Tat-based immunomodulatory compositions and methods of their discovery and use
A method for identifying new immunomodulatory chemical entities (NICE) comprising reacting a candidate NICE with a Tat SH3 binding domain, identifying the bound candidate NICE and determining whether the candidate NICE induces monocytes to differentiate into dendritic cells (DC) or regulatory macrophages (AReg). In particular, the present invention relates to identifying NICE that are either immunostimulatory or immunosuppressive.
1. A method for identifying new immunomodulatory chemical entities (NICE) and characterizing the NICE as immunostimulatory or immunosuppressive comprising:
a. reacting a candidate NICE with a Tat SH3 binding domain wherein said Tat SH3 binding domain is bound to a solid phase to identify candidate NICE that bind to said Tat SH3;
b. identifying said candidate NICE bound to said Tat SH3;
c. adding said identified candidate NICE to a culture of purified peripheral blood monocytes;
d. adding Tat having an SH3 binding domain to said peripheral blood monocytes and candidate NICE to form a test culture;
e. incubating said test culture to allow said monocytes to differentiate into dendritic cells (DC) or regulatory macrophages (AReg);
f. removing said differentiated cells from said test culture;
g. quantifying the numbers of DCs and AReg in the differentiated cell population; and
h. determining the relative numbers of DCs and AReg in the differentiated cell population; wherein greater numbers of DCs compared to AReg identifies an immunosuppressive NICE and greater numbers of ARegs compared to DCs identifies an immunostimulatory NICE.
2. The method according to claim 1 wherein said Tat SH3 binding domain in step (a) is selected from the group consisting of native immunosuppressive human immunodeficiency virus (HIV) Tat, simian lentivirus Tat, long-term non-responder Tat, randomly mutated HIV Tat and site-specific mutated HIV Tat.
3. The method according to claim 1 further comprising the step of injecting an immunostimulatory NICE into an immunosuppressed mouse wherein said immunosuppression results from the presence of an endogenous SH3 binding domain and determining the numbers of ARegs in a sample of the mouse's peripheral blood before and after administration of the immunostimulatory NICE, wherein an increase in the number of ARegs confirms the NICE is immunostimulatory.
4. The method according to claim 3 wherein the said immunosuppressed mouse is a hairless (hr) mouse.
5. A method according to claim 1 further comprising the step of injecting an immunosuppressive NICE into a mouse and further challenging said mouse with an antigen wherein tolerance to said antigen confirms that the NICE is immunosuppressive.