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In transmembrane (TM) domains, tetraspanin KAI1/CD82 contains an Asn, a Gln, and a Glu polar residue. A mutation of all three polar residues largely disrupts the migration-, invasion-, and metastasis-suppressive activities of KAI1/CD82. Notably, KAI1/CD82 inhibits the formation of microprotrusions and the release of microvesicles, while the mutation disrupts these inhibitions, revealing the connections of microprotrusion and microvesicle to KAI1/CD82 function. The TM polar residues are needed for proper interactions between KAI1/CD82 and tetraspanins CD9 and CD151, which also regulate cell movement, but not for the association between KAI1/CD82 and alpha3beta1 integrin. However, KAI1/CD82 still efficiently inhibits cell migration when either CD9 or CD151 is absent. Hence, KAI1/CD82 interacts with tetraspanin and integrin by different mechanisms and is unlikely to inhibit cell migration through its associated proteins. Moreover, without significantly affecting the glycosylation, homodimerization, and global folding of KAI1/CD82, the TM interactions maintain the conformational stability of KAI1/CD82, evidenced by the facts that the mutant is more sensitive to denaturation and less associable with tetraspanins and supported by the modeling analysis. Thus, the TM interactions mediated by these polar residues determine a conformation either in or near the tightly packed TM region and this conformation and/or its change are needed for the intrinsic activity of KAI1/CD82. In contrast to immense efforts to block the signaling of cancer progression, the perturbation of TM interactions may open a new avenue to prevent cancer invasion and metastasis.

Aim A number of transplant centers currently use killer-cell immunoglobulin-like receptor (KIR) genotyping as criteria for donor selection for hematopoietic stem cell transplant (HSCT) and natural killer (NK) cell protocols. Several factors contribute to donor selection, including presence or absence of multiple inhibitory and stimulatory KIR, centromeric vs. telomeric KIR haplotypes, “A” vs. “B” content KIR haplotypes, and NK cell education or licensing. Recent studies at our institution demonstrated that amino acid residue 245 of the KIR2DL1 protein is important in determining the signaling capacity, and had a significant effect on the overall outcome of HSCT. Patients who received a donor graft containing KIR2DL1 alleles encoding an arginine at position 245 (KIR2DL1-R 245 ) had better survival and lower incidence of disease progression than the patients who received a donor graft containing KIR2DL1 alleles encoding a cysteine at the same position (KIR2DL1-C 245 ). The goal of this work was to validate a lab-developed clinical test to distinguish the KIR2DL1 R 245 /C 245 allelic polymorphism and to facilitate donor selection based on this criterion. Methods Our research laboratory developed a real-time PCR single nucleotide polymorphism (SNP) assay that specifically detects alleles of the KIR2DL1 gene encoding arginine or cysteine at position 245, and avoids the interference of 2DL2 alleles that share the same sequence flanking this region. The HLA laboratory used the backbone of this assay to validate it for clinical use as a lab-developed companion test to current KIR genotyping. Results The SNP assay performed according to the required qualifications, including accuracy, precision, sensitivity, and specificity. Samples tested showed 100% concordance with the expected genotyping of the “Gold Standard,” cell lines with sequenced KIR2DL1 alleles from the CIBMTR Repository. Conclusions This SNP assay was validated for clinical use in the HLA laboratory, and should be in use in the near future as a companion test to the KIR genotyping currently used for donor selection in HSCT. P. Arnold: Grant/Research Support; Company/Organization; American Lebanese Associated Charities (ALSAC). P. Chou: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC). R. Bari: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Bari is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity. W. Leung: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Leung is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity.