Shared Antigens Research Articles

Humoral immunity to commensal oral bacteria in human infants: evidence that Streptococcus mitis biovar 1 colonization induces strain-specific salivary immunoglobulin A antibodies

To define the relationship between salivary SIgA antibodies and commensal oral bacteria, we examined the reactivity of SIgA antibodies from the saliva of four infants with their own colonizing Streptococcus mitis biovar 1 (S. mitis bv 1) clones (ribotypes). Immunoblot analysis was used to examine reactivity of these antibodies with persistent ribotypes isolated from the mouths of the infants over the first year postpartum. Results showed that the pattern of SIgA antibody reactivity with the majority of clones increased in complexity after colonization but that most additional bands were common to other clones, indicating that they represented shared antigens. However, unique bands were identified in 75% of the selected persistent clones. We hypothesized that if strain-specific SIgA antibody was induced in response to colonization of a particular clone and contributed to its elimination from the mouth, then the appearance of unique bands would immediately precede the disappearance of the strain. Seventy-three percent of all unique bands identified in the study fulfilled this criterion. Because the mouth is an open, dynamic environment and multiple factors are believed to play a role in the immune response at mucosal surfaces, it may not be possible to conclusively define the relationship between SIgA antibody and commensal bacteria. However, our data provide evidence that SIgA antibody, reactive with unique antigens of their own colonizing strains, is produced in infants and may point to a role of this antibody in regulating colonization by S. mitis bv 1.

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

Exosomes are small membrane vesicles that are secreted by a multitude of cell types. The exosomes derived from dendritic cells (Dex), tumor cells (Tex), and malignant effusions demonstrate immunomodulatory functions, and are even under clinical trial for cancer treatments. In this study we report the phase I clinical trial of the ascites-derived exosomes (Aex) in combination with the granulocyte–macrophage colony-stimulating factor (GM-CSF) in the immunotherapy of colorectal cancer (CRC). The Aex isolated by sucrose/D2O density gradient ultracentrifugation are 60–90-nm vesicles that contain the diverse immunomodulatory markers of exosomes and tumor-associated carcinoembryonic antigen (CEA). Totally 40 patients (HLA-A0201+CEA+) with advanced CRC were enrolled in the study, and randomly assigned to treatments with Aex alone or Aex plus GM-CSF. Patients in both groups received a total of four subcutaneous immunizations at weekly intervals. We found that both therapies were safe and well tolerated, and that Aex plus GM-CSF but not Aex alone can induce beneficial tumor-specific antitumor cytotoxic T lymphocyte (CTL) response. Therefore, our study suggests that the immunotherapy of CRC with Aex in combination with GM-CSF is feasible and safe, and thus can serve as an alternative choice in the immunotherapy of advanced CRC.

The Specific Immune Response to Tumor Antigen CP1 and Its Correlation With Improved Survival in Colon Cancer Patients

The present study was undertaken to determine the expression of a newly identified tumor antigen cancer-placenta 1 (CP1) in colorectal carcinoma (CRC) and explore the CP1-specific immune response in CRC patients and its correlation with patient survival. CP1 expression was determined by reverse-transcription polymerase chain reaction, immunohistochemistry, and Western blot analysis. Serum antibodies against CP1 were detected by enzyme-linked immunosorbent assay, and T-cell response was measured by interferon-gamma/granzyme-B release enzyme-linked immunospot assays. The HLA-A2-restricted epitopes in CP1 were predicted by bioinformatics and then experimentally validated by enzyme-linked immunospot assay. CP1 expression was detected in a significant number of CRC tissues, reaching 47.6% at the messenger RNA (mRNA) level and 28.6% at the protein level. Of patients with CP1 mRNA(+) tumors, more than 50% had CP1-responsive CD4(+) and CD8(+) T cells and 30% spontaneous-occurring antibodies against CP1. Further studies revealed 2 dominant HLA-A2-restricted epitopes in the CP1 antigen: p31-39 and p58-66. In a follow-up study up to 33 months after surgery, 9 of the 10 patients with CP1-specific CD8 T-cell response survived, whereas 6 of the 8 nonresponders died. Kaplan-Meier analysis indicated a significant correlation between T-cell response and patient survival. CP1 represents a new class of tumor-specific shared antigen. Its high expression in CRC tissues, prevalence of CP1-specific immune responses in CP1 mRNA(+) CRC patients, and positive correlation with survival suggest that the antigen may be a useful target for cancer immunotherapy.

Dendritic cell based personalized immunotherapy based on cancer antigen research

Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8+ CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intratumoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.

Increasing Functional Avidity of T Cell Receptor (TCR)-Redirected T Cells by Removing Defined N-Glycoslyation Sites in the Constant Domain of Introduced TCR Chains.

Adoptive transfer of T lymphocytes transduced with a TCR to impart tumor reactivity has been reported as potential strategy to redirect immune responses to target cancer cells. However, the affinities of most TCRs specific for shared tumor antigens that can be isolated are usually low, in part reflecting the nature of the targeted tumor antigens which are self-proteins. Thus strategies that can increase the affinity or functional avidity of TCRs to be used in therapy to transduce T cells might be therapeutically beneficial. However, current strategies for increasing TCR affinity require extensive and usually random mutagenesis followed by screening the many derived mutations, and must be individualized for each TCR. Since glycosylation impacts the flexibility, movement, and interactions of surface molecules, we tested if removing conserved N-glycoslyation sites in the constant regions of TCR a or b chains can increase the functional avidity of T cells transduced with such modified TCR. We observed enhanced functional avidity and improved recognition of tumor cells by CD8 T cells harboring partially N-deglycosylated as compared to wild type (wt) TCR chains. T cells transduced with wt or N-deglycosylated TCR chains bound tetramer equivalently at 4°C, but tetramer-binding was enhanced at 37°C, reflecting predominantly reduced tetramer dissociation, suggesting an energy-dependent conformational change and/or enhanced clustering of N-deglycosylated TCR chains. The enhanced avidity was evident in the presence or absence of the CD8 co-receptor. This strategy was effective with murine and human TCRs specific for different antigens, and thus should be readily translated to TCRs with any specificity. Thus, transfer of partially deglycosylated TCR chains is a generally applicable strategy that can be used to increase functional avidity of TCR-transduced T cells.

Immunologic and Clinical Outcomes of a Randomized Phase II Trial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting

Human melanoma cells express shared antigens recognized by CD8(+) T lymphocytes, the most common of which are melanocytic differentiation proteins and cancer-testis antigens. However, peptide vaccines for melanoma usually target only one or two MHC class I-associated peptide antigens. Because melanomas commonly evade immune recognition by selective antigen loss, optimization of melanoma vaccines may require development of more complex multipeptide vaccines. In a prospective randomized clinical trial, we have evaluated the safety and immunogenicity of a vaccine containing a mixture of 12 peptides from melanocytic differentiation proteins and cancer-testis antigens, designed for human leukocyte antigen types that represent 80% of the melanoma patient population. This was compared with a four-peptide vaccine with only melanocytic differentiation peptides. Immune responses were assessed in peripheral blood and in vaccine-draining lymph nodes. These data show that (a) the 12-peptide mixture is immunogenic in all treated patients; (b) immunogenicity of individual peptides is maintained despite competition with additional peptides for binding to MHC molecules; (c) a broader and more robust immune response is induced by vaccination with the more complex 12-peptide mixture; and (d) clinical outcome in this peptide vaccine trial correlates with immune responses measured in the peripheral blood lymphocytes. These data support continued investigation of complex multipeptide vaccines for melanoma.

