T and B cell immune response to a 55-kDa endothelial cell-derived antigen in severe asthma

Current concepts on the pathogenesis of chronic asthma emphasize the role of several inflammatory cell populations and their respective mediators that interact in a complex network. However, beside inflammatory cells, lymphocytes are also present in asthmatic airways. Although little is known about their involvement in asthma, it has been suggested that lymphocytes may participate in the development of chronic inflammation either through lymphokine secretion or through antibody production. In this study, we describe circulating IgG autoantibodies, directed against a common 55-kDa antigen shared by platelets and cultured endothelial cells, and found in 34 out of 97 asthmatic patients. Among epidemiological, clinical and biological characteristics of these asthmatic patients, the anti-55-kDa antigen antibodies are mainly restricted to patients with negative cutaneous prick tests (p = 0.0014), and corticosteroid-dependent asthma (p = 0.0036). These antibodies were also detected in a few patients with autoimmune disorders like systemic lupus erythematosus (3/30) or rheumatoid arthritis (2/36). Both platelet and endothelial cell antigens were cross-reactive, had an isoelectric point between 8.0 and 9.0, were unsensitive to reducing agents such as 2-mercaptoethanol. and were not present on either platelet or endothelial cell surface, as determined by immunostaining assay. [³H] Thymidine incorporation assay with peripheral blood mononuclear cells from patients in the presence or in the absence of 55-kDa antigen, purified from nitrocellulose sheets demonstrated a specific incorporation in 6 out of 13 patients with circulating anti-55-kDa antigen antibodies, with index values ranging from 12 to 3. Such a T cell reactivity has also been observed in 3 out to 17 patients without detectable serum anti-55-kDa antigen antibodies. Moreover, a significant correlation was found between index values of antigen-specific T cell reactivity and the forced expiratory volume in one second (r = 0.544, p = 0.003). Our data indicate that the detection of such antibodies allows to distinguish a subgroup of asthmatics in terms of severity and to suggest a relationship between clinical severity and T and B cell autoreactivity to the 55-kDa platelet/endothelial cell antigen.     

T and B cell responses following immunization with tetanus toxoid in IgA nephropathy.

The B and T cell responses were investigated in IgA nephropathy before and after immunization with tetanus toxoid (TT). Both IgA and IgG anti-tetanus toxoid antibodies were elicited, but the IgA antibodies were significantly greater in patients (92.6 +/- 11.7 ELISA units) than in the controls (49.2 +/- 7.5 ELISA units). This was associated with a significantly greater proportion of IgA+ B cells in patients than controls before immunization. However, a significant increase in the proportion of IgA1 binding CD4 and CD8 cells was also found. The proportion of CD3 cells with gamma delta T cell receptors (CD3+TCR gamma delta +), was significantly greater before immunization in the IgA nephropathy patients (37.0% +/- 2.4), compared with controls (10.0% +/- 2.3; P less than 0.001). Immunization with TT further enhanced the CD3+TCR gamma delta + cells in patients to 45.8% +/- 7.2 compared with controls (16.3% +/- 4.5), with a corresponding decrease in CD3+TCR alpha beta + cells in the patients (P less than 0.001). CD3+TCR gamma delta + cells are upregulated by common microbial antigens and clinical exacerbations of IgA nephropathy are frequently associated with mucosal infections and a rise in serum IgA concentration. The increased TCR gamma delta expression may be responsible for the enhanced IgA antibody response in IgA nephropathy. The increase in IgA antibodies may than exert a controlling effect by binding to augmenting T cells and thereby inhibiting their function.     

Lentivector immunization induces tumor antigen-specific B and T cell responses in vivo

Expression of the cancer/germ-line antigen NY-ESO-1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY-ESO-1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA-A2-transgenic mice elicited long-lasting B and T cell responses against NY-ESO-1. CD8+ T cells against the HLA-A2-restricted peptide NY-ESO-1(157-165) were readily detectable ex vivo and showed restricted TCR Vbeta usage. Moreover, rLV/ESO elicited a far greater anti-NY-ESO-1(157-165) CD8+ T cell response than peptide- or protein-based vaccines. Anti-NY-ESO-1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen-specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY-ESO-1. The induced CD4+ T cells were primarily directed against a single NY-ESO-1 epitope spanning amino acids 81-100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.     

Interactive effect of Gm and Km allotypes on cellular immune responses to streptococcal cell wall antigen

Serum samples from 121 unrelated, healthy Japanese individuals were typed for several Gm and Km(1) allotypes. Peripheral blood lymphocytes from these subjects were cultured with streptococcal cell wall (SCW) antigen and the incorporation of 3H-thymidine into T lymphoblasts was measured. Log-linear analysis showed a significant interactive effect of Gm1,17;13,16,21 and Km(1) on the cellular immune response to group A SCW antigen.     

