Anti-Gal IgG potentiates natural killer cell migration across porcine endothelium via endothelial cell activation and increased natural killer cell motility triggered by CD16 cross-linking

Xenoreactive antibodies (Ab) are important for the development of acute vascular rejection (AVR) of xenografts characterized by monocytes, natural killer (NK) cells and neutrophils infiltrating the graft. The mechanisms by which anti-galactose alpha 1,3galactose (alpha-Gal) IgG influence NK cell migration across porcine aortic endothelium (PAEC) were investigated. NK cell migration across PAEC increased in the presence of anti-alpha-Gal IgG. Anti-alpha-Gal IgG exposure activated PAEC as shown by an increased expression of CD62E and CD106. NK cells adhered, spread and showed motile forms on plastic surfaces coated with human IgG, IgG Fc and on mAb against CD16, but not on mouse IgG or BSA, suggesting that CD16 cross-linking can mediate increased adhesiveness. Increased NK cell motility was observed on Boyden filters coated with human IgG, IgG Fc, and mAb against CD16 and the alpha 4, alpha 5, alpha L, beta 1 and beta 2 integrin chains. No motile response was seen on mouse IgGor CD7, CD56 and alpha 6 integrin mAb. NK cell migration on human IgG and anti-CD16 Ab was blocked by anti-CD16 or anti-beta 2, but not anti-beta 1 Ab, implying that the motile response triggered by CD16 cross-linking is mediated via beta 2 integrins. Preformed or induced anti-alpha-Gal IgG may therefore contribute to AVR by stimulating innate immune cell infiltration of the graft.     

Preparation and characterization of antibodies against mouse prion protein (PrP) peptides.

Antisera were raised in rabbits against three peptides, representing amino acid sequences 150 to 159, 165 to 174, and 213 to 226 of mouse prion (PrP), which were synthesized by using a multiple antigenic peptide (MAP) system. The reactivities of these sera to PrP were examined by an enzyme-linked immunosorbent assay (ELISA), Western immunoblotting (WB), and immunohistochemical procedures. The results of both ELISA and WB showed that antisera to peptide sequence 150 to 159 (Ab150-159) did not react with purified mouse PrP. On the other hand, sera to the sequence 165 to 174 (Ab165-174) reacted weakly with purified mouse PrP, as detected by WB but not by ELISA. However, antiserum to peptide sequence 213 to 226 (Ab213-226) reacted strongly with mouse, Syrian hamster, and sheep PrP by WB and with mouse PrP as shown by the results of ELISA. Moreover, Ab213-226 clearly detected PrP immunohistochemically in mouse, Syrian hamster, and sheep brains affected with scrapie as well as in the brain of a cow with bovine spongiform encephalopathy. From these data, we conclude that rabbit antiserum against the MAP representing amino acid sequence 213 to 226 of mouse PrP is useful as a diagnostic tool for prion disease of animals.     

Human CD14 is an efficient target for recombinant immunoglobulin vaccine constructs that deliver T cell epitopes

It has been shown in the mouse that recombinant immunoglobulin (Ig) molecules with T cell epitopes inserted into the constant domain (Troybodies) can target antigen-presenting cells (APC) for efficient delivery of T cell epitopes. Here, we have extended the Troybody concept to human applications. Moreover, we show that a receptor of innate immunity, CD14, which is a part of the lipopolysaccharide receptor complex on monocyte APC, is an efficient target. For construction of CD14-specific Troybodies, we used rearranged variable(diversity)joining regions cloned from the 3C10 mouse B cell hybridoma. As a model T cell epitope, amino acids 40-48 of mouse Ckappa, presented on human leukocyte antigen-DR4, were inserted into a loop connecting beta-strands in C(H)1 of human gamma3. In the presence of monocytes, CD14-specific Troybodies were >100 times as efficient as a nontargeting control antibody (Ab) at stimulating Ckappa(40-48)-specific/DR4-restricted T cells. Presentation was dependent on the conventional processing pathway for presentation on major histocompatibility complex (MHC) class II molecules. Enhanced presentation of the Ckappa epitope was most likely a result of increased loading of MHC class II molecules, as the CD14-specific monoclonal Ab 3C10 did not induce maturation of the APC. The results show that CD14, a receptor of innate immunity, may be a promising target of recombinant Ig-based vaccines for elicitation of T cell responses in humans.     

Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca²⁺-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a moleculas mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn²⁺, but not Ca²⁺, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn²⁺-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.     

Antigen delivery via two molecules on the CD8- dendritic cell subset induces humoral immunity in the absence of conventional "danger"

Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8- DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRgamma-/- mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8- DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.     

Inhibition of T cell activation through down-regulation of TCR-CD3 expression mediated by an anti-CD90 Ab

We are trying to develop new Abs that can manipulate CD4 T cell responses and are usable as immunosuppressive agents. To this end, we performed functional screening, in which we examined the effect of an Ab on the proliferation of mouse CD4 T cells upon activation. The Ab, LP5, inhibited the activation of CD4 T cells stimulated with an anti-CD3 Ab or peptide antigen. The Ab alone had no stimulatory effect on CD4 T cells. Biochemical experiments demonstrated that LP5 recognized the Thy-1 (CD90) molecule. Interestingly, the treatment of CD4 T cells with LP5 in vitro induced a temporary down-regulation of CD3 expression at the cell surface. TCR molecules were also affected. Other anti-CD90 Abs not inhibitory to CD4 T cell activation failed to induce a reduction in CD3. Experiments in vitro revealed that the down-regulation caused by LP5 is due to an accelerated endocytosis of cell surface CD3. In addition, it was shown that CD3 down-regulation before or in the early stages of T cell activation is critical for the induction of hyporesponsiveness. Experiments in vivo showed that pre-treatment of CD4 T cells with LP5 inhibited the rejection of semi-allogeneic bone marrow transplants. Based on these observations, we propose that CD3 down-regulation without any stimulatory activity against T cells could be one approach to inhibiting T cell activation, and CD90 would be an appropriate target.     

CD27/CD70 interaction directly induces natural killer cell killing activity.

The CD27 molecule is expressed on a portion of natural killer (NK) cells as well as T and B cells. To provide the functional capacity of CD27 molecule on NK cells, we here highly purified CD3- CD56+ NK cells by flow cytometry (purity > 98%), and investigated their NK cell activity via CD27/CD70 interaction using a CD70-transfectant by a 4 h 51Cr-release assay. The enhancement of NK activity by purified NK cells in the presence of interleukin-2 (IL-2) or interleukin-12 (IL-12) against CD70/Nalm-6 was not recognized as compared to against mock/Nalm-6. However, after a coculture with the fixed CD70/300-19, the purified NK cells increased the NK cell activity against K562, the value being 10 to 20% higher than coculture with the mock/300-19 in the presence of IL-2 or IL-12. The enhancement of NK activity was blocked by the addition of anti-CD70 monoclonal antibody (mAb). In addition, conjugation of NK cells to the target was increased by coculture with the CD70/300-19 without increased expression of adhesion molecules. In the parallel experiment, there was no increase in the killing capacity of NK cells. These results strongly show that CD27/CD70 interaction directly enhances NK activity in the presence of IL-2 or IL-12 by increasing the effector and target conjugate formation, indicating that CD27/CD70 interaction plays an important role in the cytotoxic function of NK cells.     

The murine homologue of the T lymphocyte CD2 antigen: molecular cloning, chromosome assignment and cell surface expression

The human T lymphocyte antigen CD2 (T11, sheep erythrocyte receptor) is expressed on all peripheral T cells and all but the most immature thymocytes. Experiments with monoclonal antibodies against CD2 suggest that CD2 is the cell surface receptor for a natural ligand involved in T cell proliferation. Clarification of the functional role of CD2 would be facilitated by the identification of CD2 in the mouse. However, antibodies that recognize the murine homologue have not been described. An alternative approach to the identification of the murine homologue was to use cross-species DNA hybridization, employing human CD2 cDNA as a probe. Clones encoding the murine homologue were isolated from a murine T helper cell cDNA library. The murine cDNA sequence encoded a predicted mature polypeptide of 322 amino acids that showed 54% identity with the predicted human sequence. As with the human polypeptide, the cytoplasmic domain was large, and rich in proline and basic residues. CD2 mRNA was expressed in murine thymus and spleen, and in the T cell line EL4. The murine CD2 gene was assigned to chromosome 3 by Southern blot analysis of mouse-hamster somatic cell hybrids. A rabbit antiserum raised against purified human CD2, precipitated from surface-labeled mouse thymocytes a glycoprotein of Mr 55,000-66,000 which decreased to Mr 35,000 on digestion with endo-beta-acetylglucosaminidase F. These sizes are consistent with those predicted for the murine CD2 antigen from the cDNA sequence.     

