Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network.     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

Suppression of antibody responses to ricin A chain (RTA) by monoclonal anti-RTA antibodies

Balb/c mice treated with an immunotoxin constructed by conjugation of murine monoclonal antibody 791T/36 via a disulfide linker to ricin A chain generate a pronounced antibody response to peptide epitopes on ricin A chain. Monoclonal anti-RTA antibodies which recognize peptide epitopes have been developed and these have been used to down-regulate anti-RTA antibody responses in 791T/36-RTA immunotoxin-treated Balb/c mice. Of the five MAB tests, two (608/7 and 596/134) proved most effective, inhibiting anti-RTA antibody formation by up to 73%. MAB treatment was effective when initiated up to 3 days after immunotoxin treatment. Pharmacokinetic studies with 791T/36-RTA have shown that the immunotoxin is rapidly eliminated from the circulation, with no more than 4% remaining in blood after 24 hr. It is proposed that the down-regulation of anti-RTA antibodies is effected by MAB interfering with antigen processing.     

Homogenous expression of killer cell immunoglobulin-like receptors (KIR) on polyclonal natural killer cells detected by a monoclonal antibody to KIR2D

The activity of human natural killer (NK) cells is in part regulated by the expression of killer cell immunoglobulin (Ig)-like receptors (KIR) that recognize major histocompatibility complex (MHC) class I and can inhibit NK cell cytotoxicity. A monoclonal anti-KIR antibody was established and designated Lig1. Lig1 was shown to be specific for KIR in cell-surface staining and to react with all KIR2D, except KIR2DL4 which lacks a D1 domain, but not with KIR3D molecules in an enzyme-linked immunoadsorbent assay (ELISA) and Western blotting. Unlike other anti-KIR antibodies, Lig1 did not inhibit binding of KIR-Ig-fusion proteins to MHC-class I expressing cells nor did it interfere with KIR-mediated inhibition of NK cell cytotoxicity in a functional assay. Lig1 reacted with all NK cells in polyclonal NK populations from different donors, demonstrating that all NK cells express at least one KIR2D receptor.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5^? B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human μ chain antibody (a-μ-Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5⁺ B cells and subsequently fractionated on Percoll density gradients. While resting CD5⁺ B cells proliferated and produced IgM molecules in response to a-μ-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5^? B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5⁺ B cells had the typical phenotype of mantle zone B cells (IgM⁺ IgD⁺ CD39+ CD38^? CD10^? CDw75^dim), whereas resting CD5^? B cells were CD38^? CD39^? CD 10^? CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38⁺ CD10⁺ CD39^? CDw75^bright, IgG⁺) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5^? B cells. However, some of the data collected suggest possible relationships between CD5^? B cells and germinal center cells. The CD5^? B cells isolated from the 50 % Percoll fraction proliferated in response to a-μ-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5⁺ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5⁺ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5^? B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5^? B cells did not express CD5. The CD5^? B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro, possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro. The present study underlines the phenotypic and functional heterogeneity of CD5^? B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.     

Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19).

Bispecific antibodies (BsAb) can be used to retarget T cells irrespective of their specificity to certain target cells inducing target cell lysis. We have tested the efficacy of the BsAb SHR-1, directed against the T cell antigen CD3 and the B cell antigen CD19 to induce (malignant) B cell kill by T cells as measured in a 51Cr-release assay. Two cytotoxic T cell clones (CTL), expressing TCR alpha beta or TCR gamma delta, were effective in killing CD19 expressing B cell lines at different stages of differentiation in the presence, but not in the absence, of the BsAb. CD19- target cells were not killed. Fresh CD19+ leukaemia/lymphoma cells were also efficiently killed by SHR-1 preincubated CTL clones. In addition, phytohaemagglutinin (PHA) or CD3-activated IL-2 expanded peripheral blood mononuclear cells (PBMC) of normal donors did so after 2 weeks of stimulation. A concentration of 100 ng/ml of the BsAb was sufficient to obtain optimal lysis of all target cells tested. These results show that fresh human leukaemia/lymphoma cells, freshly derived from active lymphoblastic leukaemia (ALL) as well as non-Hodgkin's lymphoma (NHL) patients, can be effectively killed in the presence of this BsAb by activated T cells.     

