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Immune recognition by T lymphocytes and natural killer (NK) cells is in large part dependent on the identification of cell surface MHC molecules bearing peptides generated from either endogenous (MHC I) or exogenous (MHC II) dependent pathways. This review focuses on MHC I molecules that coordinately fold to bind self or foreign peptides for such surface display. Peptide loading occurs in an antigen presentation pathway that includes either the multimolecular peptide loading complex (PLC) or a single chain chaperone/catalyst, TAP binding protein, related, TAPBPR, that mimics a key component of the PLC, tapasin. Recent structural and dynamic studies of TAPBPR reveal details of its function and reflect on mechanisms common to tapasin. Regions of structural conservation among species suggest that TAPBPR and tapasin have evolved to satisfy functional complexities demanded by the enormous polymorphism of MHC I molecules. Recent studies suggest that these two chaperone/catalysts exploit structural flexibility and dynamics to stabilize MHC molecules and facilitate peptide loading.

Intracellular loading of major histocompatibility complex class I (MHC-I) molecules occurs within the peptide loading complex and is accomplished through the concerted action of several proteins, including the chaperone/catalyst tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. To gain a mechanistic understanding of tapasin’s function we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSta1 and PaSta2, and investigated their binding to human recombinant soluble tapasin by surface plasmon resonance methods and X-ray crystallography. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. PaSta1 and PaSta2 recognize distinct epitopes as tapasin captured on a surface immobilized with one PaSta Fab binds the other PaSta Fab with nanomolar affinity. However, tapasin captured on a PaSta2 Fab surface is not able to bind peptide/HLA-B*44:05/beta-2 microglobulin complexes while tapasin captured on a PaSta1 Fab surface can, indicating that PaSta2 sterically competes for the MHC-I binding site. This finding was echoed in the structural characterization of PaSta Fab-tapasin complexes by X-ray crystallography. PaSta1 binds to an extended N-terminal region of tapasin, a site accessible even in the tapasin/ERp57 complex. By contrast, PaSta2 binds at the junction of the N-terminal and C-terminal domains of tapasin, where tapasin may interact with HLA-I molecules. Comparison of conformations of tapasin in complex with PaSta1, PaSta2, HLA-B*44:05, and ERp57 reveals the dynamic nature of tapasin, a versatile chaperone and catalyst. Supported by the intramural research program of the NIAID, NIH

MHC-I molecules loaded with peptide for subsequent cell surface expression are critical for adaptive immunity. Tapasin, a component in the Peptide Loading Complex (PLC), serves as an MHC-I chaperone, and it facilitates peptide loading onto MHC-I in the endoplasmic reticulum (ER). Although the structure of TAPBPR (a homolog of tapasin) complexed with MHC-I was reported several years ago, the detailed mechanism of peptide loading by tapasin remains unclear. Here, we report the structure of a complex of tapasin with the MHC-I molecule, HLA-B* 44:05. The model of tapasin/HLA-B*44:05 compared with that of unliganded HLA-B*44:05 and to the structure of tapasin bound to ERp57 reveals dramatic changes that refect both chaperone and catalytic activities. Tapasin cradles the MHC-I molecule through contacts with the N-terminal alpha1 and alpha2 domains, as well as with alpha3 and beta-2 microglobulin interaction via its C-terminal IgC domain. Thus the MHC-I molecule is stabilized in a peptide accessible form. We compare this tapasin/HLA-B*44:05 structure with our previously determined structure of TAPBPR/H2-D(d). Although the general dispositions of tapasin and TAPBPR are the same in their complexes with MHC-I, the structural details of their interactions differ. Analyses of tapasin mutants that disrupt the interaction with MHC-I are consistent with the X-ray crystal structure. Thus, the two MHC-I dedicated chaperones, tapasin and TAPBPR, although acting at distinct stages of the MHC-I loading pathway, show broadly conserved binding modes and similar mechanisms of peptide loading in antigen presentation. Supported by the intramural research program of the NIAID, NIH