Differential pattern of T cell recognition of the 65-kDa mycobacterial antigen following immunization with the whole protein or peptides

The 65-kDa stress protein from Mycobacterium bovis (Bacillus Calmette Guérin) elicited T cell proliferation and antibody responses in seven B10 congenic mouse strains with different H-2 haplotypes. To analyze T cell determinants on this antigen, seven peptides corresponding to six predicted T cell epitopes, and one defined B cell epitope were synthesized. Mice were either immunized with the whole antigen and the specificity of the response was ascertained in respect of the six peptides, or mice were immunized with seven of the peptides and tested for proliferative responses to the whole molecule. The results showed that three peptides carried epitopes to which mice responded following injection of the whole molecule and that immunization with two additional peptides could prime for in vitro stimulation with the native antigen. The latter result indicates the feasibility of generating T cell responses to "cryptic" epitopes on proteins by immunizing with peptides. The peptide-specific T cell responses were distinctly influenced by the H-2 haplotype of mouse strains. However, two peptides were recognized by several H-2-disparate mouse strains, and one peptide could be presented by both I-A and I-E molecules. Immunization with several peptides induced a cross-reactive T cell proliferative response to the homologous GroEL protein isolated from E. coli. The amount of cross-reactivity was influenced by the extent of sequence homology between mycobacterial and E. coli proteins and the major histocompatibility complex class II molecule used to present the peptide.     

Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen.

The accumulation of somatic mutants in splenic B lymphocytes early after primary immunization with the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) coupled to chicken gamma globulin (CG) was determined. Rearranged V186.2 heavy chain genes were amplified by the polymerase chain reaction from genomic DNA and subjected to nucleotide sequence analysis. Somatic antibody mutants become detectable on day 6 after immunization, and most of the somatic mutations accumulating in the memory compartment are introduced until day 14. At this time strong selection for mutants expressing high binding affinity for NP is apparent. Extrapolation from the mutation frequency increases between day 6 and day 14 to the previously determined mutation frequency at week 6 (Weiss. U. and Rajewsky, K., J. Exp. Med. 1990, 172: 1681) leads to the prediction that the process of mutant generation ceases to operate around day 22 after primary immunization.     

Involvement in the initiation of T cell responses and structural features of an 85-kDa membrane activation antigen

A monoclonal antibody, 7F7, directed at a recently described membrane activation antigen of 85 kDa was found to inhibit the T cell proliferation induced by an anti-CD3 antibody, phytohemagglutinin A and concanavalin A. The T cell response to allogenic stimulator cells was also weakly inhibited. The inhibition of these T cell responses was only obtained if the antibody was added within the first 8 h (first 24 h in the case of the concanavalin A response) of culture. In addition the antibody inhibited the formation of cellular aggregates seen in stimulated cultures when added within the first 8 h. The membrane glycoprotein recognized by 7F7 is shown to have a slightly different molecular mass on cells of different lineage, a protein core of 55 kDa and to contain 30-50% of N-linked as well as a small amount of O-linked carbohydrates and sialic acid residues. This study suggests that the highly glycosylated membrane activation antigen defined by antibody 7F7 could contribute to the contact between those cells which are involved in the initiation of T cell responses.     

The architects of B and T cell immune responses

Published work links adult lymphoid tissue-inducer cells (LTi) with T cell-dependent antibody responses. In this issue of Immunity, Tsuji et al. (2008) associate LTi with T cell-independent IgA antibody responses in the gut.     

B cell growth factor (BCGF) secreting human T cell clones reactive to a soluble glycoprotein antigen, streptococcal antigen (SA)

A panel of human T cell clones bearing exclusively the helper (T4) phenotype and showing reactivities to a soluble glycoprotein antigen (185,000 dalton Mol. Wt. Streptococcal antigen, SA) is described. Two of these clones namely, SA 1.53 and SA 1.82, are found to co-produce B cell growth factor (BCGF) and interferon-gamma (IFN-gamma) in the absence of interleukin 2 (IL2) upon stimulation with phytohaemagglutinin (PHA) or the specific antigen in the presence of irradiated autologous antigen-presenting cells (APC). Secretion of the lymphokines is genetically restricted in part by DR molecules that are expressed on the cloned cells and APC. Produced BCGF is differentiated from the BCGF-promoting property of IFN-gamma in that only IFN-gamma activity, but not BCGF activity is removed and inhibited by anti-IFN-gamma antibodies. Exogenous IL2 induces secretion of BCGF and IFN-gamma of the cloned cells, an observation which involves interaction of IL2 with IL2 receptors. An analysis of the proliferative responses to antigen of the T cell clones shows that BCGF-producing clones, unlike those that secrete IL2, fail to proliferate significantly to specific antigen restimulation.     

