Influence of Ly49 inhibitory receptors and MHC class I on T cell and NK cell function

Abstract

Natural Killer cells (NK) can kill virus infected cells and tumor cells but do not harm normal surrounding cells. The function of Ly49 receptors on NK cells is important in maintaining selftolerance since they transmit inhibitory signals when bound to endogenous MHC class I molecules. Their role in T cells is however unclear. We showed that a Ly49A transgene expressed on all T and NK cells altered T cell development in an in vivo environment, where a ligand for Ly49A was expressed. Ly49A transgenic mice that co-expressed an NIM ligand for Ly49A, H-2Dd , developed a severe inflammatory disorder that resulted in death within the first weeks of age. These data indicated that the expression of Ly49A on T cells altered T cell selection and allowed survival of potentially self-reactive T cells.

Furthermore, using Ly49A/TCRalphabeta transgenic mice, we observed a quantitative influence on cellular responses that depended upon the activating signals received through the TCR and the inhibitory signals received through Ly49A. Low activation signal resulted in T cells that were unresponsive for target cells that expressed the Ly49 ligand H-2Dd, but responded against target cells without H-2Dd. However, this inhibition could be overcome by increasing the concentration of peptide for which the T cells were specific, i.e. the activation signal. Thus, rather than behaving as simple "off' switches, our data indicated that Ly49 receptors modulate T cell signaling so that higher amounts of activating signals are required for effector-cell responses.

A prediction from the "missing-self' hypothesis is that down-regulation of MHC class I on resting haematopoietic cells should be sufficient to make them susceptible to NK cell killing. We used a method enabling kinetic and quantitative assessments of NK cell-mediated rejection responses in vivo, and showed that resting haematopoietic cells from beta2-microglobulin-deficient (beta2m-/-) mice were rapidly rejected in unmanipulated C57BL/6 (B6) mice. The signaling adaptor K-ARAP/DAP12 was dispensable for rejection of beta2m-/- cells (lacking MHC) but critical for rejection of BALB/c cells (mismatched MHC) in unmanipulated B6 recipients while pre-activated B6 mice rejected BALB/c cells in a KARAP/DAP12-independent fashion. Loss or mismatch of MHC class I in resting cells was thus sufficient to convey susceptibility to NK cell rejection. However, activation of the effector or the target enhanced rejection and shifted the balance between different signaling pathways involved.

Using the same method we injected beta2m-/- and control B6 spleen cells into mice with a nonfunctional CD1d1 gene (CD1d1-/-(LVK)) and hence no NKT cells. CD1d1-/-(LVK) mice lacked the ability to reject beta2m-/- cells in vivo after 48 hours. They were also defecitive in rejection of TAP1/2-/- cells and inhibition of RMA-S tumor outgrowth, but were able to reject allogeneic cells (BALB/c) similar to B6 mice. To determine if the CD1 molecule per se was involved we tested another CD1-deficient mouse strain (CD1-/-). These mice rejected beta2m-/cells as well as B6 mice, indicating that the CD1 molecule was not important for rejection of cells lacking MHC. Thus, the CD1d1-deficient mice displayed an additional defect, unrelated to the targeted CD1d1 gene.

Interestingly, rejection of targets with allogeneic MHC class I molecules remained intact. Further investigation of these mice will potentially provide an answer as to how NK cells are activated to sense "normal" cells and certain tumor cells not displaying known activating ligands. Additionally, these mice strongly displays why it is necessary to critically evaluate data from genetically altered mice.

Together, these studies contribute to our understanding of how Ly49 inhibitory receptors and the MHC class I regulate T cell selection and function and NK cell activity and tolerance.