T cell tolerization in vitro: modulation of helper and suppressor cell activities.

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.

Xid mouse lymphocytes respond to TI-2 antigens when co-stimulated by TI-1 antigens or lymphokines.

Spleen cells from male (CBA/N x DBA/2) F1 hybrid mice do not significantly respond to in vitro stimulation by trinitrophenyl-conjugated polyacrylamide beads (TNP-PAA), whereas the same antigen elicits high PFC responses in female F1 hybrid cells. Therefore, this antigen could be classified as a T-independent type 2 (TI-2) antigen. When male spleen cells were co-stimulated by TNP-PAA and TI type 1 antigen, either LPS or Brucella abortus, they produced vigorous anti-TNP responses. A similar increase of the in vitro responsiveness of male F1 hybrid spleen cells to TNP-PAA antigen was provoked by the addition of supernatants from P 388-D1 cells stimulated by muramyl-dipeptide (MDP) mainly containing interleukin-1 (IL-1) or supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated EL-4 cells that contained T-cell factors. The PFC response to another TI-2 antigen, TNP-Ficoll, was also significantly enhanced after co-stimulation by P 388-D1 supernatants. The response to TI-2 antigens being macrophage dependent, the influence of supernatants of peritoneal macrophages from male and female F1 hybrids incubated with TNP-Ficoll on the PFC response of normal DBA/2 mouse spleen cells to sheep erythrocytes was assessed. It was found that macrophage supernatants from female hybrids regularly increased by more than two times this anti-SRBC PFC response, whereas macrophage supernatants from male F1 hybrids did not. Moreover, in a specific proliferation test measuring IL-1 activity, when macrophage supernatants from female F1 produced a 13-fold increase of thymidine incorporation, supernatants from male F1 only produced a three-fold increase. It is concluded that, in addition to the known defects of B cells from Xid mice, their macrophages are also defective.

Simultaneous responses to TD, TI-1 and TI-2 antigens originate from both specific and multipotent precursors.

The classification of antigens into TD, TI-1 and TI-2 varieties raises the question of whether responses to these antigens are produced by distinct or identical subpopulations of B cells. In the present study we have examined the extent of intraclonal specificity variation in the progeny of PFC appearing after stimulation with two unrelated antigens. Mouse lymphoid cells were stimulated with pairs of TD and TI antigens, PFC were individually cultured and daughter PFC examined for their specificity. In all combinations used, PFC responding to TD antigen engendered, after 48 h of culture, a high frequency of PFC daughters expressing one or the other antibody specificity, notwithstanding the specificity of parental PFC. However, PFC responding to TI antigens seemed less subject to variation in specificity, and PFC daughters engendered after a 48 h culture period were, in the majority, of the parental specificity. These results are analysed in relation to different subpopulations of B cells.

Modulation of helper T-cell activity by macrophage products.

Mouse spleen cells cultured with muramyl dipeptide (MDP) release factors (SMF, or splenic macrophage factors) which enhance the in vitro plaque-forming cell (PFC) response to the T-dependent antigen SRBC (sheep red blood cells). The present study substantiates previous results and shows that T cells were the target cells of such conditioned medium. We investigated which T-cell subpopulations had their activities modified by these factors. In the presence of SMF, T cells educated in vivo elicited enhanced PFC responses to both specific educating antigen and an unrelated antigen (during a bystander response). This could indicate that SMF potentiates Th2-cell activity. Moreover, in vivo matured but unprimed thymocytes, which are unable to cooperate with virgin B cells in an in vitro T-dependent response, are able to do so when SMF is added in cooperative cultures. It is concluded that MDP-conditioned medium allows the maturation/differentiation of a subset of T cells to the point where they can be stimulated by the antigen.

Selection of BW 5147 subclones devoid of non-specific suppressive activity for use in cell hybridization.

