CD8 Coreceptor-Independent T Cell Stimulation Induces High Avidity CTL In the Presence of IL-21
Authors: Imataki Osamu , Ansén Sascha , Tanaka Makito , Butler Marcus O. , Berezovskaya Alla , Kuzushima Kiyotaka , Nadler Lee M. , Hirano Naoto 
Published: October 2019

Abstract 2086

CD8+ T cells play a primary role in rejecting pathogens and tumors. Studies of CD8 null mice show that CD8 coreceptor is critical for the development of MHC class I-restricted CD8+ T cells in the thymus. However, while these mice possess low numbers of CTL with limited clonality, they are highly avid and contain acute and chronic infections. In humans, CD8 deficiency leads to no or only mild symptoms of immunodeficiency. These results suggest that the CD8 coreceptor is not absolutely necessary for the generation of antigen-specific CTL and that there exists a compensatory mechanism for the loss of CD8 expression.

Common γ chain receptor cytokines (e.g. IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) transmit STAT-mediated signaling in T cells. Importantly, it has been demonstrated that crosstalk between TCR and STAT signaling occurs in CD8+ T cells. For example, weak or partial TCR-initiated signaling via the Ras/MAPK pathway can be complemented by IL-2 induced STAT activation. This mechanism enables common γ chain receptor cytokines to modulate optimal and supplement suboptimal TCR signaling.

Recently, we reported the generation of K562-based artificial APC (aAPC) expressing HLA-A2 as a single HLA allele in conjunction with CD80 and CD83 (wt A2-aAPC). This aAPC can prime naïve CD8+ T cells ex vivo and generate antigen-specific CD8+ CTL with a central memory~effector memory phenotype. It has been demonstrated that the D227K/T228A mutations of A2 molecules (mut A2) inhibit the CD8/MHC interaction without affecting TCR/pMHC interactions. In this human ex vivo study, using a modified aAPC which expresses mut A2 molecules (mut A2-aAPC), we tested our hypothesis that CD8 coreceptor-independent T cell stimulation in the presence of complementary adaptive cytokines preferentially stimulates high avidity antigen-specific CTL.

When freshly isolated A2+ CD8+ T cells were restimulated against recall antigen with mut A2-aAPC, both antigen specificity measured by tetramer positivity and number of expanded antigen-specific CD8+ T cells were significantly lower compared with wt A2-aAPC. Similar results were obtained when naïve CD8+ T cells were primed ex vivo with wt-A2 aAPC in the presence of CD8 coligation and then restimulated with mut-A2 aAPC in the absence of CD8 binding. When naïve CD8+ T cells were initially primed with mut A2-aAPC in the absence of CD8 binding, subsequent restimulation using wt A2-aAPC in the presence of CD8 coligation was not able to induce the proliferation of antigen-specific CD8+ CTL. As expected, when naïve CD8+ T cells were both primed and restimulated with mut A2-aAPC in the complete lack of CD8 coligation, antigen-specific CD8+ CTL did not grow. However, adding IL-21 overcame this deficiency. When CD8-independent T cell priming and restimulation by mut A2-aAPC was supplemented with IL-21, antigen-specific CD8+ CTL expansion with high functional avidity occurred.

Lack of CD8 binding to MHC results in partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of MHC/TCR. While the responses of IL-21R- Jurkat and its Lck-null mutant, J.CaM1.6, to IL-21 were minimal, both showed robust IL-21 responses when stably transduced with IL-21R. Intracellular staining revealed that IL-21 induced robust phosphorylation of STAT3 but not STAT1 upon stimulation in both IL-21R-transduced Jurkat and J.CaM1.6. When IL-21R-transduced Lck-null J.CaM1.6 cells were stimulated in the presence of IL-21, T cell responses were completely abrogated by STAT3 inhibition. In contrast, the MAPK inhibitor only partially blocked the T cell responses. The combination of suboptimal doses of the STAT3 inhibitor and the MAPK inhibitor completely nullified the T cell responses, indicating an additive effect of STAT3 and MAPK inhibition. These results suggest that STAT3 but not STAT1 is critically involved in IL-21 signaling that rescues the defective T cell responses caused by the lack of Lck.

Taken all together, this study suggests that CD8 ligation is critical for the expansion of post-thymic peripheral antigen-specific CTL in humans. However, STAT3-mediated IL-21 signaling can complement partial TCR signaling caused by the lack of CD8 association and expand CTL with high functional avidity. Since high functional avidity CTL are optimal effectors for the clearance of pathogens and tumors in vivo, our findings may be important for generating high avidity CTL ex vivo for effective adoptive therapy.

Disclosures:

No relevant conflicts of interest to declare.