一株新型鼠抗人PD-L2单克隆抗体的研制及其生物学特性的鉴定

为了研制特异性识别人PD-L2(B7-DC,CD273)的鼠单克隆抗体,并对其生物学特性和PD-L2分子的表达特性进行初步分析。以高表达人PD-L2分子的基因转染细胞L929/PD-L2作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L2作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-L2单克隆抗体的杂交瘤细胞株;采用Western blot、Ig亚型快速定性试纸法、间接免疫荧光法和竞争结合抑制实验对单抗进行生物学特性的分析,继而利用该单抗进行免疫荧光标记和流式细胞术检测PD-L2在肿瘤细胞株和免疫细胞上的表达特性。结果显示,通过多次融合和反复筛选,成功获得一株特异性鼠抗人PD-L2(B7-DC)的杂交瘤,该杂交瘤分泌的单克隆抗体能特异识别人PD-L2分子。继而利用上述研制获得的单克隆抗体8F2进行免疫荧光标记和流式细胞术检测发现,PD-L2上调性表达在成熟的树突状细胞和调节性T细胞上。提示,成功地获得一株特异性鼠抗人PD-L2单克隆抗体,并对其生物学特性和表达谱进行初步分析,证明其识别抗原表位不同于商品化抗体,是一株新型鼠抗人PD-L2单抗,这为进一步研究PD-1/PD-L2信号通路在免疫应答中的生物学作用提供了有价值的物质基础。     

Generation of human monoclonal antibody-producing cell lines by Epstein-Barr virus (EBV)-transformation of B lymphocytes and somatic cell hybridization techniques

Summary The use of Epstein-Barr virus to immortalize human B cells and generate monoclonal antibody-producting B cell lines is described.     

Identification and typing of herpes simplex virus types 1 and 2 by monoclonal antibodies, sensitivity to the drug (E)-5-(2-bromovinyl)-2'-deoxyuridine, and restriction endonuclease analysis of viral DNA

With development of antiviral drugs, the need to identify a virus as to drug sensitivity becomes increasingly of importance. The compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been shown to be much more inhibitory to the replication of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus as opposed to herpes simplex virus type 2 (HSV-2). We have typed over 170 isolates, using an immunofluorescent technique and sensitivity to the drug BVDU. These results were then compared to the typing of isolates by analysis of viral DNA after restriction endonuclease digestion (EcoRI). Without exception the results were in agreement between the monoclonal antibody results and sensitivity to the drug BVDU. Furthermore, the typing with monoclonal antibodies was also in excellent agreement with the DNA analysis. Only those isolates inhibited with BVDU showed DNA characteristics of HSV-1 and reacted only with the S-200 antibody. On the other hand, those isolates which reacted with the monoclonal antibody S-141 were insensitive to BVDU, and again this was in agreement with the DNA analysis. These results could provide the basis for developing a diagnostic test using the two monoclonal antibodies to type either isolates or direct smears and to use the results as a basis for possible drug therapy.     

Suppression of human anti-porcine natural killer cell xenogeneic responses by combinations of monoclonal antibodies specific to CD2 and NKG2D and extracellular signal-regulated kinase kinase inhibitor

