Immunologic tolerance for immune system-mediated diseases.

Induction of long-term, antigen-specific immunologic unresponsiveness holds great promise for the treatment of many immune system-mediated diseases, including asthma, allergies, autoimmune diseases, and transplant rejection. Unlike current immunosuppressive treatments, immunologic tolerance therapies would affect only the undesired immune responses, leaving protective immunity intact. A variety of approaches to immunologic tolerance induction are being taken, reflecting the molecular and cellular complexity of immune system activation and regulation. The presentations summarized in this report represent promising strategies, some of which are being evaluated in advanced animal models and human clinical trials. Approaches presented include the following: interference with costimulatory signals in T-cell induction, T-cell receptor antagonism by altered peptides, exploitation of antigen-induced apoptosis to eliminate undesired T cells, opposition of inflammation by the induction of regulatory cytokines, induction of transplant tolerance by mixed chimerism, and deviation from deleterious allergic antibody responses by use of immunostimulatory DNA sequences. These multifaceted approaches are strongly supported by knowledge of basic immune mechanisms, which should facilitate the rational development of these therapies for controlling immune-mediated diseases.

Activated T hybridomas induce upregulation of B7-1 on bystander B lymphoma cells by a contact-dependent interaction utilizing CD40 ligand.

The upregulation of costimulatory molecules of antigen presenting cells (APC) resulting from interaction with activated T cells was studied in an in vitro system composed of well-characterized murine T hybridomas and B cell lymphomas. Increased B7-1 expression was induced on both MHC-matched and -mismatched (bystander) B lymphoma cells present in cultures of activated T hybridomas. Identical results were obtained with T hybridomas activated by either the appropriate peptide presented by MHC-matched APC or by mitogen stimulation in the absence of MHC/TCR cognate interactions. Soluble factors alone did not lead to upregulation of B7-1; B lymphomas cultured on the opposite side of a transwell membrane from an ongoing T cell stimulation response, or in supernatants of activated T cells, did not exhibit enhanced expression of B7-1. Antibodies to the CD40 ligand (CD40L) of T cells inhibited the increased appearance of B7-1 on B lymphomas. Significant B7-1 upregulation on the population of bystander B cells could be achieved even when they were present at a 10:1 excess over MHC-matched APC. These data indicate that B7-1 upregulation results from contact between bystander B cells and activated T hybridomas in vitro by CD40-CD40L interaction, without the requirement for TCR/MHC interaction.

A study of complexes of class II invariant chain peptide: major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody.

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP:MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP:MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Li peptide bound to the peptide-binding groove.

Characterization of epitopes recognized by hapten-specific CD4+ T cells.

Although protein-derived nominal Ags have, in many instances, been precisely determined, the epitopes recognized by hapten-specific CD4+ T cells responsible for contact sensitization have not been defined. To better understand the nature of the precise epitopes generated after hapten interaction with Langerhans cells (LC), we assessed the ability of TNP-modified I-Ak- and I-Au-binding peptides to activate hapten-specific CD4+ T cells obtained respectively from TNCB-primed C3H (H-2k) and PL/j (H-2u) mice. Using LC as APC, I-Ak-restricted TNP-specific CD4+ T cells proliferated in the presence of the synthetic peptide hen egg lysozyme 52-61 derivatized with TNP at position 56, and less so when TNP was coupled at positions 53 or 59. Similarly, I-Au-restricted TNP-specific CD4+ T cells from PL/j mice were triggered by the synthetic I-Au-binding 13 mer poly(A)-Y5-R6 TNP-modified at position 4, and to a limited extent with TNP coupled in positions 7 or 10. Our results indicate that hapten-modified MHC class II binding nonautologous peptides are recognized by hapten-specific CD4+ T cells and that precise positioning of hapten molecules on peptides binding MHC class II molecules is required for optimal CD4+ T cell recognition. These findings provide insight into the manner in which haptens are recognized by T cells involved in contact sensitivity and should facilitate the study and design of specific therapies for the manipulation of hapten-specific CD4+ T cell responses.

Identification of overlapping class I and class II H-2d-restricted T cell determinants of influenza virus N1 neuraminidase that require infectious virus for presentation.