Class I MHC molecules (MHC-I) provide crucial cell surface elements that signal health or status of cells for recognition by T cells via their T cell receptor or natural killer (NK) cells via various NK receptors. MHC-I dependent antigen presentation thus influences TCR and NK recognition in immunity to infection, in cancer, and in autoimmunity. Antigen presentation in the MHC-I-dependent pathway depends, in part, on the loading of peptides, derived from proteolysis, aberrant translation, or protein splicing onto MHC-I molecules in the endoplasmic reticulum (ER). The classical pathway of peptide loading and exchange involves components of the peptide loading complex (PLC): the transporter TAP1/2; a lectin, calreticulin; an oxidoreductase, ERp57; a chaperone, tapasin; as well as the peptide-receptive MHC-I molecule and its light chain, b2-microglobulin (b2m). To understand the mechanism of peptide loading and exchange, several laboratories have explored functional and structural aspects of MHC-I interactions with a tapasin homolog, TAP binding protein, related (TAPBPR). We previously reported the structure of a TAPBPR/MHC-I complex at 3.4 Å resolution. However, details of the direct interaction of tapasin with MHC-I are more desirable. Here we report the X-ray crystal structure of tapasin/MHC-I complexes at higher resolution. We discuss similarities and differences between the tapasin/MHC-I and TAPBPR/MHC-I interactions and their implications for MHC-I dependent T cell and NK cell recognition.

Antigen presentation is a cellular process that involves a number of steps, beginning with the production of peptides by proteolysis or aberrant synthesis and the delivery of peptides to cellular compartments where they are loaded on MHC class I (MHC-I) or MHC class II (MHC-II) molecules. The selective loading and editing of high-affinity immunodominant antigens is orchestrated by molecular chaperones: tapasin/TAP-binding protein, related for MHC-I and HLA-DM for MHC-II. Once peptide/MHC (pMHC) complexes are assembled, following various steps of quality control, they are delivered to the cell surface, where they are available for identification by αβ receptors on CD8+ or CD4+ T lymphocytes. In addition, recognition of cell surface peptide/MHC-I complexes by natural killer cell receptors plays a regulatory role in some aspects of the innate immune response. Many of the components of the pathways of antigen processing and presentation and of T cell receptor (TCR)-mediated signaling have been studied extensively by biochemical, genetic, immunological, and structural approaches over the past several decades. Until recently, however, dynamic aspects of the interactions of peptide with MHC, MHC with molecular chaperones, or of pMHC with TCR have been difficult to address experimentally, although computational approaches such as molecular dynamics (MD) simulations have been illuminating. Studies exploiting X-ray crystallography, cryo-electron microscopy, and multidimensional nuclear magnetic resonance (NMR) spectroscopy are beginning to reveal the importance of molecular flexibility as it pertains to peptide loading onto MHC molecules, the interactions between pMHC and TCR, and subsequent TCR-mediated signals. In addition, recent structural and dynamic insights into how molecular chaperones define peptide selection and fine-tune the MHC displayed antigen repertoire are discussed. Here, we offer a review of current knowledge that highlights experimental data obtained by X-ray crystallography and multidimensional NMR methodologies. Collectively, these findings strongly support a multifaceted role for protein plasticity and conformational dynamics throughout the antigen processing and presentation pathway in dictating antigen selection and recognition.

Cell surface major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity to microbes and tumors by presenting intracellular peptides for recognition by CD8+ T cells. Since the repertoire of bound peptides is crucial to MHC-I stability as well as to T cell and NK cell recognition, the molecular mechanism by which peptides are selectively loaded onto MHC-I molecules is of considerable interest. A key role is played by the well-characterized chaperone tapasin as indicated by reduced cell surface MHC-I levels and altered peptide repertoires in tapasin deficient cell lines and mice. Despite extensive studies of the functional role of tapasin in shaping peptide repertoires, direct structural information on its interaction with MHC-I is lacking. Following the recent biochemical and crystallographic studies of the interaction of the tapasin homolog, TAPBPR, with MHC-I, we have engineered soluble versions of tapasin for comparison with TAPBPR. Using surface plasmon resonance assays of purified, untethered proteins, we observe that tapasin binds directly to an MHC-I molecule loaded with truncated peptides but with lower apparent affinity than TAPBPR. Similarly, using fluorescence polarization, we find that tapasin facilitates peptide exchange onto MHC-I with a lower efficiency than TAPBPR. These experiments permit direct comparisons of tapasin/MHC-I and TAPBPR/MHC-I interactions and help elucidate the functional relationship of these two distinct chaperones in shaping the MHC-I peptide repertoire.