抗CMTM4多克隆抗体的制备、纯化和鉴定

目的:制备抗CMTM4的多肽抗体并鉴定其性能.方法:通过TMHMM、DNAStar等软件对CMTM4的3种剪接体,CMTM4-v1,v2和v3的蛋白序列进行分析,选取了4段序列合成多肽并与钥孔戚血蓝素(KLH)偶联.其中第1、2、3条多肽为3种剪接体共有,混合后免疫家兔,制备Ab1;而第4条多肽为CMTM4-v1所特有,单独免疫家兔,制备Ab2.ELISA法检测抗体效价.免疫亲和层析法纯化后,进行Western blot和免疫组织化学检测,鉴定这2种抗体的特异性与适用范围.结果:得到兔抗人CMTM4多肽抗体Ab1和Ab2,效价分别达1:105和1:106.纯化后的Ab1用于Western blot可特异识别超表达的CMTM4-v1和v2,并可用于免疫组织化学实验;而Ab2仅可用于Western blot,特异性识别超表达的CMTM4-v1.Ab1还可特异识别小鼠内源性Cmtm4.结论:用多肽免疫家兔制备的2种抗CMTM4抗体不仅具有较高的效价以及特异性,并且Ab1还可以特异识别小鼠Cmtm4,为CMTM4的功能研究提供了有力的支持.     

Dissection of an antibody paratope into peptides discloses the idiotope recognized by the cognate anti-idiotypic antibody

Using methods of parallel synthesis, the complete amino acid sequence of an Ab 1 antibody (Tg 10, an anti-human thyroglobulin monoclonal antibody) was made in the form of a set of 100 synthetic overlapping peptides. This set of immobilized peptides was allowed to react with the cognate Ab2 (AI 10, a highly purified rabbit anti-idiotypic polyclonal antibody to Tg 10). A dominant peptide idiotope, INTFSGVPTYA, was thus mapped, which corresponds mainly to the CDR2 region from the V(H) domain of the Tg 10 mAb. A synthetic peptide replica of this idiotope was found to bind to AI 10 with an affinity (K(D) in the 10(-8) M range, as measured using BIACORE technology) which represents a significant part of the affinity of the complete Tg 10 antibody (K(D) in the 10(-9) M range). The synthetic peptide also elicited anti-idiotypic antibodies in rabbits that recognized specifically the Ab1 antibody in an Ab1- and antigen-inhibitable manner. The peptide idiotope was further characterized chemically by the identification of residues important for binding to the Ab2 and by modelization of its structure. Our approach makes it readily possible to map and characterize functional, continuous-type idiotopes that could be further used to manipulate the immune response by peptide technologies.     

OX40 controls effector CD4+ T‐cell expansion,not follicular T helper cell generation in acute Listeria infection

To investigate the importance of OX40 signals for physiological CD4⁺ T‐cell responses, an endogenous antigen‐specific population of CD4⁺ T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S‐specific CD4⁺ T cells, but only on a subset that co‐expressed effector cell markers. This population was specifically expanded by Ab‐ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S‐specific effector cells. OX40 was not expressed by 2W1S‐specific memory cells, although it was rapidly up‐regulated upon challenge whereupon Ab‐ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4⁺ T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.     

Close localization of mouse CD14 and CD32/16 in the cell surface of monocytic cell lines.