The requirement for endogenous viral protein synthesis in helper and cytotoxic T cell (CTL) recognition of influenza virus was studied at the level of individual epitopes. The viral envelope glycoprotein neuraminidase (NA) contains class I and class II major histocompatibility complex (MHC)-restricted T cell determinants that are presented by virus-infected antigen-presenting cells (APC). We had previously shown that recognition of NA by class II I-Ed-restricted T cells required either active viral infection of APC or introduction of uv-inactivated virus to the cytosol, similar to the well-established requirements for class I-restricted responses. Detailed mapping of T cell epitopes was undertaken using vaccinia virus vectors encoding truncated segments of the influenza NA molecule and by synthetic peptides. Class I MHC-restricted CTL were found to recognize two regions of NA: residues 69-89 in the context of Dd and 191-201 presented by Kd. Analyses of T cell proliferation and T hybridoma clones revealed that class II-restricted responses recognized the same two regions as the CTL, presented by I-Ed and I-Ad, respectively. Interestingly, both class I and class II MHC-restricted T cells showed similar requirements for endogenously synthesized antigen, responding poorly or not at all to endocytosed uv-inactivated virus. This extends previous observations that specific epitopes can be preferentially presented by class II molecules from endogenously synthesized antigens and shows that the same antigenic determinants can have access to both class I and class II antigen presentation pathways.

Influenza virus infection elicits class II major histocompatibility complex-restricted T cells specific for an epitope identified in the NS1 non-structural protein.

A T cell epitope of the influenza virus NS1 molecule was identified and shown to be a determinant used in class II major histocompatibility complex-restricted T cell responses to infectious virus. An I-Ed-restricted BALB/c mouse T hybridoma clone recognizing influenza virus A/Puerto Rico/8/34 (PR8; subtype H1N1) but not A/Udorn/72 (subtype H3N2) secreted lymphokines in response to purified recombinant NS1 or fusion proteins containing amino acids 1 to 81 or 1 to 42 of NS1. As expected for recognition of a non-virion protein, the clone failed to respond to u.v.-inactivated virus. The antigenic determinant was localized by synthetic peptides to amino acids 13 to 32 of NS1, explaining the lack of recognition of A/Udorn/72 virus which has an alanine to valine substitution at position 23 within the determinant. A single intranasal dose of infectious PR8 virus was found to elicit T cells that responded to peptide NS1 13-32, suggesting that this determinant is a significant target of T cells in normal infections. To stimulate helper T cell responses similar to those achieved with infectious virus, influenza virus vaccines may therefore have to include NS1 in addition to virion components.

Class II MHC-restricted T cell determinants processed from either endosomes or the cytosol show similar requirements for host protein transport but different kinetics of presentation.

We have studied the role of APC protein transport in presentation of class II MHC-restricted T cell determinants of influenza virus glycoproteins that have distinct Ag processing requirements. Two I-Ed-restricted epitopes were analyzed: hemagglutinin (HA) 111-119, which is processed by the exogenous/endocytic pathway, and neuraminidase (NA) 79-93, which has a requirement for cytosolic processing. NA 79-93 is presented from infectious but not non-replicative virus under ordinary conditions. This requirement for viral biosynthesis could be bypassed by using a soluble inhibitor of NA,2,3-dehydro-2-deoxy-N-acetyl neuraminic acid (DDAN), to facilitate cytosolic introduction of virus. APC exposed to UV virus/DDAN present HA and NA determinants derived directly from proteins of the input virus particles. This allows presentation of both endocytically and cytosolically processed epitopes in the same experiment using noninfectious virus. The inhibitor brefeldin A (BFA) was used to interrupt host protein transport at various times relative to virus/DDAN addition. We observed that BFA added simultaneously with virus blocked recognition of NA 79-93 but not HA 111-119. This distinction was found to be based upon different expression kinetics of the HA and NA determinants. Expression of NA 79-93 required 6 to 9 h, whereas HA 111-119 was presented by 1 h after Ag addition. When APC were incubated with BFA at intervals before virus addition, presentation of HA 111-119 was also blocked as a function of time. Data indicate that about 5 h of BFA treatment is needed to deplete host protein pools required for presentation of I-Ed-restricted T cell determinants processed from either endosomes or the cytosol.

Immunogenic peptides of influenza virus subtype N1 neuraminidase identify a T-cell determinant used in class II major histocompatibility complex-restricted responses to infectious virus.