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I–restricted CD8+ T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 310 helix that flips from an exposed “open” position in the PR transition state to a “closed” position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46–53 of murine H-2Ld MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.

We examined TCR:MHC/peptide interactions and in vivo epitope availability to explore the Th1- or Th2-like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IA(d)-restricted peptides from the gastric parietal cell H/K-ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1-like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2-like AIG at 6-8 wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IA(d)/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IA(d)/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE-labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes.

The cell surface expression of MHC-I molecules displaying peptides derived from intracellular proteolytic processing is a critical requirement for T cell-mediated immunity. A complex series of steps, guided by the peptide loading machinery in the endoplasmic reticulum, ensures that only stably folded MHC-I molecules carrying peptides of proper length and high affinity are transported for display at the cell surface. TAP-binding protein, related (TAPBPR), has been recently shown to directly bind to certain MHC-I alleles and to facilitate peptide loading. To elucidate mechanistic details, we have examined the binding interaction between purified recombinant TAPBPR and MHC-I molecules loaded with truncated peptides that leave a portion of the peptide binding groove unoccupied. Such MHC-I molecules interact with TAPBPR with nanomolar affinities as measured by surface plasmon resonance and these TAPBPR/MHC-I complexes dissociate when offered a high affinity peptide. An extensive crystallization screen with purified TAPBPR/MHC-I complexes yielded crystals that diffracted to 3.5 Å. The structure of the TAPBPR/MHC-I complex, solved by molecular replacement, and refined at this resolution readily reveals the binding footprint and further structural details of the interaction. Data will be presented on the binding interaction and structural characterization of the TAPBPR/MHC-I complex that reveal insights into the mechanism of TAPBPR- and tapasin-mediated loading of peptides onto MHC-I molecules. (KN and JJ contributed equally to this work).

We generated transgenic mice expressing a single-chain β2-microglobulin (β2m)-H-2Dd. The cell-surface β2m-H-2Dd molecule was expressed on a β2m-deficient background and reacted with appropriate mAbs. It was of the expected m.w. and directed the normal development of CD8+ T cells in the thymus of a broad TCR repertoire. It also presented both exogenously provided and endogenous peptide Ags to effector CD8+ T cells. In tests of NK cell education and function, it failed to reveal any interaction with NK cells, suggesting that the site of the interaction of NK receptors with H-2Dd was disrupted. Thus, the sites of TCR and NK receptor interaction with H-2Dd are distinct, an observation consistent with independent modes of TCR and NK receptor evolution and function.

We have constructed a recombinant single-chain human HLA-A2.1 molecule (from A*0201) with a covalently attached beta 2m. This molecule (MSC beta A2.1) can be detected on the surface of transfected beta 2m- human cells by conformational antibodies W6/32 and BB7.2 and by anti-human beta 2m mAb BM-63. The covalent beta 2m, now a domain of the MSC beta A2.1 molecule, does not rescue endogenous Class I surface expression. Instead, it works in cis to achieve correct folding of the single-chain molecule. Immunoprecipitation shows that MSC beta A2.1 is a 60-kDa molecule with no dissociable beta 2m. The half-life of the MSC beta A2.1 molecule on transfected cell surfaces was as long as that of two-chain HLA-A2.1 molecules. The MSC beta A2.1 molecule was active in presentation of HTLV-I Tax 11-19 peptide and an endogenous peptide to specific CTL. MSC beta A2.1 molecules and wild-type HLA-A2.1 molecules on live cells can bind the HBV core peptide 18-27 with comparable affinities. These results show that MSC beta A2.1 molecules retain the functional ability to present both pulsed and endogenous antigens to the appropriate T cells, and thus may be useful components of antiviral vaccines.

