Soluble mouse major histocompatibility complex class II molecules produced in Drosophila cells

We have exploited Drosophila melanogaster. Schneider cells and compatible inducible expression vectors to produce large amounts of secreted major histocompatibility complex (MHC) class II molecules (I-E^d). A simple two-step purification protocol was developed. In the first step, recombinant molecules were enriched using a monoclonal anti-class II antibody column followed by a nickel chelate column which further purified and concentrated the recombinant protein to several mg/ml. Characterization of the purified material indicates that the molecules are correctly assembled into αβ heterodimers. Further analysis shows that the recombinant MHC class II molecules are devoid of endogenous peptides and, therefore, homogenous peptide/MHC complexes could be prepared by adding exogenous I-E^d-specific peptides at slightly acidic pH. Upon peptide addition, molecules underwent a conformational change into a more compact form revealed by gel filtration analysis. In addition, the peptide/MHC complexes were biologically active. As little as 10 ng of these complexes coated on plastic from a 100 ng/ml solution were sufficient to trigger antigen-specific T cell hybridomas. These MHC class II molecules, together with various forms of soluble T cell receptor (TcR) proteins, provide valuable tools to analyze the molecular details of TcR/antigen recognition.     

Dose-dependent T cell tolerance to an immunodominant self peptide

We have previously described a model of tolerance to self peptides in a mouse transgenic (Tg) line producing secreted hen egg-white lysozyme (HEL). The HEL cDNA was placed under the control of a ubiquitous promoter expressed early in embryogenesis, so that HEL should be present in Tg mice throughout the development of the immune system. Since individual HEL Tg mice express different amounts of serum HEL, we were previously able to show that H-2^d mice with HEL blood level > 10 ng/ml are tolerant to HEL and to the immunodominant (ID) peptide 108–116. However, autoreactive T lymphocytes recognizing the HEL subdominant (SD) peptides 74–96 and 1–18 still persist and the SD-specific responses disappear at higher blood HEL concentrations. In the present work, we have studied HEL Tg H-2^d mice with HEL serum levels < 10 ng/ml (HEL-low Tg animals). We find that 50% of Tg animals with HEL blood concentration < 2 ng/ml are responsive to HEL in T cell proliferation assays, although these responses are lower than those seen in non-Tg control mice. The HEL-specific T lymphocytes react only with 15-mer overlapping peptides encompassing the single H-2^d ID region of HEL (residues 102–122); whereas the 9-mer minimal ID peptide 108–116, which strongly triggers non-Tg T cells, is unable to stimulate auto-reactive T cells in vitro from HEL-low Tg mice. Altogether, our results suggest that T lymphocytes specific for the minimal ID peptide are deleted or inactivated, while T cell clones of lower affinity and reacting with epitopes on longer peptides persist. Thus, the high affinity ID peptide-specific T cell clones can be negatively selected even in the presence of low amounts of HEL.     

Immunological focusing by the mouse major histocompatibility complex: Mouse strains confronted with distantly related lysozymes confine their attention to very few epitopes

The gallinaceous lysozymes are a family of antigens that are distantly related to mouse lysozyme. A T cell-dependent proliferation assay was used to characterize the spectrum of reactivities to lysozyme determinants in B10-congenic mice. Cross-reactivity studies using a panel of species variant lysozymes to stimulate lymph node cells from chicken egg white lysozyme- and ring-necked pheasant egg white lysozyme-primed B10.D2 mice indicated a preferential focusing of T cell reactivity onto a single determinant containing amino acids 113-114. These data, in conjunction with results obtained by priming with cyanogen bromide cleavage fragments of lysozymes, suggested that a site common to the L3 region (amino acids 106-129) of all the lysozymes tested was a preferential anchorage site for I region-encoded la molecules on H-2^d antigen-presenting cells, leading to the limited display of a determinant containing residues 113-114. Priming with L2H (amino acids 13-105), a peptide containing the major epitopes recognized by B10.A and B10 mice, failed to stimulate any T cell proliferation by B10.D2 lymph node cells. Thus, it appears the la molecules in any one mouse strain attach to very few sites on lysozyme to effectively display antigenic determinants for T cell activation. This result points to a model of limited determinant selection even on a very “foreign” antigen based upon a shortage of appropriate amino acid residues usable by la antigen-presenting structures of a strain.     

Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecules in endocytic compartments

The current studies were carried out to examine the basis for the differences in the antigenic peptides generated from exogenous and endogenous forms of hen egg white lysozyme (HEL). The role of different intracellular compartments in the generation and binding of HEL peptides derived from two endogenous forms of HEL, either secreted (sHEL) or retained in the endoplasmic reticulum (ER, KDEL HEL), presented by MHC class II molecules was examined and compared to exogenous HEL. Initially it was found that antigen-presenting cells bearing both intracellular forms of HEL generated and presented a number of IA^k-restricted HEL epitopes to T cell hybridomas, although s HEL was processed more efficiently than KDEL HEL. There were differences, however, for some determinants between endogeneous and exogenous HEL. At equivalent antigen-presenting efficiencies, endogenous HEL-bearing cells displayed a lower surface density of IA^k-bound HEL-52-61-related peptides than cells pulsed with exogenous HEL, as detected by a specific monoclonal antibody. Neither endogenous HEL degradation nor peptide binding to MHC class II molecules occurred in the ER. Processing of sHEL and KDEL HEL appears to take place either in a post-trans-Golgi network acidic compartment or in the cytosol, whereas peptide binding to MHC class II molecules occurs in endocytic compartments . Furthermore, the peptides generated were derived from an endogenous source rather than from secreted and re-endocytosed HEL. Thus, processing of endogenous HEL is from a different pool than exogenous HEL and occurs in different compartments.     

mAb against hen egg-white lysozyme regulate its presentation to CD4(+) T cells.

Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or FcgammaR in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4(+) T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103-117 T cell epitope (I-E(d)) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116-129 and 34-45, I-A(k) restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 34-45 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (45-61, I-A(k) restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.     

Cytosolic targeting of hen egg lysozyme gives rise to a short-lived protein presented by class I but not class II major histocompatibility complex molecules

A way to study the role of intracellular trafficking of an antigen in its presentation to T cells is to target the antigen to various cell compartments of the antigen-presenting cells (APC) and compare the nature of the complexes associating major histocompatibility complex (MHC) molecules and antigenic peptides, expressed on the cell surface. MHC class I+ and MHC class II+ mouse L fibroblasts secreting hen egg lysozyme (HELs cells) or expressing HEL in their cytosol (HELc cells) were obtained after transfection with HEL cDNA and signal sequence-deleted HEL cDNA, respectively. HEL was evidenced in both HELs- and HELc-transfected cells and the former type of transfectant secreted a large amount of HEL. However, HEL produced in the cytosol exhibited a short half-life of less than 5 min. HEL-derived peptides could not be shown biochemically either in HELc- nor in HELs-transfected cells. We then studied the capacity of these cells to present HEL to HEL-specific class I- and class II-restricted T cells. Both cell types could be recognized by the HEL-specific MHC class I-restricted CTL clones. In contrast, MHC class II-HEL peptide complexes, recognized by HEL-specific helper T cell hybridomas, could be detected on MHC class II+ HELs- but not HELc-transfected cells. In vivo experiments showed, however, that HELc-transfected cells could provide host APC with HELc-derived peptides able to associate with MHC class II molecules. This was inferred from the capacity of MHC class II-HELc-transfected cells, unable by themselves to elicit any anti-HEL antibody response, to prime syngeneic and allogeneic mice against HEL. The priming was revealed by the induction of an antibody response after a boost with an amount of HEL unable itself to elicit an antibody response.     