Natural killer (NK) cells can destroy xenogeneic tissues by antibody-dependent cell cytotoxicity (ADCC) and direct lysis. Unlike ADCC, activating interactions between human NK receptors and their cognate ligands in pigs are not fully elucidated. We set up this study to identify human NK activating receptors recognizing porcine cells isolated from distinct organs, e.g., aorta, cornea and liver, and to provide a molecular basis for effective immunosuppressive regimens. Among the array of NK receptors tested, NKp46, 2B4, CD49d, CD48, CD2 and NKG2D, only CD2 and NKG2D were shown to be involved in both cytotoxicity and cytokine (interferon-γ and tumour necrosis factor-α) production against porcine targets. Simultaneous blocking of CD2 and NKG2D by combining its monoclonal antibodies further suppressed xenogeneic NK responses. Moreover, addition of a suboptimal dose of PD98059, an extracellular signal-regulated kinase (ERK) kinase inhibitor, to those cells maximally reduced NK cytotoxicity, suggesting that ERK plays an important role in NK-mediated xenoreactivity. These impairments in NK cells were tightly associated with defective intracellular calcium mobilization and the subsequent degranulation process. Therefore, our data demonstrate a distinct role of CD2 and NKG2D on human NK cells in recognizing porcine grafts and further provide a potentially efficacious combinational regimen using anti-CD2 and anti-NKG2D monoclonal antibodies with PD98059 in a pig-to-human transplantation model.     

Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth

Previously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct N_CCG-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the N_CCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtype B and C HIV-1 reference panels. The parental Fab 3674 is 10-20-fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (∼ 50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (∼ 120 kDa) across the subtype B and C reference panels.     

Estimation of hormone receptor status and HER2 in cytologic cell blocks from breast cancer using the novel rabbit monoclonal antibodies (SP1, SP2, and SP3)

The determination of estrogen (ER) and progesterone (PR) receptor status has become standard practice in the evaluation of patients with invasive breast cancer, having important prognostic and therapeutic implications. HER2 assessment is important to evaluate the response to Herceptin® (Trastuzumab) therapy for primary and metastatic breast cancer. This study is undertaken to compare rabbit monoclonal antibodies (RabMAb) for ER, PR, and HER2 against FDA‐approved monoclonal and polyclonal antibodies (FDAMpab). Cell blocks from primary and metastatic/recurrent breast carcinomas of 52 breast cancer patients were used. Immunohistochemistry was performed, following optimized epitope retrieval, with a polymer based detection system using RabMAb: ER (SP1), PR (SP2), and HER2 (SP3). FDA approved Mpab (Dako) used were: ER (1D5); PR (PgR636); and HercepTest® kit according to manufacturer's instructions. HER2 immunostain is correlated with FISH results. Overall, positive, and negative agreement is as follows: 88.5, 88.9, and 88.2% for ER; 84.6, 70.5, and 91.4% for PR; 58.3, 100, and 50% for HER2. There is substantial to moderate agreement between RabMAb and FDAMpab for ER (κ = 0.75) and PR (κ = 0.64), respectively. There is poor agreement (κ = 0.25) between RabMAb (SP3) and FDApab (HercepTest®). SP3 shows better concordance (93.8%) than HercepTest® (46.9%) with FISH results. RabMAb SP clones are almost comparable with FDA‐approved ER and PR, with fair to moderate agreement. Both are as sensitive as their FDA‐approved clones. SP3, on the other hand, is superior to HercepTest® for detecting HER2 overexpression, with an excellent concordance with FISH. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

A human B cell differentiation antigen selectively expressed on mature B cells identified by a monoclonal antibody (HB-2)

A monoclonal IgM antibody (HB-2), produced against a membrane antigen on the Raji, B cell line, reacted by indirect immunofluorescence with 2 to 40% of lymphoblasts from the B cell lines, Raji, Daudi, SN-1036, and SB but not with other types of cell lines, including pre-B, myeloid, melanoma, or T cells. HB-2 antibody reacted with 10 ± 3% of normal blood mononuclear cells, and was unreactive with monocytes, granulocytes, platelets, or erythrocytes. Two-color immunofluorescence revealed that HB-2 antigen expression was confined to cells bearing surface Ig. An interesting finding was the fact that 25% of plasmablasts induced by pokeweed mitogen also expressed the HB-2 antigen. However, pre-B and plasma cells from normal bone marrow did not express the HB-2 antigen either on their membrane surface or in their cytoplasm. Analysis of 85 leukemias revealed that the HB-2 antigen was expressed on acute and chronic B cell leukemias and Burkitt's lymphomas, but not on malignacies of pre-B, T, myelocytic, or plasma cells. The results suggest that expression of the HB-2 antigen is confined to mature B cells.     