TAPBPR (TAP Binding Protein-Related), a tapasin homolog, functions as a peptide editor independent of the classical peptide loading complex (PLC) that consists of tapasin, transporter associated with antigen processing (TAP), ERp57, and calreticulin. Using insect-cell expressed recombinant luminal human TAPBPR, we previously reported interaction with HLA-A*02:01 molecules freed of peptide following photolysis of HLA-A2/beta2m complexes prepared with photolabile peptides. Here, we extend our analysis of the TABPBPR/MHC interaction by examining the binding of a set of MHC/peptide complexes prepared with different MHC-I molecules refolded with a variety of peptides. Using surface plasmon resonance, we show that a variety of refolded complexes, with or without photolysis of the bound peptide, bind to TAPBPR, raising the possibility that TAPBPR may interact either with a small proportion of peptide free molecules found in some MHC/peptide preparations, or with molecular conformations containing peptide in dynamic equilibrium with the lowest energy state. We extend our analysis of TAPBPR by determination of the low resolution small angle X-ray scattering structures of both TAPBPR and tapasin luminal domains, revealing expected structural similarities.

CD8 can serve as a co-receptor or accessory molecule on the surface of CTL. As a co-receptor, CD8 can bind to the alpha3 domain of the same MHC class I molecules as the TCR to facilitate TCR signaling. To evaluate the role of the MHC class I molecule alpha3 domain in the activation of CD8(+) CTL, we have produced a soluble 227 mutant of H-2D(d), with a point mutation in the alpha3 domain (Glu227 --Lys). 227 mutant class I-peptide complexes were not able to effectively activate H-2D(d)-restricted CD8 T cells in vitro, as measured by IFN-gamma production by an epitope-specific CD8(+) CTL line. However, the 227 mutant class I-peptide complexes in the presence of another MHC class I molecule (H-2K(b)) (that cannot present the peptide) with a normal alpha3 domain can induce the activation of CD8(+) CTL. Therefore, in order to activate CD8(+) CTL, the alpha3 domain of MHC class I does not have to be located on the same molecule with the alpha1 and alpha2 domains of MHC class I. A low-avidity CD8(+) CTL line was significantly less sensitive to stimulation by the 227 mutant class I-peptide complexes in the presence of the H-2K(b) molecule. Thus, low-avidity CTL may not be able to take advantage of the interaction between CD8 and the alpha3 domain of non-presenting class I MHC molecules, perhaps because of a shorter dwell time for the TCR-MHC interaction.

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.

This note follows up on an observation concerning a possible frameshift in the DNA sequence of exon 2 of rabbit interleukin 4 (IL4) made in a comparative study of the rabbit Th2 cytokine region sequences from two different rabbits. One was from the tuberculosis-susceptible Thorbecke strain, whose whole genome was sequenced at Broad Institute (OryCun2.0), and the other a normal NZW rabbit studied as part of the ENCODE project. If present, a frameshift could have resulted in exon skipping and production of IL4δ2 protein. We resequenced DNAs of the Thorbecke OryCun2.0 donor rabbit, another rabbit of the same strain, and a third rabbit of the inbred B/J strain in the region in question. All three had sequences identical to the normal NZW in the ENCODE ENm002 assembly in that region and hence no frameshift. The sequence information was submitted to GenBank and assigned accession numbers JQ687218 and JX073284.

A mouse model of autoimmune gastritis with Th2-like phenotype in which BALB/c mice expressing a transgenic αβ TCR spontaneously develop the disease was developed in our laboratory. The TCR recognizes a 12-mer peptide from the alpha subunit of gastric parietal cell H/K ATPase presented in the context of IAd. The disease is characterized by slow and incomplete penetrance, anti-gastric mucosal cell antibodies, inflammatory eosinophilic infiltrates, parietal cell destruction and elevated Th2 cytokines. To investigate the contribution of MHC/peptide interactions to the initiation and maintenance of AIG, we generated a soluble recombinant version of the IAd/H/K ATPase889-900 complex for biophysical and crystallographic analyses. The 2.5 Å X-ray structure of the complex reveals rare features not seen in other IAd/peptide complexes: cysteine in P1, an imino acid in P6, and a large acidic residue in P9. This first structure of IAd in complex with an autoimmunogenic peptide reveals a previously unrecognized peptide binding motif for IAd with large anchor residues and with GLU in P9 encapsulated by a web of polar contacts that may contribute to stabilization of the complex and to maintenance of antigen presentation in disease. This research was supported by the Intramural Research Program of the NIH, NIAID.