Structural and functional characterization of HLA-DR molecules circulating in the serum

HLA molecules are highly polymorphic glycoproteins with a single binding site for immunogenic peptides. The complex formed by HLA-DR molecules and peptides is the entity specifically recognized by the antigen receptor of CD4+ helper T lymphocytes. This biological function has been linked to the constitutive cell surface expression of HLA molecules on antigen-presenting cells which provide immunogenic peptides through denaturation or fragmentation of antigen. Here, we report that HLA-DR molecules can be identified in a soluble form circulating in the serum. Biochemical characterization and recognition of such soluble HLA-DR molecules by a monoclonal antibody reactive to conformational determinants on the alpha-beta HLA complex indicates that they are circulating as intact heterodimers. Upon activation, human T cells and B cells release soluble HLA-DR molecules. The expression and secretion of HLA-DR by T lymphocytes is greatly enhanced in the presence of macrophages indicating that the production of HLA-DR molecules might be regulated and controlled through factors derived from antigen-presenting cells. An alloreactive human T cell clone can be stimulated to proliferate in response to soluble HLA-DR molecules purified and enriched from serum suggesting that the soluble form of the natural ligand of the T cell receptor might have immunoregulatory functions.     

Characterizing immunodominant and protective influenza hemagglutinin epitopes by functional activity and relative binding to major histocompatibility complex class II sites

In the present study the analysis of functional activity and major histocompatibility complex (MHC) binding of two adjacent MHC class II-restricted epitopes, located in the C-terminal 306–329 region of human influenza A virus hemagglutinin 1 subunit (HA1) conserved with subtype sequences and not affected by antigenic drift, was undertaken to explore the hierarchy of local immnodominance. The functional activity of two T cell hybridomas of the memory/effector Th1 phenotype in combination with in vivo immunization studies provided a good tool for investigating the functional characteristics of the T cell resonse. The in vitro binding assays performed with a series of overlapping, N-terminal biotinylated peptides covering the 306–341 sequence enabled us to compare the relative binding efficiency of peptides, comprising two distinct epitopes of this region, to I-E^d expressed on living antigen-presenting cells. Our studies revealed that (i) immunization of BALB/c mice with the 306–329 H1 or H2 peptides resulted in the activation and proliferation of T cells recognizing both the 306–318 and the 317–329 epitopes, while the 306–329 H3 peptide elicits predomonantly 306–318-specific T cells, (ii) the 317–329 HA1 epitope of the H1 and H2 but not the H3 sequence is recognized by T cells and is available for recognition not only in the 317–329 peptide but also in the extended 306–329 or 306–341 peptides, (iii) the 306–318 and the 317–329 hemagglutinin peptides encompassing the H1, H2 but not the H3 sequence bind with an apparently similar affinity to and therefore compete for I-E^d binding sites, and (iv) the 317–341, the 317–329 peptides and their truncated analogs show subtype-dependent differences in MHC binding and those with lower binding capacity represent the H3 subtype sequences. These results demonstrate that differences in the binding capacity of peptides comprising two non-overlapping epitopes located in the C-terminal 306–329 region of HA1 of all three subtype-specific sequences to MHC class II provide a rationale for the local and also for the previously observed in vivo immunodominance of the 306–318 region over the 317–329 epitope in the H3 but not in the H1 or H2 sequences. In good correlation with the results of the binding and functional inhibition assays, these data demonstrate that in the H1 and H2 subtypes both regions are available for T cell recognition, they compete for the same restriction element with an appearently similar binding efficiency and, therefore, function as co-dominant epitopes. Due to the stabilizing effect of the fusion peptide, peptides comprising the 306–341 or 317–341 H1 sequences are highly immunogenic and elicit a protective immune response which involves the production of antibodies and interleukin-2 and tumor necrosis factor producing effector Th1 cells both directed against the 317–329 region. Based on the similarity of the I-E^d and HLA-DR1 peptide binding grooves and motifs, these results suggest that amino acid substitutions inserted to the H3 subtype sequence during viral evolution can modify the relative MHC binding capacity and invert the local hierarchy of immunodominance of two closely situated epitopes that are able to bind to the same MHC class II molecule.     

