2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.
The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.
Carreño
Roberto
R
From the Section of Transplantation Immunology, Department of Stem Cell Transplantation and Cellular Therapy, Houston, Texas 77030.
Brown
Wells S
WS
Department of Immunology, Houston, Texas 77030.
Li
Dan
D
From the Section of Transplantation Immunology, Department of Stem Cell Transplantation and Cellular Therapy, Houston, Texas 77030.
Hernandez
Jessica A
JA
Department of Melanoma Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Wang
Yang
Y
From the Section of Transplantation Immunology, Department of Stem Cell Transplantation and Cellular Therapy, Houston, Texas 77030.
Kim
Tae Kon
TK
Adult Stem Cell Transplant Program, University of Miami Sylvester Comprehensive Cancer Center, Miami, Florida 33136.
Craft
John W
JW
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204.
Komanduri
Krishna V
KV
Adult Stem Cell Transplant Program, University of Miami Sylvester Comprehensive Cancer Center, Miami, Florida 33136.
Radvanyi
Laszlo G
LG
Department of Melanoma Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Hwu
Patrick
P
Department of Melanoma Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Molldrem
Jeffrey J
JJ
From the Section of Transplantation Immunology, Department of Stem Cell Transplantation and Cellular Therapy, Houston, Texas 77030.
Legge
Glen B
GB
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204.
McIntyre
Bradley W
BW
Department of Immunology, Houston, Texas 77030.
Ma
Qing
Q
From the Section of Transplantation Immunology, Department of Stem Cell Transplantation and Cellular Therapy, Houston, Texas 77030. Electronic address: qma@mdanderson.org.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002413:Cations, Divalent / Q000276:immunology / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000276:immunology; D004220:Disulfides / Q000276:immunology / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007111:Immunity, Cellular / Q000187:drug effects / Q000276:immunology; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology* / Q000378:metabolism; D019169:Jurkat Cells; D020014:K562 Cells; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology* / Q000378:metabolism; D008670:Metals / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016853:Muromonab-CD3 / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods; D014471:Umbilical Veins / Q000166:cytology / Q000276:immunology
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D004220:Disulfides; D016169:Lymphocyte Function-Associated Antigen-1; D008670:Metals; D016853:Muromonab-CD3; D021122:Protein Subunits; D018799:Intercellular Adhesion Molecule-1