relaying a signal from CD8 into the cell is Lck, we determined the effect of the Src kinase inhibitor PP2 on CTL veto activity. Pretreatment of CTLs with PP2 significantly reduced killing (21% ±2.3, p&lt;0.01) compared to PP3 treated control CTLs (54% ±5%). For Src kinase signaling to elicit granule secretion, activation of protein kinase C (PKC) would be necessary. As activated PKC 𝛉 is recruited to the cell-cell contact site during TCR dependent killing by CTLs, its localization during veto activity was studied. Confocal imaging revealed that in 39% of conjugates CTL PKC 𝛉 is clearly recruited to the contact area with the 2C cell. Remarkably, blocking Src kinase signaling completely eliminated this recruitment. Collectively, these results show that CTLs are capable of granule mediated killing of T cells directed against them and that this killing originates from a wide-ranging signaling event initiated in the absence of TCR engagement, yet involving both Src kinase signaling and PKC activation.