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The aim of this contribution is twofold: to honour Ivan Lefkovits with a short recollection of our scientific collaboration in the years 1972-1985 and a summary of our joint contribution to studies of the mechanisms and functions of the immune system and to acknowledge our long-lasting friendship. Ivan's limiting dilution microculture method was adapted to rabbit peripheral blood lymphocytes (PBL). The antibody produced in the responding cultures was shown to be electrophoretically homogeneous and, in rabbits heterozygous at the b locus, to express either one or the other allele. Thus, the antibody released in single microcultures was indeed the product of single B-cell clones and allelic exclusion, once achieved, was maintained throughout clonal proliferation. In the response to streptococcal polysaccharides, analysis of the clonotypes triggered in vitro provided information on mechanisms of clonal dominance. A two-stage culture system was established, where rabbit PBL were precultured at low cell density with antigen before being partitioned in limiting dilution cultures. This method provided a new tool for studies of various cellular aspects of the immune response. Moreover, it allowed the application of the limiting dilution analysis to PBL from unprimed animals. Later, the method was extended with success to human PBL, leading to studies of regulatory aspects of immunity in this species.

The relative distribution of IgA and IgG subclass-producing immunocytes was examined by two-colour immunohistochemistry in normal human distal ileum including Peyer's patches (PP), regional mesenteric lymph nodes (MLN), and peripheral lymph nodes. IgA2 cells predominated slightly over IgA1 cells in the PP dome area. There was a decreasing median proportion of IgA2 cells in the order of PP (52%), distant ileal lamina propria (40%), MLN (32%), and peripheral lymph nodes (11%). The reverse was true for IgA1 cells in independent enumerations. These results support the notion that PP-derived B cells after stimulation are seeded mainly to the lamina propria of the distal gut, but that there is a substantial retention and terminal differentiation of this migrating population in regional MLN. The median subclass proportions of IgG-producing cells in the PP dome area were in independent determinations 68% IgG1, 23% IgG2, 8% IgG3, and 9% IgG4. This distribution was fairly similar to that seen in other tissue categories, except for a trend towards increased IgG1 and reduced IgG2 proportions in peripheral lymph nodes and reduced IgG1 along with increased IgG3 in normal palatine tonsils. The data suggested an association between the expression of IgG2 (and possibly IgG4) and IgA2 in intestinal mucosal immune responses.

The induction of immunoglobulin production by rabbit spleen cells is markedly inhibited by the presence of normal rabbit serum during cell culture. A similar inhibition is observed when spleen cell populations in which T cells have been inactivated are temporarily incubated with normal rabbit serum before being reconstituted with T cells by adding thymocytes. In contrast, no inhibition was observed upon temporary incubation of thymocytes with normal serum prior to addition of T cell-inactivated spleen cell populations. Removal of adherent cells did not affect the induction of immunoglobulin production or its inhibition by normal serum. Lipopolysaccharide-enhanced immunoglobin production was also inhibited by normal serum, thereby providing additional confidence that bone-marrow derived (B) cells are the target of the normal serum inhibitor.

It is possible to induce idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi (S.a.e.). Ten months after priming we injected the rabbit with anti-idiotypic serum prepared against its own antibodies to S.a.e. and, 3 weeks later, gave it a booster injection of bacteria. A new anti-idiotypic serum was preparedwith the serum to S.a.e. collected after this boost and was used for the following isiotypic suppression attempt made 10 months after the first one. Using this procedurewe succeeded in two successive idiotypic suppression attempts in the same rabbit. Inthe three attempts we carried out, idiotypic suppression was totally effective, i.e. idiotypes detected by the serum used for the suppression totally disappeared after thesuppression, and the suppression lasted during the life of the rabbits (maximum 10 months). This observation is consistent with a suppression resulti-g from an interaction of anti-idiotypic antibodies with the complementary receptors at the surface of memory cells. This suppression was without effect on antibody to S.a.e. titre and on IgG concentration. Idiotypes detected by the anti-idiotypic serum prepared withthe serum to S.a.e. collected after the suppression. These idiotypes were different from those detected by the anti-idiotypic serum used for the suppression. This observation confirms that idiotypic recognition is confined to a limited number of clonal products, despite the fact that a very heterogeneous antibody population was used forthe anti-idiotypic immunization. Thus we did not observe the appearance of new idiotypes produced previously silent cell clones. All the different idiotypes we detected during the successive idiotypic suppression attempts carried determinants shich remained peculiar to each individual rabbit.

When immune spleen cells of mice immunized to a hapten carrier preparation 4 days previously were transferred to normal syngeneic hosts, they began to produce 7S antibody (presumably of the IgG class), provided that relatively small numbers of cells (about 1/10 spleen equivalent) were transferred. Increasing the number of transferred cells resulted in less IgG antibody formed. Depletion of the immune spleen cells of T cells by treatment with anti-theta serum and complement prevented IgG antibody formation. IgG antibody production by untreated and anti-therta serum-treated immune spleen cells could be enhanced (reinduced) by addition of small numbers of cells enriched for carrier-activated T cells. These suggest that T cells are necessary to stimulate antigen-activated B cells into IgG antibody production. Larger numbers of 'carrier-activated T cells' depressed IgG antibody production. Both enhancement and depression could be demonstrated to be antigen-specific. IgG antibody production high numbers of transferred immune spleen cells could be induced by treating the cells prior to transfer with suboptimal amounts of anti-theta serum and complement. It is argued that this results from the elimination of a T cell-dependent suppressor influence arising during a normal immune response.

Experiments are described which demonstrate that a single intraperitoneal (i.p.) injection of antigen in Freund's complete adjuvant results in the appearance of IgA-specific antibody-containing cells (ACC) in the intestinal lamina propria of sheep and that these cells reach the intestine via the intestinal lymph and blood circulation. Following intraintestinal administration of antigen to sheep immunized i.p. 2 weeks previously, an enhanced ACC response occurs in the intestine but cannulation and drainage of the intestinal duct does not interfere with this enhancement. Evidence is presented which suggests that the enhanced ACC response may be accounted for by antigen-induced local proliferation of ACC in the lamina propria of the intestine.

The mitogenic activity of Mycoplasma pulmonis towards both rat B and T lymphocytes has been demonstrated. The data summarized in this report show that spleen cells obtained from T X BM rats were extensively activated by M. pulmonis. Furthermore, M. pulmonis has been demonstrated to induce the development of antibody producing cells, as attested by the appearance of direct plaque forming cells against SRBC and TNP-SRBC in spleen cultures exposed to this mitogen. It was also demonstrated that rat thymus cells and a part of the lymph node T-cell population responding to either Con A or PWM, were stimulated by M. pulmonis, the response being weaker than that of rat B-cell populations. It was thus concluded that M. pulmonis activates both rat B and T lymphocytes. This mitogenic stimulation, however, is not equally exerted on both these populations, being strongly effective upon B cells and less so on T cells.

During the growth of the L2C leukaemia in strain 2 guinea-pigs there is excreted in the urine a monoclonal light chain of lambda class. This is believed to arise from synthesis by the leukaemic cells of a small molar excess of the light chains incorporated into their surface IgM. Antibodies to this light chain have been raised in rabbits and separated by immunosorption into those directed against idiotypic determinants (anti-Id), and those against determinants also present on other lambda chains (anti-lambda). Both antibodies react with L2C cells via the surface immunoglobulin. In accord with prediction anti-Id is specific for L2C cells while anti-lambda also reacts with 7 per cent of normal nodal lymphocytes. L2C cells are agglutinated strongly by anti-Id, weakly by anti-lambda. Both antisera inhibit migration of the cells, and both can kill them by invoking complement or K-cell cytotoxic mechanisms. The results emphasize the potential of anti-idiotype sera for identifying and specifically attacking neoplastic B lymphocytes.

Thoracic duct lymphocytes (TDL) from (AS2 x AS)F1 rats previously injected with human serum albumin (HSA) were transferred to 900 r irradiated syngeneic recipients which were challenged with various doses of soluble HSA (s-HSA). TDL from partially tolerant rats, which were deficient in high avidity B cells, produced a maximum PFC response to the largest challenge dose (1 g s-HSA). In contrast, high avidity B cells from primed donors were optimally stimulated by 1 microgram and maximally inhibited by 1 mg s-HSA (day 7 PFC). An additional increase in antigen concentration by 1000 fold failed to diminish the PFC numbers further. Plaque inhibition profiles indicated that these antibody forming cells resisting inhibition were of the same high avidity as those triggered by low doses of antigen. The inability of s-HSA to completely inhibit antibody synthesis is discussed with regard to current views on B cell inactivation. Evidence is also presented which indicates that the standard haemolysis-in-gel test may fail to detect many low avidity antibody forming cells to proteins.

The relationship between cellular (B-cell) responses and the development of casein-induced amyloidosis was explored. The main findings are: (1) B-cell abnormalities are more pronounced in amyloid susceptible CBA/J than in amyloid resistant A/J mice; these include increased mitogen responses to SIII, PI:C and DXS, and enhanced primary immune responses to T-independent antigens; (2) CBA/J amyloid spleen cell suspensions appear to be enriched with antibody dependent killer cells and precursors of antibody-forming cells; these abnormalities are not seen in casein treated A/J mice. This hitherto unrecognized proliferation of immunoblasts in casein-treated CBA/J animals raises the possibility that B cell-macrophage interaction may play an important role in the pathogenesis of amyloid disease.

The activity of Nocardia water-soluble mitogen (NWSM) and LPS were compared in several experimental systems, since both compounds are B-cell mitogens and polyclonal activators in vitro. The results reported here demonstrated that NWSM like LPS also has a strong adjuvant activity in vivo if administered in saline with a strong antigen (heterologous red blood cells) or even with a weak immunogen such as theta alloantigen. However, in contrast to LPS, NWSM administered to mice failed to induce in vivo proliferation of lymphocytes, polyclonal activation and PFC against syngeneic bromelain-treated erythrocytes and thymocytes. It is possible therefore, that different mechanisms may be responsible for adjuvant activity of NWSM and LPS.

The occurrence of plaque-forming cells (PFC) in mouse bone marrow was studied during primary and secondary responses to the thymus-independent antigen Escherichia coli lipopolysaccharide (LPS). Anti-LPS responses were induced by various doses of LPS. During the primary response, doses of 1 and 10 mug LPS intravenously (i.v.) were found to evoke a distinct PFC response in both spleen and bone marrow. The spleen contained the majority of PFC until about 5 days after immunization. During the course of the reaction the number of PFC in the bone marrow rose to a level which equalled or surpassed the level in the spleen. LPS doses of 0-001, 0-01 and 0-1 mug i.v. only induced a PFC response in the spleen. Apparently there is a minimal threshold dose of LPS of about 1 mug for PFC to appear in the bone marrow. The secondary response was studied in mice primed with 1 mug LPS i.v. and boosted with either 0-001, 0-1 or 10 mug LPS i.v. 3 months later. After each dose tested the PFC activity in the spleen was several times higher than during the primary response. As was observed in the primary response doses of 0-001 and 0-1 mug LPS i.v. did not evoke a PFC response in the bone marrow. After boosting with 10 mug of LPS i.v. a significant PFC response was found in spleen, bone marrow, thymus, lymph nodes, Peyer's patches and blood. From about 5 days after the booster injection the number of PFC in the bone marrow exceeded the total number found in all other lymphoid organs. The results are discussed in relation to the bone marrow PFC response to the thymus-dependent antigen sheep red blood cells. To this antigen a clear PFC response in the bone marrow is found only during the secondary response.

Evidence is presented which suggests that T cell-dependent nonspecific factor (NSF) cannot be the sole mediator of 'co-operation' for all types of thymus-dependent antibody response. NSF can replace the requirement for T cells in responses to hapten conjugated to particulate carriers, but cannot do so in responses to hapten conjugates of soluble thymus-dependent carriers.

Lymphocytes from mice immunized by painting with the contact sensitizing agent picryl chloride have been shown to produce regulatory effects on the DNA synthesis and contact sensitivity responses of normal mice painted with picryl chloride. This report describes the effect of these cells on antibody responses of normal mice to picryl chloride. Lymph node cells taken 5--7 days after painting increased early IgM antibody responses of normal mice to picryl chloride. Spleen cells were not effective. The increase was mediated by T cells as judged by anti-theta treatment and nylon wool filtration and could not be produced by killed, irradiated or allogeneic cells. A similar activity could be demonstrated in cells from mice painted with another contact sensitizing agent, oxazolone. The effect was strictly specific and cells from mice painted with picryl chloride or oxazolone would only increase responses to picryl chloride or oxazolone, respectively, even when presented with a mixture of the two antigens. The cel-s increasing antibody production were different from T cells previously shown to mediate contact sensitivity and T cells shown to regulate DNA synthesis in that they could not be generated in adult-thymectomized mice.

Production of IgA anti-hapten antibodies in vitro was studied in suspension and fragment cultures of spleens from mice immunized with NIP-CG (4-hydroxy-5-iodo-3-nitrophenylacetyl coupled to chicken globulin) a few weeks earlier. IgA anti-hapten production tended to be of much shorter duration than IgG anti-hapten production. IgA produced in fragment cultures had sedimentation coefficients of 9S, 11S, and 13S. 9S IgA anti-hapten was more prominent later during the in vitro response than earlier. In suspension cultures IgM anti-hapten production preceded IgA and IgG anti-hapten production. Very little IgM anti-hapten was seen in fragment cultures, although the responses on a cell-to-cell basis tended to be much higher in fragment than in suspension cultures.

Quantitative studies on B cells require a direct assay for antibody-forming cell (AFC) progenitor function, in which the number of AFC produced bears a simple, linear arithmetic relationship to the number of progenitors present. This might be expected under conditions where helper T-cell and accessory cell requirements are by-passed, or provided in excess. This possibility has been tested using as antigen the hapten NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid) on the carrier POL (polymerized bacterial flagellin), in adoptive transfer of normal and nude mouse spleen cells to irradiated recipients, and in cell culture. Primary and secondary IgM responses to this antigen are "T cell-independent'. The secondard IgG response is T cell-dependent but this function can be provided by 'carrier-primed' irradiated recipients. However in no case did the cell dose response curve show a linear, arithmetic relationship between cells transferred or cultured, and AFC produced. If less than 10 X 10(6) cells were adoptively transferred or cultured, a sigmoid curve was obtained, approximately linear with a slope of around 1-6 on a log-log scale. In adoptive transfer, a plateau was then seen above 10 X 10(6) cells, followed by a second sharp rise beginning around 15 X 10(6) cells. Addition of irradiated spleen cells as 'fillers' to maintain cell numbers constant produced a linear (arithmetic scale) dose response curve for the primary IgM responses, both adoptive and in culture. Lipopolysaccharide injection of recipients also produced linear regions in the adoptive transfer system. These techniques provide more direct, quantitative assay systems for the primary IgM responses to this antigen. However, arithmetic linear cell dose response curves were still not obtained for the secondary IgG responses, using irradiated filler cells.

The number of plaque-forming cells (PFC) per spleen is reduced in mice experimentally infected with 200 Trichinella larvae at 14 and 28 days after infection, whereas no difference is shown at days 7 and 56. The worms of the three different phases of the life cycle of Trichinella have been isolated and kept alive in vitro at 37 degrees in the inner compartment of a Marbrook chamber. The outer compartment of the chamber contained normal spleen cells and sheep erythrocytes. After 4 days, the spleen cells were removed and assayed for PFC. The results indicate that the new-born larvae are capable of producing substances which can diffuse through a Millipore filter and affect the spleen cells forming antibody to sheep erythrocytes. The suppression of antibody to sheep red blood cells in mice infected with Trichinella is a transitory phenomenon and can be related to the migrating phase of the life cycle of the parasite.

Foetal lambs were immunized orally 6-15 days before birth by introducing horse spleen ferritin into the amniotic fluid. Immunized and non-immunized lambs were killed at birth, usually before they had suckled, blood and intestinal contents were collected and single cell suspensions were prepared from spleen, mesenteric lymph nodes and jejunum. Specific antibody was detected in serum and intestinal contents of all immunized lambs which had not suckled. Specific antibody was usually not detected in samples from non-immunized lambs. In immunized lambs antibody activity in serum was associated with IgM and in intestinal contents with IgA and IgM. In agreement with these findings, the levels of IgM and IgA in serum and intestinal contents of immunized lambs were relatively high. Generally, immunoglobulins were not detected in samples from non-immunized lambs. Relatively high proportions of cells secreting specific antibody were present in the tissues of immunized but not non-immunized lambs. In the spleen most of the cells were secreting IgM antibody, in mesenteris lymph nodes IgM cells predominated and small numbers of IgA cells were detected, and in the jejunum approximately equal numbers of IgA and IgM cells were secreting specific antibody.

Supernatants were collected from keyhole limpet haemocyanin (KLH) primed spleen cells which had been incubated in tissue culture medium with their priming antigen KLH. These non-specific factor (NSF) containing supernatants were then tested in a microculture system for their ability to facilitate an anti-SRBC response of nu/nu or AT times BM spleen cells. It was concluded that:(a) NSF was able to engage a large number of the available pool of sheep erythrocyte (SRBC) specific B cells into an antibody response (b) this response was characterized by the development of clones expressing plaque-forming cells (PFC), the number of PFC produced within a clone being dependent upon the amount of NSF available in that culture; (c) NSF probably acted directly on B cells, and not via other accessory cell types.

Type III pneumococcal polysaccharide (SIII) is able markedly to increase the adoptive IgG ANTI-DNP antibody response of B cells primed to DNP-flagellin and stimulated with DNP conjugated to the heterologous carrier, rabbit globulin, provided the latter has anti-SIII activity. The stimulatory effect is apparently accessory cell-dependent as well as being unequivocally T cell-dependent. Although no positive evidence is available, the possibility exists that non-specific T-cell activation is involved in the stimulating effect of anti-SIII plus SIII.

Lymphocytes are heterogeneous with respect to their life-span. Typical B cells, bearing on their membranes immunoglobulin receptors, easily detectable by immunofluorescence, belong mainly to the long-lived population: this can be observed using combined autoradiography and immunofluorescence. However, when primed mice receive (-3H) thymidine before a boosting injection of tobacco mosaic virus (TMV), many plasma cells appearing in the spleen during the secondary response are labelled. In irradiated recipients repopulated with spleen cells from donors primed with TMV and injected with tritiated thymidine 2 hours before killing, the majority of plasms cells appearing in the spleen after antigen injection were labelled. If irradiated mice were repopulated simultaneously with spleen cells from donors primed with TMV and injected with (-3H) thymidine, and from donors primed with haemocyanin, most of the anti-TMV plasms cells were labelled, while most of the anti-haemocyanin plasma cells were unlabelled. These results allowed us to exclude non-specific reutilization of labelled thymidine as the main reason of our observations. It is concluded that either plasma cells derive from shortlived precursors or they receive material from a labelled cell able to co-operate specifically with plasma cell precursors.

Radioresistant T lymphocytes (RRTL) were derived from spleens of CBA mice after gamma-irradiation (2000 rad). RRTL comprise 2.2-3.5% of the total T-cell population. Adult thymectomy and treatment with ATS or cortisol do not affect the yield of RRTL. The Thy-1 antigen content on RRTL surfaces exceeds that on thymocytes and splenic T lymphocytes. 51Cr-labelled RRTL fail to home into lymph nodes: a considerable number of labelled RRTL persist in the blood. In an adoptive transfer system RRTL display poor helper activity. The adoptive response of normal B cells plus RRTL was reduced when RRTL donors were preimmunized with the same antigen. It is not clear if the unusual properties of RRTL were induced by irradiation or if they pre-existed.

In the present study we have investigated the biological effects on thymus lymphocytes resulting from Escherichia coli lipopolysaccharide (LPS) treatment in young adult mice. It has been established that LPS induces the following effects: (a) a dose-dependent reduction of thymus weight contemporaneous with a rise in the anti-LPS antibody response; (b) an increase of killer activity of thymus cells; (c) an enhancement of thymocytes helper activity; (d) a reduction of theta-positive cells in the thymus; (e) a cellular depletion in the thymus cortex. These data, indicating that LPS selects in the thymus a population of cells more efficient in expressing both killer and helper functions, are interpreted as caused by an increased rate of cortisol secretion induced by the LPS treatment.

The subcutaneous application of soluble antigen resulted in generation of helper T lymphocytes in the draining lymph nodes, tested 4 days after priming in the adoptive transfer system. Four days later a specific helper T-cell population was detected in the spleen. B-cell activity could be demonstrated 4-5 days after helper T-cell activity in both organs. We investigated the early helper T-cell population and memory T cells with respect to differences in the expression of antigen receptors. No such differences could be detected, either by anti-immunoglobulin-coated Degalan columns or by an antigen-specific rosetting method.

The distribution of plaque-forming cells (PFC) throughout the lymphoid system of CBA mice was followed with time after a primary intraperitoneal injection of hen egg white lysozyme emulsified in Freund's complete adjuvant (HEL-CFA) and after a secondary soluble injection. Throughout the primary response (predominantly IgG) and during the first week of the secondary response (exclusively IgG), the highest density of PFC was found in the draining parathymic lymph nodes, followed by the local spleen and mesenteric lymph nodes. The antibody-forming activity of the bone marrow increased as the immune response progressed, so that by the 3rd week of the secondary response this compartment provided the majority of the PFC. PFC first appeared in the accessory axillary, brachial or inguinal lymph nodes and in the thymus a few days after the secondary injection but accounted for only 1-5% of the total activity during the entire course of the secondary response. The specificity of the antibody produced in the spleen, parathymic and mesenteric lymph nodes was identical as judged by plaque inhibition by seven chemically related lysozymes which implies that these PFC were well mixed. It is postulated, therefore, that the change in distribution of PFC from an early local response to a general systemic response, and finally to a predominantly bone marrow response, was due to the migration of memory cells from the draining parathymic lymph nodes and spleen throughout the lymphoid system with an ultimate settling of the cells in the bone marrow.

The differentiation of Ig+ B cells into plaque-forming cells is dependent on antigen and factors produced by T cells and/or macrophages. We describe here the production of T-cell factors termed lymphocyte promotor factors (LPF). A foetal calf serum-specific T-cell line and its clones synthesize LPF, which is defined as factors that polyclonally stimulate normal spenic T cells to differentiate into cytotoxic T lymphocytes (T-LPF) and normal splenic B cells to differentiate into plaque-forming cells into (PFC) (B-LPF) in the apparent absence of specific antigen. The proliferation of and the B-LPF production of all T-cell clones tested were foetal calf serum-specific and I-Ab-restricted. Some of these clones produced only T-LPF, some clones produced only B-LPF, and some clones produced both T-LPF and B-LPF. B-LPF stimulate the polyclonal differentiation of Ig+ B cells into PFC without the apparent need for helper T cells, is different from T-LPF, and induces almost exclusively IgM PFC. The B-LPF described in the present paper are compared with previously described T-cell factors, which stimulate antigen-specific B-cell responses or bystander B-cell responses. The conclusion is that B-LPF are probably different from B-cell growth factors, T-cell replacing factors, allogeneic effector factors, and interleukin 2.

The light (L) and heavy (H) chain and the idiotypic (Id) composition of the antibody (Ab) plaque-forming cells (PFC) and serum Ab specific for alpha-1,3 dextran have been characterized in murine strains exhibiting the CH-Ig-lb to e allotypes and in their F1 hybrids with Ig-la1, BALB/c prototypes. The Ab response of the Ig-lb to e mice to the alpha-1,3 dextran was low in the kappa (kappa) L chain class with only a minor, sporadic Ab in the lambda (lambda) L chain class discernible after prolonged immunization. Two of a total of sixty-eight C57Bl/6 Ig-lb mice, in a total of 318 Ig-lb to e mice tested, exhibited, in late responses, a significantly elevated Ab in the lambda L class at both serum and PFC levels, equalling at the PFC level the total non-specific lambda PFC values. An Id analysis showed this lambda Ab and the kappa Ab to lack the Id relatedness to the three BALB/c alpha-1,3 dextran-binding myeloma proteins (MP) Ab prototypes, J-558, 104 E, and UPC-102, exhibited by the lambda Id+ Ab of the Ig-la1 BALB/c prototypes and their F1 hybrids with the Ig-lb to e prototypes. Furthermore, affinity differences could be detected by alpha-1,3 nigerodextrans, PFC inhibition analysis, between the late C57Bl/6 anti-alpha-1,3 dextran lambda Id--Ab PFC and the lambda Id+ Ab PFC of the BALB/c and their F1 progeny.

Lymphoid cells from unprimed high responder (C57BL/10) and low responder mice (B10.Br, B10.A, CBA) to (T,G)-A-L and high responder (B10, B10.A) and non-responder (B10.G, DBA/I) mice to GAT can be induced to form antigen specific T-helper cells in vitro under identical culture conditions. The helper cells induced from high and low or non-responder mice appear to be identical in efficiency, antigen concentration requirement for induction and induction kinetics.

Changes in the concentration and composition of serum immunoglobulin A (IgA) and deposits of IgA in tissues are well-known characteristics of alcoholic liver disease. We investigated whether these changes also accompany IgA synthesis by peripheral blood mononuclear cells (MNC), by counting immunoglobulin-producing cells using a solid-phase enzymatic 'spot' test, and by analysis of immunoglobulin content in lysed cells with culture supernatant using conventional enzymatic methods. Patients with alcoholic liver disease exhibited a significantly higher number of spontaneously IgA-producing cells than did normal healthy controls (1.7 X 10(6) cells/blood and 0.5 X 10(6) cells/blood, respectively, P less than 0.01). The IgA content of MNC directly after isolation was also higher (38 and 13 ng/10(6) MNC, respectively, P less than 0.01), as was the IgA production during an unstimulated 6-day culture period (520 and 95 ng/10(6) MNC put into culture, respectively, P less than 0.001). The spontaneously IgA-producing cells assessed directly after isolation of mononuclear cells correlated with the IgA production during an unstimulated culture (P less than 0.01). We conclude that in alcoholic liver disease, B lymphocytes circulate which may have been activated in vivo.

The relationship between T cells involved in cell-mediated immunity and antibody response of C57Bl/6J mice towards DBA/2 mastocytoma cells was investigated. Spleen cells primed with viable mastocytoma cells demonstrated marked cell-mediated cytotoxicity (CMC) in vitro, but antibody response of these cells to a hapten (TNP) conjugated to the allogeneic tumour cells in vitro was suppressed as compared with that of normal spleen cells. In contrast, spleen cells primed with frozen-thawed mastocytoma cells showed no CMC, but antibody response to the hapten in vitro of these cells was enhanced. C57Bl/6J mice primed with frozen-thawed mastocytoma cells produced more cytotoxic antibody than non-primed mice when immunized with viable mastocytoma cells. These results indicate that T cells involved in cell-mediated immunity and those involved in the antibody response to allogeneic mastocytoma cells are distinct.?222

The mitogenicity, immunogenicity and tolerogenicity of various DNP-levan (DNP-LE) conjugates were compared using in vitro methods. Anti-DNP antibody synthesis induced by DNP-LE conjugates was related to the epitope density of DNP, BUT WAS NOT AFFECTED BY Macrophage dependent and was not influenced by the degree of hapten conjugation. These results imply that mitogenicity of an antigen is not necessarily related to the specific triggering of B cells.

The production of antibody by human colostral cells was assayed by the hemolysis in-gel technique. When sheep erythrocytes coated with O antigens from frequently encountered Escherichia coli bacteria were used as detector cells and anti-IgA serum was added for development, numerous plaque-forming cells (PFC) were demonstrated in all samples tested. In contrast, plaques were rarely seen in the presence of anti-IgG developing serum. The direct (IgM) plaques occasionally noted with both antigen-coated and uncoated sheep erythrocytes were mainly due to the production of heterophil antibodies, since they were not formed when human erythrocytes were used as O-antigen carriers. A strikingly high number of the colostral lymphocytes formed antibodies to the E. coli antigens, up to 8%. This suggests that these cells represent a rather selective population--possibly cells from the gastrointestinal tract exposed to enteric bacteria. The large number of plaques observed, the predominance of the cells forming IgA antibodies, and the marked changes in PFC number in relationship to parturition pose a number of questions relevant to the antibody-producing colostrum cells and their relationship to the secretory immune system.

The synthesis of DNA, as measured by the incorporation of [3H]-thymidine into the nuclei of cells of cultured explants of rabbit spleen, has been studied and correlated with estimation of DNA content of these nuclei using Feulgen densitometry, with and without colcemid arrest. Observations of the proportion of nuclei labelled with [3H]thymidine and their DNA content indicated that many cells were incorporating this precursor for DNA synthesis without the concomitant increases in DNA expected of cells in division cycle. Colcemid arrest failed to produce the changes in DNA content expected of a cell population as rapidly dividing as the labelling kinetics suggested. The results indicated that [3H]thymidine-labelled material, possibly DNA, was being transferred between cells and this accounted for the increased labelling index seen after pulsing with [3H]thymidine. The suppression of [3H]thymidine uptake by hydroxyurea indicated that this was not repair synthesis of damaged DNA.