Highly immunogenic tumor variants are generated by in vitro or in vivo treatment of L5178Y murine lymphoma cells with triazene derivatives. Most of these variants express new transplantation antigens which are not present on the original L5178Y tumor cells. In this study, a polyclonal syngeneic antiserum raised to one such variant (L5178Y/DTIC) was employed in immunoprecipitation studies of cell surface and metabolically labeled L5178Y/DTIC cells. One- and two-dimensional electrophoretic analyses of the immunoprecipitates detected a surface antigen of approximately 80 kDa. Additionally, a 45-kDa component was detected in the lysate of [35S]methionine-labeled cells. Anti-xenotropic MuLV gp70 serum precipitated material whose electrophoretic pattern was similar to that of the 80-kDa surface antigen. Sequential immunoprecipitation analysis revealed that the molecules reactive with the variant-specific antiserum were removed by the anti-xenotropic gp70 antibodies, whereas immunodepletion was only partial when the cell extract was first treated with the variant-specific antibodies. After Western blotting, the 80- and 45-kDa antigens precipitated by the variant-specific antibodies were injected intrasplenically into recipient mice. Only the animals sensitized with the 80-kDa antigen developed specific immunity to L5178Y/DTIC cells in that they displayed an increased frequency in CTL precursors (CTLp) to the variant cells. Sera from mice sensitized to the 80-kDa protein specifically inhibited the development of a primary CTL response to L5178Y/DTIC cells.

A possible requirement for class II major histocompatibility complex (Ia) molecules in the initial activation of cytotoxic T lymphocyte precursors (CTLp) for allocytotoxic responses was investigated. To avoid possible interaction with other alloreactive cell types, a highly purified population of Lyt-2+ splenocytes was used as a source of CTLp. In the light of preliminary results indicating that Lyt-2+ CTLp, even in the presence of interleukin 2 (IL2), could best be triggered into mature CTL in vitro by cells known to be Ia+, we examined whether an interaction of CTLp with Ia antigens (either on syngeneic accessory cells or on allogeneic stimulators) played a role in the development of allocytotoxicity. Results from experiments done with C57BL/6 Lyt-2+ splenocytes co-cultured with P815 stimulator cells and IL 2 showed that the early activation of CTLp was independent of Ia+ syngeneic accessory cells: (a) flow microfluorometry analysis of the responder population at the beginning or after 1 or 3 days of co-culture did not reveal the presence of Ia+ cells; (b) procedures for removal of residual Ia+ cells or of dendritic cells from the responder population before co-culture did not affect the development of cytotoxicity; (c) co-culture with monoclonal antibodies against syngeneic Iab antigens did not inhibit the CTLp activation. By comparing an Ia+ P815 tumor line with its Ia- clone as allogeneic stimulator cells, it was found that the CTLp activation was also independent of Ia alloantigen on the stimulator cells. The response against both the Ia+ and the Ia- stimulator cell types was not inhibited by monoclonal anti-L3T4 present in the co-culture, indicating that these responses were not affected by residual L3T4 helper cells.

An antiserum specific for cytotoxic T lymphocytes (CTL) and their precursors has been prepared by immunization of strain 13 guinea pigs with a partially purified population of splenic CTL from BALB/c mice immunized with the C57 T cell lymphoma EL4. After absorption with EL4 cells, this antiserum (in the presence of C) totally abolished the activity of in vivo generated CTL and CTL precursors, although it killed only 20% of total Ig- spleen cells. Antibody activity was independent of the H-2 or Ly allotypes of the cells being treated. Helper activity and proliferative responses to concanavalin A and to phytohemagglutinin were unaffected. There was a variable effect on the mixed lymphocyte reaction, and a partial inhibition of graft-vs-host activity.