A shared antigen among Babesia species: ribosomal phosphoprotein P0 as a universal babesial vaccine candidate

Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was previously identified as a cross-protective antigen against Babesia microti infection in mice. Interestingly, the same protein showed considerable antigenicity when tested with serum samples collected from Babesia-infected animals. Moreover, the polyclonal antibody raised against the recombinant BgP0 (rBgP0) recognized the P0 homologues from other Babesia species either by immunoblotting or by immunoscreening. The P0 genes from Babesia caballi, Babesia equi, and Babesia bigemina were then cloned and sequenced. The phylogenic analyses based on the amino acid sequences indicated that BgP0 has high identities with B. caballi P0 (88.1%), B. bigemina P0 (85.6%), Babesia bovis P0 (81.4%), and B. equi P0 (64.9%). Western blot analyses revealed that the corresponding native proteins ranged between 31 and 34 kDa, consistent with predicated molecular weight of Babesia P0. Furthermore, the immunogenic property of anti-rBgP0 IgG was evaluated against a B. bovis in vitro culture. The growth of B. bovis parasites was restricted by anti-rBgP0 IgG in a concentration-dependent manner, and significant reductions in parasitemia were observed only at 1 mg/ml in the culture. Taken together, these data suggest that P0 is a conserved protective antigen among Babesia species and might be a potentially universal vaccine candidate for babesiosis.

Schistosomiasis and malaria: another piece of the crossreactivity puzzle

The recent discovery that individuals living in endemic areas have antibodies in their sera that are crossreactive for both helminth and malaria parasites raises important questions both of the interpretation of existing immunoepidemiological data and of the basic biology of the host and the parasites. One such shared antigen (SmLRR) has now been cloned and has, therefore, opened up an intriguing and exciting field of research for immunologists and parasitologists.

Childhood Onset Demyelination and Graves' Disease: Shared Antigen or Autoimmune Clustering?

We describe a case of concomitant Graves' disease and demyelination, with optic neuritis and cerebellar dysfunction, in an adolescent girl. Autoimmune thyroid disease with encephalopathy is the hallmark of Hashimoto's encephalopathy, linking thyroid and central nervous system dysfunction. However, to our knowledge, Graves' disease has not been previously reported in association with clinically isolated demyelinating disease in childhood.

Dendritic cells fused with allogeneic breast cancer cell line induce tumor antigen-specific CTL responses against autologous breast cancer cells

Dendritic cell (DC)/tumor cell fusion vaccine has been revealed as a promising tool for the antitumor immunotherapy. Previous research has shown that fusion hybrids of human DCs and autologous tumor cells can induce cytotoxic T lymphocyte (CTL) responses against autologous tumor cells in animal models and human clinical trials. However, a major restriction factor for the clinical use is the difficulty for preparation of sufficient amount of autologous tumor cells especially for the patients with metastasis cancer whose primary tumor lesion is not clear or has been resected. In this study, allogeneic breast cancer cell line MCF-7 cells were electrofused to autologous DCs from patient with breast cancer as a strategy to deliver shared breast cancer antigens to DCs. Fusion cells generated by autologous DCs and allogeneic MCF-7 were able to induce autologous T lymphocytes proliferation, high levels of IFN-gamma production and CTL responses. CTLs induced by DCs/allogeneic MCF-7 fusion cells were able to kill autologous breast cancer cells in an antigen specific and HLA restriction manner. Our study may provide the experiment basis for the use of allogeneic breast cancer cell line in the DC/tumor cell fusion cell vaccination strategy.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Sudden Sensorineural Hearing Loss and Hemodialysis

The etiology of sensorineural hearing loss (SNHL) associated with renal failure and hemodialysis is controversial. Possible mechanisms include a shared antigenicity between the kidney and the labyrinths, osmotic alteration caused by hemodialysis, and the ototoxic effect of diuretics. We present 2 cases of SNHL associated with renal failure and its treatment. One patient was a 35-year-old man who developed profound SNHL after 5 sessions of hemodialysis, and the other was a 36-year-old woman who developed severe to profound SNHL after 7 sessions. It is our impression that both hearing losses might have been attributable to osmotic disequilibrium in the labyrinth or to an acute labyrinthine injury caused by contamination of the blood by the degraded product of an old cellulose acetate hemodialyzer membrane; the hemodialyzer had been in use for 15 years.