The binding of a rat anti-mouse CD14 monoclonal antibody (mAb) (rmC5-3) was inhibited by pretreatment of a mouse monocytic cell line WEHI-3 cells with anti-mouse CD32/16 mAb (2.4G2), whereas that of 2.4G2 was not inhibited by pretreatment of WEHI-3 cells with rmC5-3. An enzyme-linked immunosorbent assay showed that rmC5-3 detected peptide 9 corresponding to amino acid position 308-322 of CD14 but 2.4G2 did not. A Western blot analysis of sera revealed that rmC5-3 and 2.4G2 detected the bands thought to be soluble CD14 and CD32/16, respectively. rmC5-3 reacted with mouse CD14-transfected CHO cells, CD14-CHO-K1 cells, but 2.4G2 did not. Lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha release was enhanced when a monocyte cell line (J774) was pretreated with rmC5-3. The enhancement was abolished by pretreatment with 2.4G2. The release of TNF-alpha was observed following treatment of J774 cells with 2.4G2 followed by anti-rat IgG F(ab')2.     

Induction of central T cell tolerance: recombinant antibodies deliver peptides for deletion of antigen-specific (CD4+)8+ thymocytes

In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self-antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen-presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II-specific Troybodies with the lambda2(315)T cell epitope were injected into lambda2(315)-specific TCR transgenic mice, a profound deletion of (CD4+)8+ thymocytes was observed. MHC class II-specific Troybodies were 10-100-fold more efficient than non-targeting peptide Ab, and 500-fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II-specific Troybodies with the OVA(323-339) T cell epitope were injected into OVA-specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II-specific Troybodies for 4 weeks, had reduced antigen-specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases.     

Detection of human auto-anti-idiotypic antibodies (Ab2). II. Generation of Ab2 in atopic patients undergoing allergen immunotherapy

The present study demonstrates the cross-reactivity of a murine monoclonal antibody (MoAb) directed against purified ragweed antigen E (AgE) with human Ab1. This antiragweed AgE MoAb (clone SC7H.1G, IgM kappa) was used to detect Ab2 in the sera obtained from the three groups of subjects. Utilizing an ELISA assay, we found that immunoglobulins from 12 nonatopic subjects bound to a significantly greater extent to SC7H.1G (mean +/- SE; OD = 0.353 +/- 0.052) than immunoglobulins from 14 untreated ragweed atopics (OD = 0.149 +/- 0.020). However, immunoglobulins from 9 immunotherapy-treated patients (OD = 0.395 +/- 0.120) bound to the mouse anti-AgE MoAb to the same extent as nonatopic sera. Furthermore, an additional 7 patients with ragweed-allergic rhinitis were studied prospectively while undergoing immunotherapy and Ab2 levels were found to increase with time posttreatment. We also found an inverse correlation between Ab1 and Ab2 levels in the nonatopic and untreated atopic groups. These data indicate that immunotherapy stimulates the production of Ab2 in atopic patients and may be involved in the regulation of the antiragweed IgE response.     

Induction of specific immunity to human colon carcinoma by anti-idiotypic antibodies to monoclonal antibody CO17-1A

Anti-idiotypic antibodies (Ab2) to murine monoclonal antibody (mAb) CO17-1A (Ab1), which defines an antigen associated with human colon carcinoma, were produced in goats. The Ab2 inhibited binding of Ab1 to colon carcinoma cells, and purified tumor antigen inhibited binding of Ab1 to Ab2. The Ab2 elicited in rabbits anti-anti-idiotypic antibodies (Ab3) that bound to cultured human cells of various tissue origins with a binding pattern identical to that of Ab1. Moreover, both Ab1 and Ab3 bound to the isolated tumor antigen, and the Ab3 lysed human colon carcinoma cells in culture in the presence of rabbit complement. Thus, Ab2 raised to mAb CO17-1A are candidates for use in immunotherapy trials in cancer patients.     

Cellular distribution of a natural killer cell tumour recognition-related surface antigen in purified human lymphocytes.

Natural killer (NK) cells are large granular lymphocytes capable of human leucocyte antigen (HLA) unrestricted killing of tumour cells. A putative NK cell tumour-recognition molecule (NK-TR) was previously isolated and cloned. The predicted primary structure of the NK-TR revealed that the amino terminus of the protein shared high homology with cyclophilin proteins. In this study, we used rabbit antibodies directed against synthetic peptides corresponding to amino acids 476-497 of the NK-TR protein, to examine the expression of the NK-TR antigen in freshly purified human lymphocytes. Cell-surface staining experiments using these peptide antibodies indicated the presence of the NK-TR protein on the surface of human CD3+ T-cell populations purified from peripheral blood. There were individual donor differences in the levels of cell-surface expression of this antigen ranging from 35 to 90% in T lymphocytes and, NK cells purified from different healthy volunteers. The immunoreactivity of our peptide antibodies in immunoprecipitation showed that the NK-TR-related protein expressed in purified T cells is similar to that expressed in NK cells in terms of its electrophoretic mobility. Cell-surface staining experiments using the peptide antibodies revealed that the NK-TR-related protein is more abundantly expressed on the surface of purified T cells compared with NK cells. Northern blot analysis of the mRNA species transcribed in human lymphocytes revealed abundant expression of NK-TR-specific mRNA species in purified T cells. Furthermore, another mRNA species smaller than 7 kb was detected in both NK and T-cell populations of lymphocytes freshly isolated from peripheral blood. Expression at the cell surface of a cyclophilin-homologous protein in purified human T lymphocytes may indicate another function for the reported NK-TR protein, that is, distinct from tumour-cell recognition and cytosis.     

Levels of autoantibodies, unlike antibodies to all extrinsic antigen groups, fall following B cell depletion with Rituximab

Many autoantibodies have variable-region sequences indicating their production in an affinity-matured antibody response involving germinal centers (GC). Plasma cells from GC can be long-lived, do not express CD20 and thus should not be depleted by a therapeutic monoclonal Ab against human CD20 - Rituximab. Nevertheless, autoantibody titers often fall following Rituximab treatment. To test if this reflects exclusive production by short-lived plasma cells in extrafollicular Ab responses, we monitored, after Rituximab treatment, levels of natural Ab and Ab against extrinsic antigens that do not induce productive GC. Eleven patients with active vasculitis and anti-proteinase-3 (PR3) Ab were assessed before and during 5 months after Rituximab therapy. Blood B cells were undetectable within 2 wk, and all patients achieved clinical remission. Levels of natural Ab - isohemagglutinins and anti-phosphorylcholine Ab - and Ab levels against thymus-independent and thymus-dependent extrinsic antigens were little affected. By contrast, 5 months after Rituximab, IgG autoantibody against PR3 had fallen to a median of 22% of pretreatment values. While the kinetics of this fall do not suggest an intrinsically short lifespan of autoantibody-producing cells, the data are consistent with Rituximab causing loss of sites within inflammatory tissues that selectively sustain autoantibody-producing cells.     

Various expressions of a unique anti-human thyroglobulin antibody repertoire in normal state and autoimmune disease

Polyclonal anti-human thyroglobulin (hTgb) antibodies (Ab) were purified from sera of rabbits immunized with human thyroglobulin, normal humans and patients suffering from Graves' disease, Hashimoto's thyroiditis and thyroid carcinoma. The avidity of the various Ab preparations for hTgb ranged from 0.3 X 10(10) -2.2 X 10(10) M-1. By using well characterized mouse monoclonal antibodies (mAb) directed against hTgb, it was shown that the fine specificities of induced anti-hTgb Ab in rabbits, natural Ab in normal subjects and autoantibodies in diseased patients were similar; however, they differed from that of rabbit anti-bovine and anti-porcine thyroglobulin Ab which were able to inhibit the hTgb binding of only a few of the mAb. Anti-hTgb in rabbits and in patients with thyroid carcinoma varied from those in normal subjects only by uniformly elevated serum titers. In contrast, patients with Graves' disease and Hashimoto's thyroiditis showed an increased concentration essentially restricted to Ab reacting with few of the antigenic determinants recognized by the mAb. Our data suggest that the repertoire of anti-hTgb Ab is similar in mouse, rabbit and human. Furthermore, the finding of identical fine specificities for anti-hTgb Ab in normal and pathological conditions implies that autoantibodies are produced in normal subjects and held to a low level by regulatory processes which fail with respect to selected epitopes in autoimmune diseases.