Summary Reticulated polyester polyurethane foam was found to be a suitable matrix for the immunoadsorption of cells. The column used to support the foam was mounted in a horizontal position and slowly rotated about its long axis in order to permit maximal contact between cells and the foam surface while at the same time allowing free passage of unbound cells through the column. Bound cells were easily removed, intact, by gently squeezing the foam. Untreated foam had a high affinity for guinea pig erythrocytes suspended in physiologic medium at pH 7.4. This nonspecific affinity was greatly reduced by pretreating the foam with various polyanions. Immunospecific binding of erythrocytes was achieved by adding anti-erythrocyte antibody to a gum arabic solution used to protect the foam. A 38-fold increase in binding of the cells was thus obtained. A single antibody-coated foam disk having a diameter of 1 in. and a thickness of ½ in. was capable of binding up to 3 × 108 guinea pig erythrocytes. The high specificity of this method was further demonstrated by the finding that erythrocytes coated with haptens were bound specifically to foam coated with their corresponding anti-hapten antibodies, with a low background of cross-reactivity.

Antibody-forming cells (1) and their precursors (2) readily bind to antigen, presumably through immunoglobulin receptors on their surfaces (3). Absorption occurs at 4°C, within minutes (4). On the other hand, thymusderived lymphocytes (T-cells),1 which have been implicated in cellular immune phenomena such as cell-mediated killing of allogeneic target cells (5), and in the graft-vs-host (GVH) reaction (6), do not have readily demonstrable immunoglobulin on their surfaces (7), and specific binding to cell surface antigen in vitro has generally required incubation at 37°C for cytotoxic effector cells (5, 8), or in vivo absorption for GVH antigen-reactive cells (GVH-ARC) (9). On immunization across a strong histocompatibility barrier in vivo (10, 11) or in vitro (12), there is a large increase in the level of cytotoxic activity (10) but little or no change in GVH activity (11, 12), implying that different populations of T-cells mediate these two reactions.

Summary The ability of early (7 day) and late (7 week) rabbit 7 S and 19 S antibodies to differentiate between two intratypic herpes simplex virus (HSV) antigenic variants was studied by carrying out neutralization kinetic tests. The early and late 19 S antibodies could differentiate between these HSV variants in the presence of complement (C′), and the late 19 S antibody could also differentiate between these variants in the absence of C′. However, neither the early nor the late 7 S antibodies could differentiate between these HSV variants in the presence of C′, but the late 7 S antibody could differentiate between these variants in the absence of C′. The ability of the late 7 S antibody to differentiate between these HSV variants in the absence of C′ was destroyed by treatment of the antibody with 2-mercaptoethanol (2-ME), although 2-ME treatment did not reduce the neutralization titers of the late 7 S antibody.

Spleen cells (from BALB/c mice immunized with the C57BL/6 lymphoma EL4, or from non-immune BALB/c) were incubated on monolayers of [C57BL/6 × BALB/cF1 (B6CF1) spleen cells on polylysine-coated polystyrene Petri plate, for ½ hr or for 1 hr at 37°C followed by centrifugation of the monolayers for 5 min at 70 × G to 110 × G at 34 to 37°C. Control monolayers were BALB/c spleen cells. As measured by the Simonsen spleen weight assay in neonatal mice, graft-vs-host (GVH) activity was partially depleted in cell populations nonadherent to B6CF1 monolayers. Residual GVH activity of these nonadherent cells was about half that of cells incubated on the control syngeneic monolayers (the mean of eight experiments was 49% ± 11% S.D.). Two or three consecutive cycles of incubation and centrifugation did not significantly diminish the residual GVH activity, suggesting that spleen cells with GVH activity are heterogeneous with respect to binding to allogeneic target cells under the above conditions. Cell populations nonadherent to third-part [A × AL]F1 monolayers retained full activity, and cell populations partially depleted of GVH activity in B6CF1 neonates had full activity in third-party [BALB/c × AL]F1 neonates.

Summary Spleen cells from mice immunized with sheep erythrocytes were passed through a horizontal rotating reticulum of open pore plastic foam to which sheep erythrocytes had previously been bound by means of specific antibody. Cells forming hemolytic antibody plaques were preferentially and specifically bound, which resulted in partial separation and a sixfold increase in their concentration.