Six nonoverlapping peptides of the neuraminidase (NA) glycoprotein of influenza virus A/Puerto Rico/8/34 (H1N1) (PR8 virus) were found to be immunogenic for proliferating T cells when injected into BALB/c mice in Freund adjuvant. T cells elicited by peptide immunization could recognize PR8 virus in vitro. However, only one of these peptides, corresponding to residues 79 to 93 of NA (NA 79-93), was able to restimulate T cells of mice immunized with infectious virus. T cells that recognized this peptide were uniformly I-Ed restricted, yet infectious influenza virus was required for responses. NA 79-93-specific T-hybridoma clones raised by immunization either with whole virus or with the synthetic peptide alone each responded to replicative virus and not to UV-inactivated virions. These data suggest that the NA 79-93 T-cell determinant which is commonly presented during an encounter with influenza virus in vivo is processed preferentially from NA synthesized within antigen-presenting cells.

Class I H-2d-restricted cytotoxic T lymphocytes recognize the neuraminidase glycoprotein of influenza virus subtype N1.

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) that recognize the neuraminidase (NA) glycoprotein of subtype N1 influenza A viruses have been demonstrated in BALB/c mice. Responses to NA were obtained only in protocols that use two in vivo inoculations of virus, including a recombinant vaccinia virus containing the NA of subtype N1 influenza virus (NA-VAC) to prime or boost. Restimulation in vitro was also required for CTL recognition of NA and strongly depended on the specific N1 virus used. Influenza viruses A/Puerto Rico/8/34 (H1N1), A/CAM/46 (H1N1), J1 (H3N1), and JAP/BEL (H2N1), but not A/Bellamy (H1N1) or MEM/BEL (H3N1) virus, were able to stimulate NA-specific memory T cells in vitro. Single or double in vivo inoculation of any of the N1 viruses or a single injection of NA-VAC failed to elicit restimulatable NA-specific CTL. Lysis of NA-VAC-infected cells at low effector/target ratios was comparable to that observed toward other influenza virus proteins known to be major targets of CTL in BALB/c mice, indicating that antigenic determinants of the subtype N1 NA molecule can be efficiently presented in the context of major histocompatibility complex class I.

Virus entry and antigen biosynthesis in the processing and presentation of class-II MHC-restricted T-cell determinants of influenza virus.

Receptor-mediated uptake of influenza virus is responsible for efficient introduction of virus particles to APC. This leads to the effective presentation to T-cells of very small concentrations of proteins entering on the intact virus. Endocytosed virus transits rapidly to the endosome compartment. Entry into this environment appears to greatly affect the fate of T-cell determinants. While promoting the presentation of determinants which require extensive antigen processing, the intracellular environment appears also to lead to destruction of labile determinants, such as those of NA. The same NA determinants are efficiently presented by actively infected cells, indicating that newly biosynthesized viral proteins need not be subjected to the same handling as internalized viral particles. In a similar way, site 3 of HA, which, in a single pulse of noninfectious virus or isolated HA protein is expressed with a relatively short half-life, has greatly improved levels of duration and expression on actively infected APC. Since certain T(H) determinants are unavailable or poorly expressed when introduced on nonreplicative influenza virus, vaccination with inactivated virus might have limitations in stimulating T(H) as well as class-I responses. Finally, individual T-cell determinants of the same protein can exhibit distinct patterns of expression and persistence on APC surfaces. These different half-lives of T(H) determinants may be influential in determining immuno-dominance of T-cell sites. Determinants that are longer-lived on APC may have a greater probability of interacting with appropriate T(H) precursors, which could lead to an enhanced T-cell response to that region of the viral protein.

Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes.

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-expressing APC. This indicates that HA endogenously synthesized within these cell lines is available for Ag processing by an exogenous route.

Class II major histocompatibility complex-restricted T cells specific for a virion structural protein that do not recognize exogenous influenza virus. Evidence that presentation of labile T cell determinants is favored by endogenous antigen synthesis.