Culture supernatants of BW 5147 cells widely used for T-cell hybridization often manifest non-MHC-restricted, non-antigen-specific regulatory activities on the mixed lymphocyte reaction (MLR) of mouse cells. This report demonstrates that, whereas supernatants of BW 5147 cells grown at low concentrations (2 X 10(5)/ml) enhanced MLR, high cell concentration (2 X 10(6)/ml) supernatants markedly inhibited this reaction. BW 5147 cell-free extracts significantly inhibited MLR and in vitro antibody production (PFC), as well as the mitogenic response to lipopolysaccharide E. coli (LPS) of mouse spleen cells, but did not affect the response to an optimal dose of phytohaemagglutinin (PHA). Both supernatant and cell-free extract inhibitory activities were located in 60,000 MW fraction. Inhibitory material of low MW (less than 12,000) was also found in high cell concentration supernatants. A similar suppressive activity was exerted by cell-free extracts of P3 X 63 NS cells used for B-cell hybridization. The suppressive activity seemed to stem from some kind of interaction between BW 5147 cells and the fetal calf serum (FCS) of the culture medium. Supernatants from subclones of BW 5147 cells obtained in selected batches of FCS and maintained in the same serum, even at high cell concentrations, did not affect MLR, whereas the supernatants from the same subclones maintained in other batches definitely suppressed this reaction. Thus, provided that culture conditions are chosen carefully, subclones of BW 5147 devoid of effect on in vitro immune reactions can be obtained.

Activation of the human classical complement pathway by a mouse monoclonal hybrid IgG1-2a monovalent anti-TNP antibody bound to TNP-conjugated cells.

A mouse hybridoma selected and cloned for anti-TNP specificity produced three distinct monoclonal antibody species that were separated on protein A-Sepharose by stepwise acid elution. The IgG1 kappa product of the parental myeloma was eluted at pH 6.0. An IgG2a kappa bivalent anti-TNP antibody was eluted at pH 4.5, whereas elution at pH 5.0 yielded a hybrid IgG1-2a kappa monovalent anti-TNP antibody. The IgG2a molecules agglutinated TNP-conjugated sheep erythrocytes (TNP-ES) and lysed TNP-ES in the presence of normal human serum (NHS). Hybrid IgG1-2a antibody was also capable of lysing the cells in NHS, although it did not agglutinate TNP-ES. A threshold in monovalent antibody input was necessary for the lysis of TNP-ES, indicating a requirement for a minimal density of bound monovalent IgG to trigger complement activation. Lysis occurred in NHS-VBS++ but not in NHS-MgEGTA, and it was associated with a dose-dependent consumption of C1, C4, and C2 hemolytic activities. Quantitation of the antibody bound to TNP-ES when using radiolabeled rabbit anti-mouse Fab antibody demonstrated that for similar inputs, 5.4 times as much bivalent as monovalent antibody bound to TNP-ES. When similar amounts of antibody were effectively bound to TNP-ES, monovalent hybrid IgG1-2a was five times less efficient than bivalent IgG2a to yield 50% cell lysis in the presence of NHS. These results indicate that neither bivalent binding nor the presence of two identical heavy chains are necessary requirements for antibody-dependent activation of the classical complement pathway.

[Recovery of the in vitro reactivity of splenic cells of CBA/N mice toward type 2 thymus-independent antigens]. / Restauration de la réactivité des cellules spléniques des souris CBA/N in vitro envers des antigènes thymo-indépendants du type 2 (TI-2).

CBA/N mice bearing a chromosome X linked immunological deficiency (Xid) cannot respond to type 2 thymus independent antigens (TI-2). However, when their spleen cells are in vitro simultaneously stimulated by both a TI-2 (Fluorescein conjugated polyacrylamide, Flu-PAA) antigen and a type 1 thymus independent (Trinitrophenyl conjugated Brucella abortus, TNP-BA) antigen, their capacity to respond to the TI-2 antigen is recovered. On the contrary, thymus dependent (TD) Sheep red blood cells (SRBC) antigen did not produce any significant increase of the anti-TI-2 response.

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin.

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.

Human myeloma light chains with increased molecular weight: high frequency among lambda chains.