T cell-mediated proliferative and cytotoxic responses occur in vitro to syngeneic tumor cells antigenically altered by mutagen treatment. One such xenogenized variant of the murine L5178Y lymphoma elicits IgG antibodies reactive with determinants on variant cells that are not expressed at detectable levels on parental or normal cells of the same H-2d haplotype and are also unrelated to public specificities of H-2b or H-2k histocompatibility antigens. In the present study we investigated the effect of those antibodies on development of cell-mediated responses in vitro to the xenogenized cells used for induction of the humoral response. The proliferative reaction, generation of cytolytic activity and target cell lysis were all inhibited by the anti-xenogenized tumor immune serum, whereas the corresponding reactions to the parental cells by syngeneic or allogeneic effector lymphocytes were not. In order to investigate the possible H-2 association of T cell-mediated responses to xenogenized cells, we also examined the effect on those reactions of antibodies specific for Class I or Class II products of the H-2d complex. The results obtained suggested a role for I-Ad molecules in the T cell proliferative response to the xenogenized cells, and also indicated a preferential association of the cytotoxic response with H-2Kd determinants.

A convenient, preparative scale, nonlytic separation of mouse T lymphocytes into Lyt2.2+ and Lyt2.2- populations is reported. Immunoglobulin-negative (Ig-) spleen cells, Ig- lymph node cells, and peanut lectin-unagglutinated (PNA-) thymocytes were incubated under sterile conditions at 0 degree C with monoclonal mouse antibody to the Lyt2.2 T cell differentiation antigen. The antibody-treated cells were washed and placed in polystyrene tissue culture dishes that had been precoated with antibody to mouse Ig. Nonadherent populations were depleted to Lyt2.2+ cells and were essentially devoid of cytotoxic T lymphocyte precursors (CTLp), but contained helper activity for in vivo T-dependent IgM, IgG and IgA antibody formation. Adherent cell populations were enriched for Lyt2.2+ cells and for CTLp. The graft-vs.-host activity of the separated, adherent (Lyt2.2+) and nonadherent (Lyt2.2-) cells in the Simonsen spleen assay in neonatal (C57BL/6 x BALB/c)F1 mice was less than of unfractionated cells, but the activity of remixed Lyt2.2+ plus Lyt2.2- cells was higher than the sum of the contributions of these cells tested separately, and equal to that of the unfractionated cells. PNA- thymocytes were also separated into Ly2.2+ and Lyt2.2- populations by fluorescence-activated cell sorting. Nonlytic separation allows the recovery of the Lyt1+2+ population, which is lost in cytotoxic elimination experiments. Under the conditions described for the plate separation, the purity of the separated cells and recovery of activity approaches that of cells separated by sorting. Therefore, the plate separation offers a convenient alternative to fluorescence-activated cell sorting when large numbers (i.e. up to 5 x 10(7) positively selected cells) are needed, as in studies of in vivo cell-mediated immune reactions.

We have developed a soluble macromolecular conjugation reagent, polyacrylamide-streptavidin (PASA), for the simplified preparation of multivalent protein-protein conjugates. Soluble linear polyacrylamide, with a molecular weight of approximately 10(6), has carboxyl groups generated by limited alkaline hydrolysis. It is then activated with carbodiimide, separated from excess carbodiimide, and conjugated to streptavidin. The resulting conjugate, which has approximately 20 streptavidin residues per molecule, can bind biotinylated proteins to produce homo- or heteroconjugates of known composition. We have used this technique to prepare soluble multivalent heteroligating antibody conjugates that can bind either of two antigenically distinct cell lines, as well as reagents that specifically label murine tumor cells with different MHC class I antigens. The method is potentially useful for making multivalent arrays of epitopes for measuring low affinity interactions such as that between the T cell receptor and MHC molecules, as well as for making immunotoxins, tumor labelling conjugates, and complex immunogens.

Mouse spleen cells could be preparatively separated into immunoglobulin positive (Ig+) and immunoglobulin-negative (Ig-) populations by incubating as many as 2 × 108 cells per 100 mm diameter petri plate coated with specifically purified goat anti-mouse immunoglobulin. The non-adherent population was 95% or more Ig-, and possessed graft versus host and cytotoxic effector activities, as would be expected for T cells. They could also give a mixed lymphocyte reaction and generate cytotoxic effector activity on culture in vitro. The adherent cells could not be released undamaged from plates coated with undiluted anti-Ig, but they could be released from plates coated with a 1 4 or 1 10 dilution of anti-Ig in an irrelevant antibody. The released cells were over 90% vaible by trypan-blue staining, and 94% or more of the viable cells were Ig+.