Identification of HLA‐A24 restricted shared antigen recognized by autologous cytotoxic T lymphocytes from a patient with large cell carcinoma of the lung

The aim of the present study was to elucidate the tumor-specific cellular immunological responses occurring in a patient with large cell carcinoma of the lung who had no evidence of recurrence following surgical resections of both a primary lung lesion and a metastatic adrenal lesion. We analyzed an autologous tumor-specific cytotoxic T lymphocytes (CTL clone F2b), which were HLA-A*2402 restricted from regional lymph node lymphocytes. The F2b possessed T cell receptor (TCR) using the Valpha5 and Vbeta7 gene segment. The existence of precursor CTL (pCTL) against autologous tumor cells (A904L) was analyzed using CTL clone-specific PCR. Lymphocytes with the same TCR as F2b were detected in the primary tumor tissue, regional lymph node and the peripheral blood collected from the patient 3 years after the operation. Using the F2b, we identified a cDNA clone encoding the tumor antigen using cDNA expression cloning method. The gene was found to encode splicing variant of the Tara gene. Finally, we identified the 9-mer Ag peptide, using constructions of mini-genes. The F2b recognized 3 out of 7 HLA-A24 positive allogeneic tumor cell lines and in 1 out of 7 HLA-A24 negative allogeneic tumor cell lines when transfected with HLA-A24. This peptide is therefore considered to be potentially useful for performing specific immunotherapy in a significant proportion of lung cancer patients bearing HLA-A24.

Coimmunization with Antigens Shared between Neoplastic and Normal B Cells Suppresses Induction of Strictly Tumor-Specific Cytotoxic T Cells through In Situ Activation of Regulatory T Cells.

CD4+CD25+ regulatory T cells (Treg) recognize autoantigens and inhibit autoreactive immune responses in a cell contact-dependent manner. In cancer-bearing patients, expansion and functional aberrations of Treg may inhibit immune responses against the tumor. The available evidence suggests that such Treg recognize self antigens expressed by the tumor and argues that induction of anti-tumor T cell responses might be more successful if true tumor-specific rather than lineage-restricted or shared antigens are used for active immunotherapy. Indeed, we have observed a preferential recognition of tumor-individual over shared epitopes by vaccination-induced T cells after immunization of B-NHL patients with recombinant lymphoma idiotype (Bertinetti et al., Cancer Res. 2006). To study this phenomenon in an exemplary fashion, we immunized BALB/c mice with dendritic cells loaded with H-2K-restricted peptides of the immunoglobulin of the A20 lymphoma. A J region-derived peptide served as a model for a shared antigen; a heteroclitic peptide from the CDR3 region represented a tumor-specific antigen. Both peptides bind H-2Kd with similar affinity. Compared to a highly immunogenic influenza HA peptide, the CDR3 peptide was similarly efficient in inducing specific cytotoxic T cells as analyzed by tetramer staining, IFNγ release to peptide stimulation, and in vitro and in vivo cytotoxicity assays with CFSE-labelled, peptide-loaded splenocytes. In contrast, no effector cells were detected with any assay after J immunization. After in vitro restimulation with peptide, however, antigen-specific IFNγ-secreting effector populations were demonstrated for each vaccination, suggesting in vivo inhibition, possibly mediated by Treg, rather than total absence of J-specific T cells. No difference in numbers and the TCR repertoire of CD4+CD25+FoxP3+ cells in the draining lymph node could be detected. However, activation of Treg by J immunization was indicated by potent suppression of antigen-specific splenic effectors compared to CDR3-immunized animals, and by a 4fold higher spontaneous proliferation of FoxP3+ cells from the draining lymph node in vitro. In contrast to CDR3-derived Treg, the addition of J-induced Treg to effector cells resulted in a dose-dependent production of IL-10 in mixed cultures, independently of the antigen specificity of the effectors. Finally, coimmunization with HA and J peptides led to inhibition of the proliferation of HA-specific CD8+ effectors in vivo as demonstrated by adoptive transfer and subsequent flow cytometry analysis of CFSE-labelled TCR-transgenic T cells. This inhibition was absent after coimmunization with HA and CDR3 peptides and could be largely abolished by prior in vivo depletion of Treg with an αCD25 antibody. These data demonstrate in a non-transgenic model that coimmunization with shared and individual, strictly MHC I-restricted tumor antigens leads to a potent inhibition of tumor-specific CD8+ T cells through rapid in situ activation of CD4+FoxP3+ Treg elicited by the shared tumor antigen. It is postulated that these Treg recognize MHC II-restricted self antigens presumably derived from non-neoplastic cells as a consequence of an aborted immune response to the shared antigen. These experiments provide direct evidence that active immunotherapy of malignant tumors exclusively with true tumor-specific antigens has a greater chance of success since the presence of shared antigens will prevent tumor-specific immune responses through Treg activation.