Selective stimulation of murine cytotoxic T cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast-derived surface antigen (S-antigen or S-protein) of hepatitis B virus (HBV). Native S-protein molecules self-assemble into stable 22-nm particles. BALB/c mice immunized with low doses of native S-particles without adjuvants efficiently generated an H-2 class I-restricted CD8⁺ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S-particle or linear epitopes of the denatured S-protein. Disruption of S-particles with sodium dodecyl sulfate and β-2-mercaptoethanol generated p24 S-monomers. Injection of an equal dose of S-monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S-protein. In vivo priming of CTL by S-particles or S-monomers required “endogenous” processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S-derived 12-mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S-antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.     

Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La (SSB) autoantigen

The aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145-164 aa (p1), 289-308 aa (p2), 301-318 aa (p3) and 349-364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental animal.     

Hapten-induced primary and memory humoral responses are inhibited by the infusion of anti-CD20 monoclonal antibody (IDEC-C2B8, Rituximab)

Blocking the elicited humoral immune response has proven useful in treating individuals with autoimmune disorders or those who are at risk of developing antibodies which might be pathologic, e.g., transplant patients. Unfortunately, humoral immunity has evaded efforts at ablation and those therapies aimed at ameliorating it have resulted in only partial success. In addition, some of the current anti-humoral therapies not only target B-cells but also cross-react with other elements of immune response, making these therapies nonspecific. Thus there is a need in the clinical arena for specific anti-humoral therapies. Here we report the impact of infusion of a chimeric monoclonal, an anti-CD20 IgG, on the primary humoral and memory response against a simple hapten (DNP) in a nonhuman primate model. Anti-CD20 IgG interfered with the elicited humoral response and with the memory response when administered prior to antigen exposure. Furthermore, we provide evidence that anti-CD20 blocks the humoral response by eliminating those B-cells capable of responding to the hapten.     

Development of the follicle‐associated epithelium and the secretory dendritic cell in the bursa of fabricius of the guinea fowl (Numida meleagris) studied by novel monoclonal antibodies

Two stromal elements, follicle‐associated epithelium and secretory dendritic cells of the bursa of Fabricius were studied by light microscopy and two novel MAbs, that were produced against splenic cell suspensions of guinea fowls. Both antigens recognized by these MAbs, designated GIIF3 and NIC2, are localized in the cytoplasm of the stromal cells, and their molecular weights are 50 and 30 kD, respectively. During embryogenesis the GIIF3 and NIC2 cells emerge in the mesenchyme of the folds before follicle formation. The GIIF3 and the NIC2‐positive cells accumulate under the surface epithelium of the plicae and migrate into the epithelium, that precedes the bud‐formation. From the bud, the GIIF3‐positive cells migrate up to the luminal surface, and they transform to distinct, highly polarized follicle‐associated epithelial cells. Single GIIF3‐positive cells are also present in the interfollicular epithelium. The NIC2 MAb recognized mesenchymal cells harbor in the lymphoepithelial compartment of the folliculus, and they elaborate cytoplasmic granules. Around Day 20 of embryogenesis large amount of NIC2‐positive substance appear extracellularly in the medulla and around it. This period well correlates with the starting up of the bursal functions; clonal expansion of B cells, and generation of immune repertoire. After hatching the NIC2 stainability diminishes, and it is restricted to the medullary bursal secretory dendritic cells. The NIC2‐positive, possibly elderly bursal secretory dendritic cells, are capable for migration into the follicle‐associated epithelium. In eight‐day old birds some cells of the follicle‐associated epithelium reveals temporary NIC2 positivity, that may prove the transport of the follicle‐associated epithelial cells into luminal direction. By 12 weeks of age the presence of NIC2‐positive substance in the intercellular space of the FAE, rather than in the cells of FAE may indicate the termination of the transport of secretory substance. In conclusion, two types of mesenchymal cells enter the surface epithelium of the bursal folds. The GIIF3‐positive cells appear on the luminal surface of the follicles and occupy the place of the follicle‐associated epithelial cells. The NIC2‐positive cells become secretory in nature and differentiate to bursal secretory dendritic cells. The follicle formation possibly, requires the joint presence of both GIIF3 and NIC2 cells in the epithelium. Anat Rec 262:279–292, 2001. © 2001 Wiley‐Liss, Inc.     

Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens

Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs^* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC^* assays, MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.     

Malignant B lymphocytes from non-Hodgkin's lymphoma induce allogeneic proliferative and cytotoxic T cell responses in primary mixed lymphocyte cultures: An important role of co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) in stimulation by tumor cells

We analyzed the stimulating capacities of malignant B cells from non-Hodgkin's lymphomas (NHL) to induce an allogeneic response in primary mixed lymphocyte reaction (MLR). T cells purified from a single healthy donor (KS) were used to compare the responses induced by either malignant or hyperplastic cells. Malignant B cells induced strong proliferation of KS cells independently of their level of expression of adhesion molecules. The KS cells after MLR were predominantly CD3⁺, CD25⁺, HLA-DR⁺, Ki67⁺ and CD45RO⁺ T cells, and the CD4/CD8 ratio was heterogeneous (from 0.8 to 2.7). To investigate the role of co-stimulatory molecules CD80 and CD86 for the stimulatory capacities of B cells, the expression of both molecules was analyzed before and during the MLR. Most fresh malignant B cells were negative for CD80 and CD86, whereas co-cultured B cells expressed high levels of both molecules. This expression was crucial for T cell proliferation, since monoclonal antibodies directed against CD80 and CD86 completely abrogated the MLR. We also report that KS responding cells at the end of co-culture were able to lyse fresh B cells used as stimulator cells to different extents (from 10 to 51%), and the level of lysis was enhanced after PMA activation of the target cells. Inhibition experiments using CD8 and CD4 mAb showed that effector cells were mainly CD8⁺. This report is the first to describe the accessory function of human malignant B cells from NHL and their sensitivity to lysis mediated by CD8⁺ T cells, and suggests new strategies for the development of antitumor immunity in NHL.     

Antigen-specific T cell responses in human peripheral blood leucocyte (hu-PBL)-mouse chimera conditioned with radiation and an antibody directed against the mouse IL-2 receptor beta-chain

A weakness of the hu-PBL-SCID model for the study of human immune functions is the appearance of anergy and the consequent loss of T cell function. We demonstrate here that human T cells retain normal functions during the early stage of chimerism. At 1 and 2 weeks post-engraftment, T cells isolated from the peritoneal cavity of hu-PBL chimeras could be activated and proliferated upon stimulation with phytohaemagglutinin (PHA) or specific antigens to which the cell donor was known to be immune. T cells derived from hu-PBL-SCID and hu-PBL-NOD/LtSz-scid (NOD/SCID) mice not only proliferated but also produced interferon-gamma (IFN-gamma) and IL-5 following in vitro stimulation with tetanus toxoid (TT) or hepatitis B surface antigen (HBsAg). These antigen-specific T cells could only be demonstrated when cognate antigen was administered together with or immediately following the PBL transfer. Without an early rechallenge with antigen in vivo, no TT- or HBsAg-specific T cell responses could be elicited, showing the vulnerability and antigen-dependence of the T cell response. Vigorous anti-TT or anti-HBs responses could be observed in all chimeras. Administration of antigen together with the PBL graft enhanced the humoral anti-TT response in SCID and NOD/SCID mice but had little effect on the anti-HBs antibody response in NOD/SCID mice. These data confirm the observation that the B cell compartment in hu-PBL-SCID chimera is largely antigen-independent and extend this to SCID/NOD.