Siglec-G Expression on Donor T Cells Controls Severity of Gvhd

Members of the sialic acid binding Ig–like lectin (Siglec) family, such as Siglec-G, have immunoreceptor tyrosine-based inhibitory motifs (ITIM) regions that negatively regulate innate immune activation induced by non-infectious damage-associated molecular patterns (DAMPs). The T-cell autonomous role of Siglec-G in modulating immunity is not known. In order to examine the role of Siglec-G on T-cells we utilized the well-established MHC disparate B6->BALB/c model of allogeneic BMT. BALB/c WT mice were lethally irradiated (850cGy) and transplanted on day 0 with 5x10⁶ WT bone marrow from either syngeneic WT-BALB/c or allogeneic WT-B6 along with and 0.5x10⁶ CD90⁺T cells from WT or Siglec-G^-/- B6 animals. The allogeneic recipients receiving donor T cells from Siglec-G^-/- animals showed significantly more severe GVHD and worse survival compared to allogeneic WT-B6 animals (p=0.01). This increased mortality was also associated with greater GVHD specific histopathology but similar levels of DAMPs such as HMGB1. Donor T cell analyses demonstrated significantly greater expansion of activated CD69⁺, Th1 (IFN-g⁺) and Th17 (IL-17A⁺) cells in the spleen and in the GVHD target organs, namely liver and gut (P<0.05). Enhanced GVHD mortality and severity was observed in multiple irradiated BMT models (MHC-matched multiple minor antigen mismatched C3H.sw->B6 and haploidentical B6->F1 models). We performed detailed phenotypic analysis of various T cell subsets in naïve Siglec-G^-/- and WT B6 animals and found similar distribution of naïve, memory, effector and regulatory T cells suggesting that alteration in T-cell subsets in the donor T-cell inoculum was not the cause for increase in GVHD. Functional analyses of naïve Siglec-G^-/- T cells, showed similar proliferation in vitro after stimulation with allogeneic DCs or CD3/CD28 antibody stimulation when compared to WT T cells. Furthermore Siglec-G^-/- Tregs as well as WT Tregs were equally suppressive in repressing either WT or Siglec-G^-/- naïve T-cells demonstrating that Siglec-G expression on Tregs is not critical for their suppressive function or on naïve T-cells to be regulated by Tregs. By contrast Siglec-G^-/- T cells showed higher proliferation after TCR stimulation (CD3/CD28) only upon with addition of DAMP (HMGB-1) demonstrating that direct regulation of T-cell response to DAMP by Siglec-G regulates its TCR driven proliferative responses. Finally, in order to explore the Siglec-G ⁺T cells interaction with its ligand in hosts, we next used CD24^-/- BALB/c animals as hosts. CD24^-/- animals also demonstrated enhanced GVHD mortality when compared to WT animals. Collectively, our data suggest, for the first time to our knowledge that Siglec-G mediated responses to DAMPs has T-cell autonomous effects and through its engagement with its ligand CD24 in the hosts is critical for mitigating GVHD.