The contribution of viral infectivity to the expression of MHC class II-restricted T cell determinants was studied. A murine I-Ed-restricted T cell hybridoma recognizing the neuraminidase (NA) glycoprotein of influenza PR8 virus was stimulated strongly by infectious virus but failed to recognize antigen introduced on noninfectious virions. Recognition correlated with the de novo synthesis of viral NA within infected APC. The effectiveness of infectious virus did not depend strictly upon the amount of NA present in cultures, since high NA concentrations could be achieved by addition of nonreplicative virus without being stimulatory for NA-specific T cells. Recognition of a determinant generated only when synthesized in murine host cells was ruled out, since, in high concentration, NA isolated from purified egg-grown virions, even if reduced and alkylated, was recognized by the T hybridoma clone. Isolated NA was recognized when added to pre-fixed APC, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. Data suggest that endogenously synthesized antigen may contribute most significantly to presentation of labile T cell determinants. In addition to NA, recognition of an I-Ed-restricted determinant of the influenza hemagglutinin (HA) molecule, shown previously to have a relatively short half-life on APC surfaces, was enhanced greatly by infectious virus. In contrast, T cell recognition of a more stably expressed I-Ed-restricted site of the same HA polypeptide was only marginally improved on infected APC.

Individual class II-restricted antigenic determinants of the same protein exhibit distinct kinetics of appearance and persistence on antigen-presenting cells.

The kinetics of presentation of class II-restricted T cell determinants of influenza virus hemagglutinin (HA) was investigated over a 48-h time course after pulsing of A20 B lymphoma APC with non-replicative virus or isolated HA. At intervals after Ag pulse, APC were fixed with paraformaldehyde to arrest Ag processing and to preserve the expression levels of the presented determinants. Expression of T cell sites at each time point was probed by a panel of BALC/c T hybridomas specific for the HA of influenza A/Puerto Rico/8/34 virus, recognizing either site 1 (residues 111 to 119), site 2 (126 to 138), or site 3 (302 to 313). Characteristic patterns of presentation were observed for each site: sites 2 and 3 achieved maximal expression by 8 h post pulse, but declined thereafter, whereas site 1 presentation continued to increase over time. The quantitative expression of each T cell site was affected by the proteolysis inhibitor leupeptin, resulting in partial inhibition of site 1, complete blocking of site 2, but enhancement of site 3. However, the expression kinetics of sites 1 and 3, which could be observed in the presence of the inhibitor, remained qualitatively unchanged. These observations indicate that some T cell determinants (e.g., HA site 1) may exhibit a greater longevity of expression on APC than other antigenic sites of the same protein. Differences in the persistence of surface expression of distinct T cell sites may be a factor in their relative immunodominance.

Acid-induced conformational modification of the hemagglutinin molecule alters interaction of influenza virus with antigen-presenting cells.

Brief exposure of influenza virus to pH 5 was found to have extensive effects upon presentation of viral Th cell antigenic determinants. This acidity, comparable to that encountered in host cell endosomes, was known to effect conformational changes in the viral hemagglutinin (HA) which alter the molecule's fusion activity, antigenicity, and susceptibility to enzymes. Three major effects of low pH upon presentation of viral T cell determinants were observed: first, acid pretreatment permitted presentation by pre-fixed APC of two of three linear T cell sites of the HA molecule, bypassing the APC activity required to present untreated virus; second, the two determinants presented in this manner disappeared rapidly from APC surfaces; third, acid-pretreated virus was not efficiently utilized by active APC in the normal pathway of viral antigen presentation. These observations suggest that the pH-induced conformational transition of HA may constitute sufficient processing for certain linear determinants of the molecule and additionally influences the processes involved in the general formation and presentation of viral T cell sites.

Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.

Influenza-specific suppression: contribution of major viral proteins to the generation and function of T suppressor cells.

Suppressor cells were generated in BALB/c mice by two sequential injections of PR8 influenza virus (A/Puerto Rico/8/34[H1N1]) and were tested for their ability to inhibit proliferative cellular responses towards multiple viral and nonviral antigens. In this way, suppression specific to PR8 as compared with purified protein derivative (PPD) and keyhole limpet hemocyanin (KLH) antigenic responses was illustrated. Experiments involving adoptive transfer of suppression to naive hosts with subfractionated lymphocyte populations demonstrated that the suppressors were Lyt-2+ T cells. Two major questions were addressed with this system. First, a determination was made of which anti-viral protein proliferative responses were affected by the PR8-induced T suppressor (Ts) cells. Ts cells were found to inhibit proliferating cells with specificities for isolated hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix (M) antigens. Second, experiments were conducted to analyze the viral proteins contributing to the induction of PR8-specific Ts cells. Inoculations with either isolated HA or a combination of M + NP proteins induced T suppression specific to proliferative responses towards PR8. These experiments illustrate the contribution of external (HA and NA) as well as internal (M + NP) viral proteins to Ts cell generation and function.

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus.

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.