Induction of immune responses to functional determinants of a cell surface streptococcal antigen.

Antibodies directed against the cell surface adhesin, termed streptococcal antigen I/II of Streptococcus mutans can protect against dental caries. Streptococcal antigen I/II (SA I/II) interacts with salivary glycoproteins and promotes adhesion to the tooth surface. Topical application of monoclonal antibodies which recognize a domain within residues 816-1213 (fragment 3) prevents colonization by S. mutans in primates. In this study the immunogenicity and antigenicity of fragment 3 was investigated in five strains of mice. Fragment 3 induced an immune response following immunization with whole cells of S. mutans in all strains of mice. Immunization with recombinant fragment 3 also induced T-cell proliferative and antibody responses both to fragment 3 and to the SA I/II. Antibody responses to the previously defined adhesion determinants (residues 1005-1044) were weak or undetectable. Immunization of three representative strains of mice with a recombinant polypeptide (residues 975-1044) comprising this adhesion epitope and an adjacent T-cell epitope (residues 975-1004) elicited both T- and B-cell responses to the polypeptide and to native SA I/II. The B-cell epitopes overlapped with the adhesion determinant. These findings provide a means of directing immune responses to functional determinants of SA I/II.     

Direct helper T cell-induced B cell differentiation involves interaction between T cell antigen CD28 and B cell activation antigen B7.

Cognate interactions between major histocompatibility complex class II antigen (Ag)-reactive CD4+ T helper (Th) and Ag-presenting B cells induce first the activation of B cells and their subsequent differentiation into Ig-secreting cells (IgSC). The Th cell-associated homodimeric glycoprotein CD28 has been implicated as an important regulator of Th activation. Recently, B cell-associated early activation Ag B7 has been identified as a ligand for the CD28 molecule. In this study, we have examined using monoclonal antibodies (mAb) the roles of CD28 and B7 molecules during the Th-B cell cognate interactions leading to the differentiation of B7+ B cells. Anti-CD28 mAb 9.3 specifically inhibited proliferative responses of CD4+ T cells to both allogeneic B cells and soluble Ag-presenting autologous non-T cells. In addition, anti-CD28 mAb 9.3 inhibited Th-induced differentiation of alloantigen-presenting B cells into ISC. Similar inhibition of both Ag-induced Th activation and B cell differentiation into ISC was observed using mAb BB1 which recognizes a B cell-associated molecule B7. In contrast, non-cognate Th-independent exogenous interleukin 6-induced differentiation of B7+ B cells into ISC was not inhibited by mAb to either molecule. These results clearly demonstrate the involvement of CD28 on Th and its ligand B7 on B cells during cognate Th-B interactions leading to the differentiation of B cells. Furthermore, these results also suggest the development of new mAb-based therapeutic approaches for exaggerated B cell activation associated with certain autoimmune diseases such as systemic lupus erythematosus.     

B族链球菌表面蛋白C5a肽酶系统及鼻内黏膜免疫的研究

目的 研究B族链球菌表面蛋白C5a肽酶(streptococcal C5a peptidase,ScpB)系统及其鼻内途径免疫后在小鼠血清及肺、直肠、阴道等黏膜部位特异性抗体水平,探讨ScpB蛋白黏膜免疫应答的效果.方法 在大肠杆菌BL21中表达ScpB蛋白,亲和层析法进行纯化,质谱分析法对所纯化的蛋白进行鉴定.小鼠随机分为5组,每组10只.分别为对照组、皮下30μg组、鼻内5μg组、鼻内10μg组和鼻内30μg组.用ELISA法检测小鼠血清及黏膜萃取液中特异性抗体水平;调理吞噬试验检测SepB蛋白抗血清的保护性功能.结果 成功表达并纯化了ScpB蛋白;质谱分析和蛋白质库比较证实其为B族链球菌表面蛋白C5a肽酶的可能性分值为175;ELISA显示ScpB蛋白皮下30μg及鼻内不同剂量(5 μg、10 μg、30μg)免疫后均可诱发小鼠产生高水平特异性IgG抗体(P＜0.01),30 μg剂量组IgG大于5μg及10μg剂量组(P＜0.05);鼻内不同剂量(5μg、10μg、30μg)免疫后均可诱发小鼠产生高水平的黏膜特异性IgA抗体,与对照组相比差异有统计学意义(P＜0.01);黏膜免疫组间无差异;30 μg ScpB蛋白皮下免疫后黏膜特异性IgA抗体与对照组相比差异无统计学意义(P＞0.05).SepB抗血清对细菌具有调理吞噬作用,吞噬指数为12.3,与对照组相比差异有统计学意义(P＜0.05).结论 B族链球菌表面蛋白C5a肽酶鼻内途径免疫小鼠后可在血清、肺及阴道、直肠等免疫近端和远端黏膜中产生相应高水平IgG及IgA抗体,上述ScpB抗体具有促进吞噬细胞吞噬B族链球菌的功能.     