A peptide binding weakly to the major histocompatibility molecule augments T cell responses

An I-A^d-derived peptide PB1 was found to enhance the reactivity of I-A^d-restricted T cells. The augmentative effect was not due to the cross-reactivity of PB1 peptide with antigens. PB1 had no effect on T cells specific for I-A^b and I-E^k, nor did PB1 increase the T cell responses to concanavalin A and staphyloccocal enterotoxin B. The strict I-A^d specificity suggests that PB1 enhances the recognition of antigen-I-A^d complex by T cell receptor. PB1 bound to I-A^d weakly. The augmentative effect could be found on other I-A^d-binding peptides in appropriate conditions; however, PB1 was distinct in its prominently augmentative effect on all the I-A^d-restricted T cells analyzed. A similar enhancing activity was demonstrated on a synthetic transferrin receptor peptide with minimum affinity for I-A^d. The unusual enhancing activity of PB1 may thus be attributed to the low I-A^d binding affinity. It was postulated that the binding of low-affinity PB1 would not only stabilize I-A^d structure, but also enhance the binding of other peptides. This was supported by the increased binding of OVA 323-339 and cI 84–98 to I-A^d in the presence of PB1. The inclusion of PB1 in the immunization mixture also enhanced T cell responses in vivo, suggesting the possibility of using low-affinity peptide to promote specific immunity.     

Cathepsin D,but not cathepsin B,releases T cell stimulatory fragments from lysozyme that are functional in the context of multiple murine class II MHC molecules

In this study, the major endosomal/lysosomal proteases cathepsin D and cathepsin B were tested on their ability to release T cell stimulatory peptides from hen egg white lysozyme (HEL) in vitro. Whereas neither enzyme could cleave unreduced HEL under mild conditions, reduced HEL was readily cleaved by cathepsin D but not by cathepsin B. Instead, cathepsin B was found to be very active in the trimming of HEL peptides after their release by cathepsin D. Following high-performance liquid chromatography (HPLC) fractionation, cathepsin D-released HEL fragments were screened for recognition by HEL-specific T cells from three strains of mice, i.e. B10. A (H-2^a), C57BL/6 (H-2^b) and BALB/c (H-2^d). Peptides in a large number of different HPLC fractions triggered significant T cell responses in all three strains. Interestingly, the response profiles of T cells from the three different strains showed marked similarities. Also, several individual synthetic HEL sequences corresponding to selected cathepsin D-released fragments were recognized by murine T cells in the context of all three major histocompatibility complex (MHC) haplotypes tested. Our data suggest that cathepsin D rather than cathepsin B may play a central role in the initial release of HEL fragments during endosomal/lysosomal processing. The relatively long HEL fragments released by cathepsin D, containing about 20—30 amino acid residues, are significantly more promiscuous in murine class II MHC binding than the shorter synthetic HEL sequences previously employed by others for the delineation of HEL epitopes. Extensive documentation of HEL epitopes in previous investigations indicate that this promiscuity cannot be explained by simply assuming that longer peptides contain additional epitopes. Rather, an increased peptide length by itself appears to promote promiscuous MHC binding.     

B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.     

Modification of the inhibitory amino acid for epitope peptide binding onto major histocompatibility complex class II molecules enhances immunogenicity of the antigen

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.     

Analysis of T cell reactivities to phosphorylcholine-conjugated hen egg lysozyme in C57BL/6 mice: hapten-conjugate specificity reflects an altered expression of a major carrier epitope.