Different Lineages of P1A-Expressing Cancer Cells Use Divergent Modes of Immune Evasion for T-Cell Adoptive Therapy

Tumor evasion of T-cell immunity remains a significant obstacle to adoptive T-cell therapy. It is unknown whether the mode of immune evasion is dictated by the cancer cells or by the tumor antigens. Taking advantage of the fact that multiple lineages of tumor cells share the tumor antigen P1A, we adoptively transferred transgenic T cells specific for P1A (P1CTL) into mice with established P1A-expressing tumors, including mastocytoma P815, plasmocytoma J558, and fibrosarcoma Meth A. Although P1CTL conferred partial protection, tumors recurred in almost all mice. Analysis of the status of the tumor antigen revealed that all J558 tumors underwent antigenic drift whereas all P815 tumors experienced antigenic loss. Interestingly, although Meth A cells are capable of both antigenic loss and antigenic drift, the majority of recurrent Meth A tumors retained P1A antigen. The ability of Meth A to induce apoptosis of P1CTL in vivo alleviated the need for antigenic drift and antigenic loss. Our data showed that, in spite of their shared tumor antigen, different lineages of cancer cells use different mechanisms to evade T-cell therapy.

Adoptive immunotherapy using autologous lymphocytes sensitized with HLA class I-matched allogeneic tumor cells

A 29-year-old female breast cancer patient with multiple bone metastases (HLA-A2) was treated with adoptive transfer using autologous peripheral blood mononuclear cells (PBMCs) activated with the HLA-A2-matched allogeneic GC022588 gastric cancer cell line and interleukin-2 plus an immobilized anti-CD3 antibody culture system. The relief of bone pain in parallel with a decrease of serum carcinoembryonic antigen levels was obtained just after the administration of GC022588-activated effector lymphocytes, and a good quality of life was accomplished for 4 months. The GC022588-activated effector lymphocytes included 44% CD4+, 77% CD8+, and 26% CD4+CD8+ phenotypes, and expressed 25% killing activity against GC022588 stimulator cells at an E/T ratio of 50:1. T cell receptor (TCR) usage analysis for the effector cells showed oligoclonal expression of TCRVbeta1, 3, 9, and 11, especially TCRVbeta5.2, 12, 13.1 and 17, and their killing activity was significantly inhibited in the presence of anti-TCRalphabeta antibody and anti-TCRVbeta12 antibody. SSCP analysis revealed clonotypic bands of TCRVbeta12. These results suggest that shared antigens exist between breast and gastric adenocarcinomas. Allogeneic tumor cells can stimulate PBMCs to generate effector cells with selected TCRCDR3 usages that recognize tumor antigens. These effector lymphocytes may be good candidates for the adoptive immunotherapy of cancer.

Antigenic Profile of Osteoblasts Present in Human Bone Tissue Sections

The antigenic profile of human osteoblasts was previously analyzed by our group using primary cultures as study samples. These studies suggested a novel functional approach to this cell population. Osteoblasts have a characteristic antigenic profile and share antigens in common with other cell populations that also originate in the bone marrow. Some of the detected antigens are constitutively expressed, while others are modulated by different factors and/or cytokines. The aim of the present study was to analyze the antigens present in osteoblasts in vivo, since the presence of certain biomolecules in fetal bovine serum may modulate the antigenic expression, compromising the results. For this purpose, human bone tissue sections were analyzed with a wide panel of mAbs and using the immunoperoxidase technique. CD10, CD44 and alkaline phosphatase antigens and IL-12, IL-18 and IFNgamma cytokines were detected in osteoblasts in the bone tissue. However, CD80 and HLA-DR antigens were not found in all samples and when present their expression was weak. The expression of CD54 antigen was moderate or weak. These results allow data obtained by the primary culture of osteoblast-like cells to be endorsed.

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.