Increasing data supported the biological significances of MHC II signaling as well as its potential as a therapeutic target [4
]. Multiple signaling pathways are activated by anti-MHC II mAbs crosslinking including cAMP production, PKC activation, calcium mobilization, tyrosine phosphorylation and AKT activation [4
]. However, it is not clear which of these signaling pathways are important for the therapeutic effect of anti-MHC II mAbs. To start address this question, we undertook analysis of class II structural requirements for transduction the death signals. We determined that it is the MHC class II α chain connecting peptide region, not the MHC class II TM and cytoplasmic domains, is required for death signaling.
MHC II contains a short cytoplasmic tail that is important for many signaling events including MHC II mAb induced PKC activation and cAMP production [16
]. We showed here that the cytoplasmic tails and the TM regions of MHC II are not required for the cell death response (, ). Thus, our results indicate that the signaling events initiated by these regions are not required for the death response. They also suggested that the death signaling must be transduced by MHC II associated cell surface molecule(s).
The MHC II-mediated death response, as well as the calcium response, is significantly impaired in the αCP mutant (&). We hypothesized that the transducer(s) that mediates death and calcium signaling maybe associated with MHC II through this region. We found in this report that the Igα/β association is through the αCP region (). Furthermore, knock-down Igβ expression by shRNA eliminated MHC II mediated calcium response (). These data confirmed early report that MHC II mediated calcium signal is dependent on its association with Igα/β[17
] and further showed that this signal is via the I-Ak
αCP region. It is noteworthy that MHC II-Igα/β association is different from the IgM-Igα/β association which is mainly mediated by IgM transmembrane region[29
Interestingly, the Igβ knock-down cells had normal cell death responses (). This suggested the impaired death signaling in αCP mutant is not due to its loss association with Igα/β. Some other MHC II associated transducer(s) must be responsible for the defect. Multiple cell surface proteins are reportedly associated with MHC II including Igα/β, CD19, CD20, CD40, and CD9-CD37-CD81 tetraspanin proteins [17
]. The Igβ knock-down cells have normal CD19 association (). However, CD19, CD20 and Igα/β are B cell specific proteins and since MHC II-mediated cell death is also observed in DC[14
], these B cell specific proteins can not be required for the death signaling. Tetraspanins have very short cytoplasmic tails and no defined signaling motifs. CD40 is found on both B cells and DC. However, CD40 actually protects cells from MHC II-mediated cell death [14
]. Thus it seems likely that other, as yet undefined, associated proteins must be responsible for transmitting the death signals. We are currently exploring this possibility and have defined a previously unknown MHC II associated tetraspan protein that contains multiple signaling motifs in its cytoplasmic tail and is required for the death response (Jin, L and Cambier, JC, manuscript in preparation).
It is interesting to notice that MHC II transduced death signaling does not require calcium mobilization, tyrosine phosphorylation (data not shown) [32
] and all other signaling events that require the cytoplasmic tails of MHC II. On the other hand, loss of Igβ expression eliminated calcium response () and tyrosine phosphorylation (data not shown) but does not affect cell death and AKT activation (). These observations suggested that MHC II engages multiple parallel signaling pathways that may be initiated by different MHC II associated membrane proteins (). Calcium signaling is transmitted by MHC II associated Igα/β. AKT is activated by MHC II-associated CD19, and the death response is initiated by some unknown MHC II-associated protein ().
Model: Parallel signaling induced by different MHC II associated accessory molecules.
The implications of the accessory molecule coupling to parallel MHC II signaling pathways model are twofold. First, most of the well-known MHC II-associated signaling proteins, including CD19, CD20, Igα/β are B cell specific. This suggests that the response to MHC II crosslinking in B cells and DCs might be different. In fact, though MHC II engagement can kill both B cell lymphoma and DCs in vitro
], MHC II-mediated antigen presentation to CD4+
T cells by DCs and B cells have different outcomes in vivo
. Mature DCs die after antigen presentation, while B cells may be activated to participate in immune response.
Second, the findings reported here have implications for the therapeutic use of anti-MHC II mAb. Most death-inducing MHC II mAbs also activate the AKT survival pathway [12
](), which may decrease the efficiency of tumor killing. AKT activation is likely a result of MHC II-associated CD19-PI3K pathway [20
]. An anti-MHC II mAb, designed to target the CD19 binding sites on MHC II could disrupt surface MHC II/CD19 association and initiate the death signals without the activation of AKT survival pathway. Such an agent might be a more efficient killer of tumor cells.
Future studies should be focused on identification of additional MHC II associated surface signaling proteins, particularly the surface co-receptor that is responsible for transmitting MHC II-mediated death signal.