F4/80 Protein Is Required for Cell Aggregation in ACAID

Abstract

Abstract: : Purpose: While it is known that F4/80 protein expression is required for both in vivo and in vitro ACAID, the function of this protein during ACAID induction is unknown. Here, we tested the following postulations: (1) F4/80^–/– APCs are capable of producing ACAID inducing signals and only lack the F4/80 protein; (2) F4/80 protein promotes cell interactions in vitro that lead to CD8⁺ regulatory (Tr) cell generation. Methods: RT–PCR analysis: F4/80^–/– and WT APC were treated with TGFß and antigen one day prior to their collection, mRNA isolation and RT–PCR analyses of IL–10, IL–12, CXCR4 and CCR7 mRNA. In vitro ACAID: The role of F4/80 protein in the formation of cell aggregates during in vitro ACAID was studied in cultured spleen cells from F4/80 ^–/–. Five days later, the media was removed and the cells remaining on the culture slides were fixed with acetone and stained with Wright–Giemsa or fluorescence antibodies to F4/80, CD4, CD8, NK1.1 and B220 to identify aggregates and cells within them, respectively. Results: We observed that TGFß/OVA treatment of F4/80^–/– or WT APC resulted in increases in IL–10, CXCR4 and a decrease in IL–12. Thus F4/80^–/– APC was capable of responding to ACAID inducing signals. However, cluster formation was diminished in in–vitro cultures containing F4/80^–/– cells, compared to in vitro cultures containing WT cells. Moreover there seemed to be a selective diminution of NKT and CD8 T cells within the few clusters that did associate in the F4/80^–/– cell cultures. Conclusions: The F4/80 protein is a marker of the "eye–derived" APC that carry the tolerogenic signal to the marginal zone of the spleen where lymphocytes aggregate with F4/80⁺ APC during ACAID. Antigen–specific regulatory CD8⁺ T cells that mediate peripheral tolerance emerge from such cell clusters. Similarly, here, we show that lymphocytes and F4/80⁺ cells aggregate with F4/80^–/ during in–vitro ACAID development. However, F4/80^–/– APCs were less able to form the cell aggregates in in–vitro ACAID cultures. Similar to WT APC, the F4/80 negative APC were able to respond to TGF ß treatment with tolerance inducing signals, We conclude that F4/80 protein functions as an adhesion molecule required for efficient cellular interaction during peripheral tolerance induction by ACAID. These data support that notion that cellular interactions required for peripheral tolerance induction are unique from those required for immune responses. This work is supported in part by NIH grants: EY: 11983; EY 13066

Keywords: ACAID • immunomodulation/immunoregulation • cell adhesions/cell junctions