Comparison of Streptomyces albus muramidase-extracted streptococcal antigen with acid-extracted M antigen and with pepsin-extracted T antigen.

Purified Streptomyces albus lytic enzyme was used in an attempt to extract type-specific antigen from a type 1, group A streptococcus. The presumably type-specific antigen was purified by ammonium sulfate fractionation followed by chromatography on O-(carboxymethyl)-cellulose columns. Comparison of the enzyme-extracted substance with acid-extracted material showed it to be serologically different from M protein. In addition, the extract obtained by enzyme treatment was resistant to trypsin as well as to the lytic enzyme. It was inactivated partially by pepsin and totally by papain. Comparison of the enzyme extract with pepsin-extracted T antigen showed these two preparations to be serologically identical. Subtle differences in their susceptibility to heat and acid treatment were noted. Immunodiffusion analyses of acid-extracted M protein and pepsin-extracted T protein, as well as with the enzyme extract, clearly established that the M-protein preparation contained a component serologically identical with one of the precipitinogens common to the other two extracts.     

The effect of B cell receptor signaling on antigen endocytosis and processing

B cell receptor (BCR)-mediated antigen processing and presentation involves both the BCR-mediated internalization and processing of cognate antigen as well as the formation and expression of antigenic peptide-MHC class II complexes. While BCR signaling is known to result in changes in the biosynthesis and intracellular trafficking of class II molecules, the effect of BCR signaling on the cell biology of antigen endocytosis and processing is less clear. Therefore, the effect of BCR signaling on the cell biology of fluid phase antigen endocytosis, processing and presentation was analyzed in both B cell lines or in normal splenic B cells. The results demonstrate that BCR signaling alters neither the global level of fluid phase antigen endocytosis nor the duration of intracellular persistence of fluid phase internalized antigen. Moreover, while BCR signal does result in an increase in the level of total cell surface MHC class II molecules as well as specific peptide-class II complexes, stimulation failed to alter the fraction of class II molecules loaded with antigen-derived peptide. These results indicate that while BCR-mediated signaling elicits an increase in the expression of antigenic peptide-class II complexes, signaling does not augment antigen presentation by profoundly altering the basic biology of antigen endocytosis and processing. These results also demonstrate that the high efficiency of BCR-mediated antigen processing (when compared to fluid phase antigen processing) is likely to occur independent of BCR signaling-induced global alterations in the biology of endocytosis, processing and presentation. This finding suggests that if BCR signaling augments the efficiency of processing of cognate antigen, it must impact unique aspects of BCR-mediated antigen processing, such as the intracellular persistence of internalized antigen-BCR complexes.     

Priming of CD8+ T cell responses following immunization with heat-killed Plasmodium sporozoites

Protective immune responses against malaria are induced by immunization with radiation-attenuated Plasmodium sporozoites. In contrast, non-viable, heat-killed sporozoites do not induce protection, emphasizing the requirement for live parasites to achieve effective immune responses. Using an experimental system with CD8+ T cells from T cell receptor-transgenic mice, we analyzed the primary CD8+ T cell responses elicited by heat-killed inactivated sporozoites. We found that the numbers of specific CD8+ T cells induced were much lower compared to when immunizing with attenuated sporozoites; however, the kinetics of activation and the phenotype of these T cells were similar in both groups. Despite their low frequency after priming, high numbers of specific CD8+ T cells were observed after boosting with a recombinant vaccinia virus. Upon induction of the recall response, the same level of protection was observed when either heat-killed or attenuated sporozoites were used for priming. We propose that live parasites are not critical for the induction of memory T cell populations against the malaria liver stages.     