We have selected several different T cell hybridoma clones reactive to hen egg lysozyme (HEL) conjugated to phosphorylcholine (PC) after fusion of PC-HEL-primed C57BL/6 lymphocytes with BW5147 parent cells. These hybridoma clones preferentially recognize PC-HEL over unconjugated HEL, but not other carrier molecules conjugated with the same hapten. All the PC-HEL-reactive clones are similarly responsive to not only p-azobenzenearsonate (ABA)-conjugated HEL (ABA-HEL) but also to a variety of other diazotized hapten-HEL conjugates. However, these clones are not stimulated by fluoresceinated or dinitrophenylated HEL beyond the level of HEL carrier alone. Therefore, the type of hapten linkage (diazonium) to the carrier molecule appears to affect T cell recognition. The hybridoma clones apparently recognize the carrier molecule alone, although the level of stimulation is relatively low compared to that induced by either PC-HEL or ABA-HEL. Interestingly, HEL unfolded by S-carboxymethylation is capable of stimulating the hybridomas to a level comparable to that obtained with PC-HEL. T cell recognition of the unfolded HEL is independent of antigen processing, which is different from that of PC-HEL. The peptide sequence corresponding to the amino acids 81-93 of HEL appears to contain the epitope region for the hybridoma clones based on testing stimulation activity with synthetic peptides. Previously, the peptides including this region (81-96) have been reported as the determinant recognized by T cells derived from C57BL/6 mice after immunization with an HEL peptide (HEL 13-105) but not with native HEL. These results suggest that a hapten conjugation via diazonium linkage modifies antigen presentation and consequently the presentation of the major T cell epitopes similar to that of the HEL fragment.     

Antigen processing and presentation by macrophages

The classical macrophage is one of the most important cells involved in presenting antigen to helper T cells, because of its ability to regulate its expression of Ia molecules and to encounter and process particulate and soluble antigens. We have summarized in this report studies examining the handling by macrophages of two different antigens, the bacteria Listeria monocytogenes and the protein hen egg white lysozyme (HEL). The purpose was to identify potential sources of immunogenic peptides. Presentation of Listeria required an intracellular processing stage sensitive to lysosomotropic drugs. The Listeria required internalization and processing, after which immunogenic molecules were recognized by T cells on the macrophage surface. Metabolic studies showed that Listeria-derived peptides were released by macrophages that had phagocytosized the bacteria. The release of these peptides was a temperature-dependent process, unaffected by inhibiting lysosomal catabolism by treatment with chloroquine. Listeria-derived peptides were also detected on the surface of the macrophage. These peptides behaved like integral membrane proteins, some of which persisted for at least 24 hr at the macrophage surface. When tested for immunogenicity, the released peptides were very weakly immunogenic. The membrane-associated peptides alone could not stimulate Listeria-specific T cells, but could be reprocessed by additional macrophages and subsequently stimulate the T cells. A defined antigen system using HEL-specific T-cell hybridomas was used to examine the processing of HEL. Presentation of HEL required a choloroquine-sensitive intracellular processing stage. In examining two T-cell hybridomas, a differential requirement for antigen processing was determined. The immunogenicity of released HEL from HEL-pulsed macrophages was also tested, and no “processed” antigen was detected capable of directly stimulating T cells; however, native HEL was released from the macrophage and could be processed and presented by other macrophages.     

Antigen presentation of hen egg-white lysozyme but not of ribonuclease A is augmented by the major histocompatibility complex class II-associated invariant chain

The influence of the class II-associated invariant chain (Ii) on the presentation of the protein antigens hen egg-white lysozyme (HEL) and ribonuclease A (RNase) was investigated. For this purpose the Ii- rat-2 fibroblasts were transfected with I-Ak genes with or without Ii. Transfectants expressing Ii were superior in the presentation of the complete HEL protein to a panel of I-Ak-restricted T hybridomas characterized by distinct specificities for different HEL peptides and by different sensitivities to antigen concentration. There appeared to be a correlation between the antigen-presenting capacity and the amount of Ii, in that transfectants expressing large amounts of Ii were the best antigen presentors. The presentation of synthetic HEL peptides was not influenced by Ii. In contrast to the findings with HEL, the presentation of RNase by the same set of transfectants was clearly independent of Ii. Both antigens, HEL and RNase, required processing in the chloroquine-sensitive compartment. However, only the presentation of HEL but not of RNase could be efficiently blocked by brefeldin A. These data confirm that presentation of HEL depends on de novo synthesized class II molecules, whereas the presentation of RNase seems to be predominantly mediated by a pool of pre-existing class II molecules whose interaction with endocytosed antigen does not depend on Ii. These results suggest different mechanisms for the presentation of HEL and RNase and they raise the possibility that different antigens intersect the class II pathway at distinct intracellular locations.     

