High Concentration of Immunoglobulin Allows Long-Lasting Suppression of Allo-Immune Reaction By Directly Inhibiting T-Cell Activation

Intravenous immunoglobulin (IVIg) has been used after allogeneic stem cell transplants (HSCT) mainly to prevent infections. In addition, some studies have also demonstrated that IVIg reduced GVHD and GVHD-related mortality, but others failed to show these effects. To answer whether IVIg could reduce GVHD and/or has preventive effects, as a first step, we compared the mixed lymphocyte reactions (MLRs) between a variety of responder and stimulator combinations (Balb/c and C57BL/6) with/without immunoglobulin (Ig). We showed that the MLR was strongly inhibited when high concentration of Ig was added to the cocluture (Stimulator (S): bone marrow-derived dendritic cells (BM-DCs) obtained from C57BL/6 using GM-CSF, Responder (R): splenocytes obtained from Balb/c). Interestingly, dose dependent inhibition was not observed only when high concentration of Ig could suppresses allo-reaction. The next concern was whether Ig could suppresse MLR via modulation of DCs or T-cell directly, or both. To test this, splenocytes were stimulated with phytohemagglutinin (PHA) in the presence of various Ig concentrations. We observed that Ig suppressed PHA-induced T-cell proliferation in a dose dependent manner. To eliminate the possibility that Ig might have direct cytotoxicity against lymphocytes and consequently allo-immune reaction seemed to be inhibited, we performed dead cell analysis by flowcytometry using 7-AAD and annexin V on T-cells in the various concentrations of Ig. Even in high concentration of Ig, the frequency of apoptotic or dead cells were observed as same levels as in low concentration or absence of Ig, suggesting that Ig inhibited T-lymphocyte activation directly. Next concern was when Ig allowed tolerogenic function to T-cells and/or DCs. That is, only pretreatment of Ig before MLR was enough to suppress allo-reaction or persistent high concentration of Ig exposure was needed during MLR. To test this, splenocytes and BM-DCs were cultured with Ig for 24 hours before MLRs, and then washed twice and we compared the MLRs. Ig pretreatment to either splenocytes or BM-DCs or both did not show any suppression of MLRs as shown in the figure below, indicating that persistent high levels of Ig during interaction were needed to induce maximum inhibiting effects. In conclusion, we have clearly demonstrated that Ig has a strong capacity to suppress allo-immune reaction. Repetitive high dose IVIg treatment would be a promising approach to reduce or prevent sever acute GVHD, only if serum Ig concentration could be kept in high level. In addition, to apply this in clinical settings, disadvantage of high dose IVIg should be further investigated.

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