Lipopeptide immunization without adjuvant induces potent and long-lasting B,T helper,and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees

We have employed a 26-amino-acid synthetic peptide based on Plasmodium falciparum liver stage antigen-3 to evaluate improvements in immunogenicity mediated by the inclusion of a simple lipid-conjugated amino acid during peptide synthesis. Comparative immunization by the peptide in Freund's adjuvant or by the lipopeptide in saline shows that the addition of a palmitoyl chain can dramatically increase T helper (Th) cell responses in a wide range of major histocompatibility complex (MHC) class II haplotypes, to the extent that responses were induced in mice otherwise unable to respond to the non-modified peptide injected with Freund's adjuvant, and that the increased immunogenicity of the lipopeptide led to high and longer lasting antibody production (studied up to 8 months). B and T cell responses induced by the lipopeptide were reactive with native parasite protein epitopes, and a lipopeptide longer than ten amino acids was endogenously processed to associate with MHC class I and elicit cytotoxic T lymphocyte (CTL) responses. Finally, the lipopeptide was safe and highly immunogenic in chimpanzees, whose immune system is very similar to that of humans. Our results suggest that relatively large synthetic peptides, carefully chosen from pertinent areas of proteins and incorporating a simple palmitoyllysine, can induce not only CTL, but also strong Th and antibody responses in genetically diverse populations. Lipopeptides engineered in this way are simple to produce and purify under GMP conditions, they are well tolerated by apes, and with the enhanced immunogenicity without the need for adjuvant that we report here, they offer a quick and relatively low-cost route to provide material for human malaria vaccination trials.     

Cross-reactivity of a normal human cell surface antigen with primate retrovirus glycoproteins

Normal human cells express a human-specific antigen, HuLy-m5 (defined by the E4.3 monoclonal antibody), cross-reactive with determinants of the primate retroviruses, MPMV(Mason Pfizer monkey virus) and GALV (gibbon ape leukemia virus). Purified virus preparations of MPMV and GALV absorbed E4.3 antibody activity while antisera to these retroviruses blocked the binding of E4.3 antibody to human target cells. Sequential immunoprecipitation and two-dimensional gel analysis both indicated that the anti-primate retrovirus sera recognize the same molecular entity (a two-chain glycoprotein of Mr60 and 69Kd) as does the E4.3 antibody. These results suggest that normal human cells express primate retroviral proteins (most probably viral envelope glycoprotein, gp69) at the cell surface.     

Cytotoxic T cell responses in patients with chronic hepatitis B virus infection undergoing HBe antigen/antibody seroconversion

Cytotoxic T cells have been identified in the peripheral blood of patients with acute hepatitis B virus infections for a short period after clinical presentation. However, in patients in whom the virus persists these have been difficult to demonstrate. In the chronic infection during HBe antigen clearance, when there has been an exacerbation of the disease, we have been able to demonstrate MHC class I-restricted cytolytic response directed against the nucleocapsid antigens. In an HLA-A2 patient this was induced in vitro with the peptide pl8-27, previously described as an HLA-A2-restricted T cell epitope. In patients of other HLA types, recombinant core antigen was used to induce antigen-specific lysis: statistical analysis of the cytolytic responses of chronically infected patients demonstrated a nucleocapsid antigen-specific lysis in patients who were seroconverting. Removal of CD4⁺ cells reduced non-MHC-restricted cytolysis, allowing an MHC class I-restricted cytolytic component to be demonstrated.     

The effect of aging on T cell responses in the horse.

Horses greater than 20 years of age exhibit alterations in their immune responses similar to those observed in other aged individuals. The purpose of this study was to characterize immunosenescence in a population of aged ponies. The peripheral blood mononuclear cells (PBMC) from aged ponies exhibited a decreased proliferative response to various mitogens that was not overcome by the addition of interleukin 2 (IL-2) to the cultures. No difference in overall expression of the IL-2 receptor was seen between young and aged ponies, though CD8(+) cells from aged ponies exhibited increased levels of IL-2 receptor expression. The kinetics of the response to both mitogen and IL-2 did not appear to be affected in the aged PBMCs. These results indicate that the age-related decrease in the proliferative response to mitogens is not due to a failure to produce or respond to IL-2 but probably involves some other process.     

Expression of CD23 antigen is not necessary for human 12-kDa B cell growth factor-mediated B cell proliferation

In an attempt to understand the relationship between the CD23 antigen and the human 12-kDa B cell growth factor (BCGF) receptor, we have undertaken studies to define the biological efficacy of 12-kDa-BCGF on CD23+ (Raji) and CD23- (P3HR1) human Burkitt B cell lines. Our results show that recombinant 12-kDa-BCGF can induce efficient [3H]thymidine incorporation and proliferation in cells of either phenotype. These results strongly suggest that a functional receptor for the 12-kDa-BCGF may exist independently of the CD23 molecule.