‘Empty’ Ld molecules capture peptides from endocytosed hepatitis B surface antigen particles for major histocompatibility complex class I-restricted presentation

Peptides recognized by CD8⁺ cytotoxic T lymphocytes in the context of major histocompatibility complex (MHC) class I molecules are usually derived from endogenous proteins synthesized within the cell. Exogenous 22-nm hepatitis B surface antigen (HBsAg) particles are taken up by many cells, and are processed in a novel peptide-transporter-independent, endosomal or lysosomal pathway for class I (L^d)-restricted epitope presentation. Here, we present evidence that ‘empty’ L^d molecules derived from the cell surface are involved in presenting antigenic peptides from endocytosed HBsAg particles. Intracellular assembly of presentation-competent, trimeric L^d molecules required endocytosis of the exogenous antigen and ‘empty’m L^d molecules. These data assign a functional role to surface-associated, ‘empty’ MHC class I molecules.     

An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV-1 envelope glycoprotein

The immunodominant H-2D^d-restricted cytotoxic T lymphocyte (CTL) response to the HIV-1 gp160 envelope glycoprotein maps to a single determinant in the V3 loop, designated p18. Using a series of peptides synthesized on pins we have determined that the minimal core sequence of this determinant required for CTL recognition comprises 8 amino acids (residues 320-327). However, 9mer and l0mer peptides containing this core sequence were more effective than the 8mer peptide at sensitizing D^d-expressing target cells. To analyze the antigenicity of endogenously synthesized p18, minigenes encoding a 10-amino acid determinant (residues 318-327) and a 67-amino acid peptide (residues 281-348; containing the V3 loop) were expressed using vaccinia virus (Vac) recombinants. Both peptides were as effective as wild-type gpl60 in their ability to sensitize target cells for lysis by gpl60-specific CTL. Immunization of BALB/c mice with Vac recombinants encoding both gp160 peptides elicited gp160-specific CTL. These data demonstrate that both the V3 loop itself and a 10-residue epitope are sufficient to prime CTL in vivo and strongly support the potential use of minigene-encoded CTL epitopes for recombinant vaccines designed to induce protective T cell-mediated immunity against HIV-1.     

Antigen presentation is a function of all B cell subpopulations separated on the basis of size

Purified splenic B cells from nonimmune mice were separated by counterflow centrifugal elutriation into 6 subpopulations containing cells of discrete sizes ranging from 119 to 200 μm³. B cells of each subpopulation were competent to process and present a native globular protein antigen, cytochrome c, to a cytochrome c-specific T cell hybrid. In all cases, the B cells' antigen-presenting function was radiation sensitive and did not require T cells or T cell products, since B cells fixed with paraformaldehyde effectively presented a carboxyl-terminal peptide fragment of cytochrome c containing the T cell determinant. Furthermore, the antigen-presenting function of B cells of each subpopulation was augmented by treatment with submitogenic doses of the F(ab')₂ fragment of rabbit anti-mouse Ig antibodies, in that 10-30-fold fewer B cells were required and higher maximal T cell responses were achieved, indicating that B cells of all sizes are capable of being regulated in their antigen presentation function through their surface Ig. In addition, B cells of each subpopulation responded to soluble factors present in the supernatants of activated T cells as evidenced by an increase in volume and by the uptake of [³H]thymidine. These results indicate that B cells, regardless of size, are able to participate in at least two essential phases of T cell-dependent antibody responses, initiating the interaction by processing and presenting antigen to helper T cells and responding to soluble helper factors secreted by activated T cells.