Human intestinal CD3⁺TCR⁺CD8⁺ intraepithelial lymphocytes (IELs) are intimately associated with epithelial cells (ECs) through binding of CD103 to E cadherin. How these two cell types functionally interact is largely unknown. IEL-EC crosstalk was determined using HT-29 cells as the model EC and IL-8 as the read-out. IL-8 was derived from both cell types and synergistically increased when the cells were combined. This synergistic effect required active transcription by both IELs and HT-29 cells. Cell contact was required as shown by the loss of the synergistic increase in IL-8 when the two cell types were separated by transwells. Specifically, IL-8 release required the binding of CD2 on the IELs to CD58 on the HT-29 cells. The association of the CD3/TCR complex with major histocompatibility antigen (MHC) class I antigens was not involved. Antibody neutralization of tumor necrosis factor-alpha (TNF), but not interferon-gamma (IFN), resulted in increased IL-8 production by the coculture. While both TNF and IFN increased IL-8 synthesis and CD58 expression by the HT-29 cells, only IFN reduced IL-8 production by IELs. IL-8 production by either cell type involved phosphorylation of p38 and JNK. In summary, the synergistic synthesis of IL-8 occurs when IELs are stimulated through the CD2 pathway by CD58 on HT-29 cells, resulting in TNF release that, in turn, augments IL-8 synthesis and CD58 expression by the HT-29 cells.