SUlTllnaryNaive T cells are selectively recruited from the blood into peripheral lymph nodes during lymphocyte recirculation. L-selectin, a lectin-like receptor, mediates the initial attachment oflymphocytes to high endothelial venules (HEV) in lymph nodes. A subsequent step involving the activation of {32 integrins has been proposed to facilitate firm adhesion, but the activating signals are poorly understood. We report here that either antibody-mediated cross-linking of L-selectin on human lymphocytes or treatment of the ceils with GlyCAM-1, an HEV-derived, secreted ligand for L-selectin, stimulates their binding to ICAM-1 through the {32 integrin pathway. Furthermore, GlyCAM-1 causes the rapid expression ofa neoepitope on {32 integrins associated with a high-avidity state. Naive (CD45RA+), but not memory (CD45P,.0 +) lymphocytes, respond to L-selectin cross-linking or GlyCAM-1 treatment. Thus, the complexing of L-selectin by specific ligands may provide key signals to naive lymphocytes, contributing to their selective recruitment into peripheral lymphoid organs.

SummaryThe lymphocyte differentiation marker CD8 acts as a coreceptor with the T cell receptor (TCR) during recognition of peptide presented by major histocompatibility complex (MHC) class I molecules. The functions of CD8 in the TCR complex are thought to be signaling through the association of CD8 with the protein tyrosine kinase p56kk and adhesion to MHC class I through the a3 domain. While the ability of the CD8 a/cx homodimer to bind to classical MHC class I molecules has been shown, it is unclear whether CD8 can also bind nonclassical molecules. Ofparticular interest is human histocompatibility leukocyte antigen (HLA)-G which is expressed on placental cytotrophoblast cells. These cells do not express HLA-A, -B and -C molecules. In this report, we demonstrate that CD8 can bind to HLA-G. It is possible, therefore, that a cell bearing CD8 may interact with HLA-G-expressing cells. I n humans three nonclassical MHC class I molecules have been described human histocompatibility leukocyte antigen (HLA)-E (1, 2), -F (3), and -G (4) which map to the MHC complex and which encode expressible proteins. While mRNA for all three genes has been detected in a number of cell lines (5), only expression of HLA-G on the cell surface was reported (6, 7). Because there are no specific antibodies against HLAE, -F, and -G, specific expression of HLAG can only be identified by analysis of labeled cell surface proteins on two-dimensional polyacrylamide gels (6) . By this approach, HLA-G was the only HLA molecule expressed on fetal cytotrophoblast cells (6) . Cytotrophoblasts invade the maternal decidualized endometrium and are directly exposed to maternal lymphoid cells yet the placenta is not normally rejected by the mother as an allograft . Thus, the ability of HLA-G to be recognized by immune cells is unclear.Because CD8 can bind to MHC class I molecules including HLAA2, we decided to determine whether CD8 could recognize and bind to HLA-G as well . Salter et al . (8) recently identified a conserved negatively charged loop on the MHC class I a3 domain that is important for binding to CD8. Single amino acid substitutions at each position in this loop from residues 223-229 severely reduced binding to CD8 in a cellcell adhesion assay. At position 228 within this loop HLAG has a valine instead of the conserved threonine found in HLA-A, B, and C. Based on this difference as well as other differences, it would not be predicted from sequence comparisons that CD8 would bind to HLA-G. To determine whether CD8 could bind to HLA-G, we obtained a cell line, LCL 221, that was null for expression of classical HLA class I molecules HLA-A, -B, -C and transferents of this cell line that expressed either HLA G or HLAA2 (6) . Using a cell-cell adhesion assay, we found that CD8 could bind to HLAG and that the binding was similar to the binding to HLA-A2 . Materials and MethodsCell Lines. The lymphoblastoid cell lines: the HLA class I null mutant 221 and HLA-A2 and HLA-G transfectants of 221 were obtained from Dr. Robert DeMars . The HLAG and HLA-A...

Monoclonal antibody (mAb) candidates from high-throughput screening or binding affinity optimization often contain mutations leading to liabilities for further development of the antibody, such as aggregation-prone regions and lack of solubility. In this work, we optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. We also extended this analysis to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.

The CD8 dimer interacts with the a3 domain of major histocompatibility complex class I molecules through two immunoglobulin variable-like domains. In this study a crystal structure-informed mutational analysis has been performed to identify amino acids in the CD8a/a homodimer that are likely to be involved in binding to Recently the crystal structure of a soluble form of the human CD8a/a homodimer was solved (12). In the N-terminal domain of each polypeptide, two sheets consisting of five and four antiparallel (3-strands form a typical immunoglobulin fold. Connecting these strands is a conserved array of loops, three of which are analogous to the complementarity-determining regions of immunoglobulin (CDR1, CDR2, CDR3). Modeling of the electrostatic surface potential of the CD8a/a homodimer has revealed that the membrane-distal, CDR-containing surface of the molecule is predominantly positively charged.The binding site on class I molecules for CD8a/a has been studied in detail. Previous mutational analyses have identifled three clusters of amino acids in the a3 domain that are important for CD8a/a-class I binding and pinpointed one cluster, residues 223-229, as critical for this interaction (1,(13)(14)(15). This cluster is conserved between mouse and human and comprises a highly exposed, negatively charged loop between strands C and D in the a3 domain. In contrast to the body of work that has contributed to the localization of the putative class I binding site for CD8, only one study has focused on defining the complementary interaction site on CD8a/a. Based on the finding that murine CD8a/a did not bind to human HLA class I in a cell-cell adhesion assay, Sanders et al. (16) performed homolog scanning mutagenesis by exchanging human amino acids with their nonconserved murine counterparts. The results ofthis work indicated a role for both the CDR1-like and CDR2-like loops in this speciesspecific interaction. However, since this study focused on the differences between two species, conserved amino acids involved in binding would not have been identified. In addition, the individual amino acids which might be potential contact points could not be determined because these mutants often contained more than one amino acid substitution.In the current study the site of interaction on CD8a/a has been further characterized by using a panel of point mutants generated with the aid of the CD8a/a crystal structure. Analysis of these mutants in a transient cell-cell adhesion assay has identified a core of amino acids that are likely candidates for interaction with class I molecules. Overall, the results support a bivalent model of interaction between one CD8 homodimer and two class I molecules. The role of electrostatic interactions between the predominantly positively charged CD8a/a and the predominantly negatively charged class I a3 domain is also supported by these findings. MATERIALS AND METHODSMutagenesis. Site-directed mutagenesis of the human CD8a cDNA in pBluescript SK(+) was as described (16). To facilitate t...

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain.