IgG Anti-tetanus Toxoid Research Articles

Development of novel fusogenic vesosomes for transcutaneous immunization

Transcutaneous immunization (TCI) is a novel vaccination strategy based on the application of antigen together with an adjuvant onto hydrated bare skin. This simple and non-invasive immunization procedure elicits systemic and cell mediated immune responses and therefore, it provides a viable and cost-effective strategy for disease prevention. In the present study, novel fusogenic vesicular carrier constructs, i.e. vesosomes were developed and evaluated for topical delivery of vaccines using tetanus toxoid (TTx) as a model antigen. Prepared vesosomes were characterized for size, shape, entrapment efficiency and zeta potential. The prepared novel systems were examined for in process antigen stability and long-term storage stability studies. In vitro skin permeation and fluorescence microscopy study were also preformed for prepared novel vesicular systems for the evaluation of skin penetration efficiency. The immune stimulating activity of these vesicles was studied by measuring the serum anti-tetanus toxoid IgG titer and isotype ratio IgG2a/IgG1 following topical immunization in three different protocols and results were compared with the alum adsorbed tetanus toxoid given intramuscularly and topically administered plain tetanus toxoid solution, plain liposomes and cationic fusogenic liposomes. Serum IgG titers after three consecutive topical administrations were significantly better ( * P < 0.05) than single administration of TTx antigen with vesosomal systems, suggesting an effective stimulation of serum immune response. Furthermore, notable serum anti-TTx antibody titers also occurred in animals primed with alum adsorbed TTx and subsequently boosted with topical administration of novel vesosomal systems. In each immunization studies, the vesosomal systems could elicit combined Th1 and Th2 immune responses following topical administration. These results suggest that the investigated vesosomal systems can be effective as topical delivery of vaccines.

Is there a causative role for tetanus toxoid vaccination in the development of allergy-like symptoms and in the increasing prevalence of atopic diseases?

Allergic diseases are a worldwide health problem. They mainly affect people living in developed countries where an increasing prevalence of allergy symptoms has been recorded in the last 20–30 years. The cause of this increase is still disputed, and, among others, the “hygiene hypothesis” supported the concept that relevant changes in lifestyle could have a relationship with the phenomenon. More recently the recorded parallel increase in autoimmune diseases has suggested to consider the “hygiene hypothesis” as a cause of a more general disregulation of the immune system leading to both allergy and to autoimmunity. Here are reported a series of observations, evidence, and data from the literature leading to a different hypothesis. The key points are: (1) the presence of two subsets of patients having allergy symptoms based on an IgE-mediated mechanism or not; (2) the positive results obtained with the autologous serum skin test in either cutaneous or respiratory affected subjects, mainly in children and adult females; (3) the presence of IgG autoantibodies against the α-chain of the high affinity IgE receptor (Fc εRIα) in non-IgE-mediated urticaria and even in respiratory subjects; (4) the cross-reactivity between epitopes of the tetanus toxoid molecule and the Fc εRIα detected by means of an α-chain affinity purified IgG fraction; (5) the positive skin reactivity obtained using IgG anti-tetanus toxoid preparations in allergic and non-allergic volunteers. The presence of IgG autoantibodies actively generated by the population-based vaccination with tetanus toxoid could induce both mediator release from activated mast cell and Th2 cytokine production early in life. There are epidemiological evidences that tetanus toxoid vaccination could be linked with an increased tendency to have allergy symptoms. The different epidemiological distribution of non-IgE-mediated symptoms, mainly affecting young infants would be in agreement with the present hypothesis. The prevalent mother-to-child relationship in terms of risk for allergy symptoms could be explained with the trans-placenta transfer of IgG. A similar transfer could also take place through the mother milk during breast feeding. It may thus be hypothesized that the increased prevalence of allergic diseases could be caused by the generalized tetanus toxoid immunization procedure, progressively extended to most of the countries worldwide in the last 30–40 years. Both the induction of non-IgE-mediated symptoms caused by the mast cell activation via the anti-Fc εRIα IgG and the long lasting Th2 inflammation of affected tissues would be the inducing mechanisms. This hypothesis would re-configure part of the allergic diseases as a Th2 phenotypic expression of an autoimmune disease.

Coexistent yellow nail syndrome and selective antibody deficiency

Yellow nail syndrome (YNS) is a rare, often underdiagnosed condition of unknown origin. The clinical features of the syndrome include yellow nails, chronic sinusitis, bronchiectasis, pleural effusion, and lymphoedema. Despite the frequent occurrence of upper and lower respiratory tract infections in patients with YNS, comprehensive analysis of their humoral immunity has not been previously reported. To present the case of a patient with YNS whose recurrent upper and lower respiratory tract infections may have been caused by an underlying selective antibody deficiency that manifests as impaired IgG antibody response to polysaccharide antigens. The patient underwent cultures of purulent sputum for Streptococcus pneumoniae and Haemophilus influenzae, bronchial washings for H. influenzae, and nail scrapings for fungi. Her serum levels of IgG, IgA, IgM, IgG subclasses, and serum titers of IgG antitetanus toxoid, anti-H. influenzae, and anti-S. pneumoniae antibodies were measured. Cultures of purulent sputum were positive on multiple occasions for S. pneumoniae and H. influenzae and bronchial washings were positive for H. influenzae. Nail scrapings were consistently negative for fungi. She had no reductions in serum levels of IgG, IgA, IgM, or IgG subclasses and had normal serum titers of IgG antitetanus toxoid antibodies. However, she demonstrated impaired IgG antibody responses following immunization with Pneumovax and an H. influenza B vaccine. This case report describes the first comprehensive analysis of humoral immune function in a patient with YNS. The finding of a selective antibody deficiency in our patient provides a potential explanation for the occurrence of respiratory infections in YNS. Accordingly, we recommend that functional antibody determinations and quantitative serum immunoglobulins be evaluated in patients diagnosed as having this unusual, enigmatic syndrome.

Organ and Non-organ Specific Autoantibody Titres and IgG Levels as Markers of Disease Activity: A Longitudinal Study in Childhood Autoimmune Liver Disease

No longitudinal study has investigated whether autoantibody titres and serum IgG levels correlate with disease activity in autoimmune liver disease. To determine this, we investigated prospectively 19 patients on 254 occasions between 10 months to 5 years from diagnosis. Nine had anti-nuclear and/or anti-smooth muscle antibody (ANA/SMA) positive autoimmune hepatitis (type 1 AIH), 5 liver kidney microsomal type 1 (LKM-1) positive AIH (type 2 AIH) and 5 ANA/SMA positive autoimmune sclerosing cholangitis (ASC). Correlation between IgG levels, titres of ANA, SMA and LKM-1 and levels of the organ specific autoantibodies anti-liver specific protein (anti-LSP), and anti-asialoglycoprotein receptor (anti-ASGPR) with biochemical evidence of disease activity, as measured by serum aspartate amino transferase (AST) levels, was sought during the course of the disease. AST levels correlated with levels of anti-LSP, anti-ASGPR and IgG in type 1 and 2 AIH, but not in ASC. Positive correlation with AST was also observed for LKM-1 titres in type 2 AIH and for SMA titres in type 1 AIH, but not in ASC. In both AIH and ASC, AST levels correlated with the T cell-dependent immune responses anti rubella IgG and anti tetanus toxoid IgG, but not with the T cell-independent IgG 2 response to pneumococcal capsular polysaccaride. Our results indicate that measurement of organ and non-organ specific autoantibodies and IgG levels may be used to monitor disease activity in AIH.

Effects of toxaphene on the immune system of cynomolgus ( Macaca fascicularis) monkeys

Toxaphene, dissolved in glycerol/corn oil, was administered at 0.1, 0.4 or 0.8 mg/kg body weight/day in gelatin capsules to groups of 10 young adult female cynomolgus monkeys ( Macaca fascicularis), while a group of five male monkeys ( Macaca fascicularis) received 0.8 mg/kg body weight/day. Control male (a group of five) and female (a group of 10) monkeys ingested the glycerol/corn oil vehicle only. Treatment continued for 75 weeks. Testing for immune effects was initiated at 33 weeks of treatment. Immunization was initiated at 44 weeks of treatment. Pairwise comparisons between each of the treated female groups to the control indicated that the mean primary (post-immunization weeks 1–4) and secondary (post-immunization weeks 5–8) anti-SRBC IgM responses were significantly reduced at the 0.4 and 0.8 mg/kg body weight/day doses compared to the control ( P⩽0.05). The mean primary (post-immunization weeks 1–4) anti-SRBC IgG response was significantly reduced compared to the control ( P⩽0.05), while the secondary (post-immunization weeks 5–8) anti-SRBC IgG was not significantly affected by treatment ( P>0.05). The mean anti-tetanus toxoid IgG response in the 0.8 mg/kg body weight/day dose group The mean primary anti-SRBC (IgM) response in the treated males was significantly different from the control ( P<0.05), while the primary anti-SRBC IgG response was not affected by treatment. The mean absolute B-lymphocyte numbers in the female group administered 0.8 mg/kg of toxaphene was significantly reduced compared to the control ( P⩽0.05). All other parameters including the natural killer cell activity, the delayed-type hypersensitivity response, the lymphoproliferative response of peripheral blood leukocytes to the mitogens Con A and PWM and the serum cortisol levels were not affected significantly by treatment ( P>0.05). The no-observed-adverse-effect level (NOAEL) for the female monkeys based on the toxaphene effects on humoral immunity was 0.1 mg/kg body weight/day.

A typical bacterial ornithine-containing lipid Nalpha-(D)-[3-(hexadecanoyloxy)hexadecanoyl]-ornithine is a strong stimulant for macrophages and a useful adjuvant.

Nalpha-[3-(Hexadecanoyloxy)hexadecanoyl]-ornithine is a typical bacterial ornithine-containing lipid (OL). The configuration of the 3-hydroxy fatty acids in the OL was proved to be D by using HPLC with chiral column. For this analysis, Nalpha-(D or L)-[3-(hexadecanoyloxy)hexadecanoyl]-L-ornithine were synthesized and used as standards. The typical bacterial OL, as well as the synthesized one, exhibited strong interleukin-1- and prostaglandin E2-inducing activities, and further, it induced the production of high IgG anti-tetanus toxoid antibodies in mice. The typical OL is expected to be utilized as a nontoxic, potent adjuvant.

A typical bacterial ornithine-containing lipid N α-( d)-[3-(hexadecanoyloxy)hexadecanoyl]-ornithine is a strong stimulant for macrophages and a useful adjuvant

N α -[3-(Hexadecanoyloxy)hexadecanoyl]-ornithine is a typical bacterial ornithine-containing lipid (OL). The configuration of the 3-hydroxy fatty acids in the OL was proved to be d by using HPLC with chiral column. For this analysis, N α -( d or l)-[3-(hexadecanoyloxy)hexadecanoyl]- l-ornithine were synthesized and used as standards. The typical bacterial OL, as well as the synthesized one, exhibited strong interleukin-1- and prostaglandin E 2-inducing activities, and further, it induced the production of high IgG anti-tetanus toxoid antibodies in mice. The typical OL is expected to be utilized as a nontoxic, potent adjuvant.

Serum immunoglobulin levels in heterozygous subjects with immunoglobulin heavy chain constant region gene deletions.

The immunoglobulin heavy chain constant region locus is a multigene family composed of nine genes and two pseudogenes, whose high homology is often responsible for meiotic mispairings leading to deleted and duplicated haplotypes. These rearrangements have a population frequency of about 1.5% and 4.5% respectively, with a significant difference between deletions and duplications (P < 0.001). Both positive selection of duplications or negative selection against deletions can account for this imbalance. Serum levels of IgG and IgA subclasses, of IgE, of isohemagglutinins and of IgG antibodies to tetanus toxoid and pneumococcal antigens were evaluated in 11 heterozygous carriers of constant region deletions. There was no gross abnormality in serum IgG and IgA subclass levels, with the possible exception of G1-deleted individuals; furthermore, isohemagglutinins and anti-tetanus toxoid and pneumococcal IgG antibodies are in the normal range, suggesting that the humoral immune response is normal in these carriers. The influence of single and multiple immunoglobulin heavy chain constant region gene deletions on the humoral response is discussed.

The role of tyrosine phosphorylation and Ca2+ accumulation in Fc gamma-receptor-mediated phagocytosis of human neutrophils.

When stimulated with an insoluble immune complex consisting of tetanus toxoid and human anti-tetanus toxoid IgG antibody, human neutrophils recognized the immune complex via Fc receptors (Fc gamma RII and Fc gamma RIIIB) on their plasma membranes, resulting in Ca2+ accumulation, and finally phagocytosed the immune complex. Ca2+ depletion experiments suggest that Ca2+ accumulation is essential for the maximal phagocytic activity. A tyrosine kinase inhibitor, genistein, inhibited the phagocytosis of immune complex. Genistein also inhibited both the later prolonged Ca2+ accumulation below 100 microM, and the initial rapid Ca2+ accumulation above 100 microM. Stimulation of human neutrophils by immune complexes through Fc gamma Rs caused tyrosine phosphorylation of multiple neutrophil proteins within 15 s, proceeding to an increase in the intracellular Ca2+ concentration. A similar pattern of tyrosine phosphorylation was observed when Fc gamma RII or Fc gamma RIIIB was aggregated by an anti-Fc gamma R monoclonal antibody and anti-mouse Ig. These results suggest that tyrosine phosphorylation mediates, at least in part, the later responses (Ca2+ accumulation and phagocytosis) of the cells and is an important early step in signaling via Fc gamma Rs.

Anti-tetanus toxoid antibodies in intravenous gamma globulin: an alternative to tetanus immune globulin.

The levels of anti-tetanus toxoid IgG antibodies were measured in 29 lots of intravenous gamma globulin (IVIG). The antibody levels varied from 4 to 90 IU/mL (geometric mean, 18.6; 90% confidence interval, 9.7-35.7). The variation from manufacturer to manufacturer accounted for most of the observed differences among lots; there was relatively little variability among multiple lots from a single manufacturer. IVIG may be an acceptable alternative to horse or human tetanus immune globulin.

Humoral Immunity in Surgical Patients With and Without Trauma

We measured antitetanus toxoid antibody responses after blunt (n = 24) and penetrating (n = 7) trauma and compared them with responses in patients without trauma (n = 55). Patients were defined as anergic or reactive on the basis of delayed type hypersensitivity response. The response to tetanus toxoid vaccination on admission of patients surviving trauma for over 2 weeks was defined as the ratio of day 14 to day 0 serum IgG antitetanus toxoid levels. Antitetanus toxoid responses were normal after both blunt and penetrating trauma. When stratified according to delayed type hypersensitivity responses, patients with trauma showed better antibody responses than patients without trauma. Major infection rates were similar between trauma groups (three of 24 with blunt trauma vs two of seven with penetrating trauma) and independent of delayed type hypersensitivity (two of 20 reactive patients vs three of 11 anergic patients), in contrast to patients without trauma (one of 19 reactive patients vs 15 of 36 anergic patients). We conclude that decreased delayed type hypersensitivity after moderate trauma is temporary, and that this transient immunodeficiency is not as strongly associated with reduced antibody responses and increased risk of infection as anergy in surgical patients without trauma.

Inhibition of human antigen-specific memory B cell response in vitro by a diphtheria toxin-related interleukin 2 fusion protein

Recombinant diphtheria toxin-related interleukin-2 fusion protein (DAB 486IL-2) is specifically cytotoxic for cells bearing the high-affinity IL-2 receptor (p55/75). We evaluated the effects of DAB 486IL-2 on the generation of tetanus toxoid(TT)-specific IgG antibody-forming cells in 6-day cocultures of human splenocytes and TT-coupled Sepharose beads. The results indicate that a significant portion (30–75%) of the anti-tetanus toxoid IgG response in vitro was susceptible to inhibition by 10 −10 M DAB 486IL-2. The inhibition required both the IL-2 portion of the fusion protein and an active toxin moiety and was greater when the IL-2 toxin was added on Day 3 as compared with Day 0 of culture. The induction of the p55 (Tac) subunit of the IL-2R was demonstrable by two-color flow cytometry on a small percentage (5%) of B cells and on a higher percentage (10%) of non-B cells 3 days after exposure to TT-coupled Sepharose. Short-term (2 hr) treatment of T and B cell subpopulations separated on Day 3 of culture followed by remixing indicated that while activated T helper cells were most strongly inhibited by DAB 486IL-2, up to 50% of the TT-specific IgG response was inhibited by treatment of B cells alone with DAB 486IL-2. Our results suggest that a strategy of eliminating human memory B cells by a combination of antigen activation and properly timed administration of a recombinant lymphokine-toxin fusion protein is feasible.

INHIBITION OF BOTH MLC AND IN VITRO IgG MEMORY RESPONSE TO TETANUS TOXOID BY RS-61443

We designed experiments to test the ability of RS-61443 to inhibit two types of human T cell-dependent responses: the mixed lymphocyte culture response of human peripheral blood leukocytes and the anti-tetanus toxoid IgG response of human splenocytes

IgG anti-tetanus toxoid antibody synthesis by human bone marrow. I. Two distinct populations of marrow B cells and functional differences between marrow and peripheral blood B cells

This investigation uses a system for inducing and detecting anti-tetanus toxoid antibody (anti-TT) synthesis to study specific antibody (Ab) synthesis by bone marrow mononuclear cells (MC). We measured the amounts of anti-TT secreted and the number of B cells secreting antibody (Ab). The ELISA plaque detects single B cells secreting specific Ab. The results show that (1) spontaneous anti-TT secretion by MC is higher than spontaneous anti-TT secretion by peripheral blood lymphocytes (PBL) using an ELISA plaque (P less than 0.01); (2) spontaneous anti-TT production by MC correlated with the serum anti-TT titers as measured by an ELISA (r = 0.75, P = 0.005); (3) two types of marrow B cells were identified--one that spontaneously secretes anti-TT and another that produces anti-TT after TT-stimulation; (4) the frequency of anti-TT-secreting B cells is higher in MC than in PBL; (5) the amount of Ab secreted per marrow B cell is not different from that secreted by a peripheral B cell; and (6) marrow B cells could be induced to produce anti-TT in vitro up to 10 months without added cytokines. These results show that bone marrow is a major repository for differentiated B cells that spontaneously produce Abs to maintain circulating Abs titers and for memory B cells that can be induced to produce specific Ab.

Probing the normal and autoimmune B cell repertoire with Epstein-Barr virus. Frequency of B cells producing monoreactive high affinity autoantibodies in patients with Hashimoto's disease and systemic lupus erythematosus.

The frequency of cell precursors producing Ig of different classes and Ag-binding activities were determined, using EBV-infection and limiting dilution assays, in healthy subjects and patients with autoimmune disease. A large proportion of circulating B cells from healthy subjects were committed to the production of IgM antibodies that were polyreactive and bound a variety of self- and exogenous Ag, i.e., IgG Fc fragment, ssDNA, thyroglobulin, thyroid microsomal Ag, insulin, and tetanus toxoid. Similar frequencies of these polyreactive antibody-producing cells were found in patients with Hashimoto's disease and SLE. In contrast, significantly higher frequencies of cell precursors producing monoreactive IgG autoantibodies to thyroid Ag (thyroglobulin and thyroid microsomal Ag) and ssDNA were found in Hashimoto's disease and SLE patients, respectively. Calculation of the Kd revealed that monoclonal polyreactive antibodies were in general low affinity (Kd, 10(-3) to 10(-7) mol/liter), whereas monoclonal monoreactive autoantibodies were high affinity (Kd, 10(-9) to 10(-11) mol/liter). The detected frequency and high affinity of the monoreactive autoantibodies in Hashimoto's disease and SLE patients were comparable to those of anti-tetanus toxoid and anti-insulin IgG mAb produced by B cell clones from vaccinated healthy subjects and insulin-treated patients with insulin-dependent diabetes mellitus, respectively. These findings support the hypothesis that the autoimmune B cell repertoire in patients with organ-specific and systemic autoimmunity is shaped by Ag-driven responses rather than merely reflecting a polyclonal B cell activation.

Immunological responses to chronic heat exposure and food restriction in rats

Immunological variables were studied in rats chronically exposed to high environmental temperature (35 degrees C). Responses were compared with those of rats at 25 degrees C both fed ad libitum and pair fed to the decreased intake found in heat-exposed rats. Heat-exposed rats showed slower delayed-type hypersensitivity responses to keyhole limpet hemocyanin. They showed lower counts of peripheral blood total T cells (OX19+) as well as helper T cells (W3/25+) and smaller numbers of splenic T cells. The thymus was decreased in size. Increased levels of serum IgG antitetanus toxoid antibodies were found in heat-exposed rats. [3H]-thymidine incorporation into Concanavalin A (ConA)-stimulated splenic lymphocytes was decreased in pair-fed rats but not significantly altered in heat-exposed rats compared with controls. Heat exposure alters some aspects of both cellular and humoral immune function in a manner different from that induced by comparable food restriction without heat exposure.

The effects of aggregated human IgG and IgG-immune complexes on the agglutination of bacteria mediated by non-immunoglobulin salivary agglutinins

Non-immunoglobulin salivary agglutinins (SBA) for bacteria which bind to Streptococcus milleri TJ7 were isolated from parotid saliva and their interactions with human IgG studied. Purified SBA showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular weight of approx. 500,000. Heat aggregated human IgG (63 °C, 30 min), but not native IgG, interacted with SBA and thereby interfered with the ability of the SBA to agglutinate Strep. milleri. Immune complexes prepared from tetanus toxoid and isolated human IgG anti-tetanus toxoid antibody also inhibited salivary bacterial agglutination by SBA; antigen (tetanus toxoid) alone or antibody (anti-tetanus toxoid antibody) alone did not have this effect. Direct-binding studies with immobilized SBA on nitrocellulose paper showed that aggregated IgG bound to immobilized SBA and that this binding was inhibited by EDTA. Thus it appears that heat or specific antigen is able to induce an aggregation of IgG which results in the binding of the aggregated form of IgG to SBA.

In vitro inhibition of anti-DNA producing cells from systemic lupus erythematosus patients by autologous anti-F(ab′) 2 antibodies

We have previously documented an inverse relationship between serum levels of anti-F(ab′) 2 antibodies and disease activity in systemic lupus erythematosus (SLE). The present study focused on the specific in vitro inhibition of anti-DNA producing cells from SLE patients by autologous anti-F(ab′) 2 antibodies. Peripheral blood lymphocytes (PBL) from eleven inactive SLE patients with no apparent disease activity were cultured in vitro to evaluate anti-DNA antibody secretion. Low levels of synthesis of anti-DNA antibody were detected in 3 of 11 SLE patients using unstimulated PBL; on the contrary, pokeweed mitogen stimulation of cultured cells increased production of anti-DNA in all SLE subjects. Parallel cultures were also performed in the presence of heterologous and autologous anti-F(ab′) 2 antibodies and results on production of anti-DNA evaluated. Lymphocytes from SLE patients in remission showed inhibition of synthesis of anti-DNA antibodies when autologous anti-F(ab′) 2 antibodies were added to the cultures, whereas production of anti-tetanus toxoid IgG by the same cells was not significantly altered under the same conditions. These data suggest that a functional anti-idiotypic role may be assigned to anti-F(ab′) 2 antibodies during clinical remission of SLE.

Designed transfer of specific immune responses with bone marrow transplantation.

Bone marrow transplant donors were immunized with tetanus/diphtheria toxoids 6-7 d before bone marrow donation to investigate the role of B cell subpopulations in reconstitution of humoral immunity. Lymphoblastoid B cells spontaneously producing IgG antitetanus and/or antidiphtheria toxoid were detected in the donor marrows at the time of transplantation. Recipients rapidly demonstrated 3-90-fold increases in serum IgG antitetanus and antidiphtheria toxoid levels. Antidiphtheria fragment A antibody in three donor/recipient pairs demonstrated spectrotypic identity indicating transfer of the donors' response. Reimmunization of three recipients 64-154 d after transplant revealed an IgG antibody response associated with reappearance of spontaneous antibody-producing B cells and an antidiphtheria fragment A response characteristics of the donor's immune response. These observations extend the understanding of the role of B cell subpopulations and provide a basis for specific modulation of immunity in the setting of bone marrow transplantation.

The transfer of antigen-specific humoral immunity from marrow donors to marrow recipients

This study shows that specific humoral immunity could be transferred from marrow donors to marrow recipients. Peripheral blood lymphocytes from long-term human marrow recipients produced IgG anti-tetanus toxoid antibody after in vitro tetanus toxoid stimulation. Antitetanus toxoid antibody biosynthesis was induced using a new tetanus toxoid-specific system employing high lymphocyte numbers, many replicate microcultures, and antigen washout. Anti-tetanus toxoid antibody in 12-day culture supernatants was detected using an enzyme-linked immunosorbent assay. Of 14 marrow recipients, 6 (5 with and 1 without chronic graft-vs-host disease) had lymphocytes that produced anti-tetanus toxoid antibody. Culturing additional numbers of marrow recipient lymphocytes increased in vitro biosynthesis of anti-tetanus toxoid antibody. The presence of circulating serum antibodies to tetanus toxoid and the production of specific anti-tetanus toxoid antibody by peripheral blood lymphocytes from marrow recipients show that engrafted donor lymphocytes can produce in vitro specific antibodies to recall antigens without postgrafting reimmunization.