The discovery of a human myeloma protein comprising a kappa L-chain with an increased mol. wt of 30,000) (Bouvet et. al., 1980) prompted investigations on the incidence of such heavier L-chains among other human myeloma proteins. In 105 samples examined, 34 were found to have L-chains heavier than normal (23,000-24,000), ranging from 25,000 up to 31,000, and five of lighter mol. wt (21,000-22,000). These mol. wt abnormalities were detected by electrophoresis in sodium dodecyl sulfate 10% polyacrylamide gels (SDS-PAGE) after reduction with 2-mercaptoethanol. The mol. wt of three of the heavier kappa or lambda chains was also estimated by filtration through a Sephadex G100 column and by sedimentation equilibrium. All three methods indicated a mol. wt increase of about 15-25% as compared with the usual mol. wt. The distribution of the high mol. wt chains among all L-chains examined was found to be 11 out of 62 kappa chains (17.7%) and 23 out of 43 lambda chains (53%) (P less than 0.001). A preferential association of such L-chains with H-chains producing multiple bands in SDS-PAGE (P less than 0.01) and an association between multiple L-chain and multiple H-chain band (P less than 0.05) were also observed. In contrast, no abnormal L-chain was found in immunoglobulins from normal subjects. Spontaneous degradation of the normal H-chains sometimes yielded fragments of 30,000 mol. wt. These fragments were easily distinguishable from abnormal L-chains. The nature of extra mol. wt in heavy L-chains was investigated for the presence of carbohydrate moiety. Four large and three normal size L-chains were examined for amino-sugar and sialic acid content. A small amount (one residue per molecule) of amino-sugar was detected only in two normal and two heavy L-chains, whereas sialic acid was only found in the heaviest (27,000-30,000) L-chains (Lh) and in small percentage (one or two residues per molecule). Total sugar estimation in one Lh chain indicated a proportion not exceeding three or four residues per L-chain (mol. wt 1,000) and this is insufficient to explain the 15-25% (3,600-6,000) mol. wt increase. It is therefore possible that, at least in some heavy myeloma L-chains, an additional peptide is expressed. Whatever the nature of the increase it would be of interest to elucidate whether this is a marker of malignant process or of an intermediate step of normal Ig synthesis.

Specification of the immune response: its suppression induced by chloramphenicol in vitro.

Specific in vitro PFC responses to trinitrophenyl conjugated to sheep red blood cells are inhibited by chloramphenicol (CAP), thiamphenicol (TAP), and diuron (DIU) by B-cell impairment. However, both mitogenic and polyclonal response to lipopolysaccharide is not affected by CAP and TAP but severely inhibited by DIU. Similarly, contact of cultured cells for the first 24 h with CAP did not much affect the 4th-day in vitro PFC response but the same incubation with DIU reduced it by 70%. Moreover, DIU used at the same concentration provoked an important mortality of cultured cells. These differences suggest that the target mechanism of CAP and TAP differs from that of DIU.

Three M components IgA lambda, + IgG kappa n + IgG kappa h in one patient. II. Structurally different kappa n and kappa h chains associated with H(gamma) chains sharing idiotypic determinants.

Structural analysis of purified IgG kappa h and IgG kappa n molecules of DA myeloma paraproteins indicated that the two IgG had different electrophoretic mobility and that L-kappa h had an unusually heavy m.w. (30,000). Peptide mapping showed the existence of additional peptides in the L-kappa h map when compared to the L-kappa n map. Total amino acid analysis showed that L-kappa h contained two additional cysteine residues and certain other amino acids than L-kappa n contained. Sequence of the first 25 NH2-terminal amino acids showed differences at positions 4, 5, 15, 18, and 21, but both L-kappa h and L-kappa n belong to the same V kappa IV subgroup. IgG kappa n but not IgG kappa h reacted with anti-gamma 1 antiserum, indicating that H chains of these two paraproteins were also different. Sera from rabbits immunized with IgG kappa n or IgG kappa h, and rendered specific for idiotypic determinants by appropriate absorption, were used for idiotype characterization of these components. Immunodiffusion, immunoelectrophoresis, and direct hemagglutination demonstrated that IgG kappa h and IgG kappa n cross-reacted partially. Inhibition tests disclosed that the main anti-idiotypic antibody was directed against a conformation structure of the complete IgG kappa h molecule, whereas cross-reaction was due to partial idiotypic similarity between H chains of the IgG kappa h and IgG kappa n. In addition, each of these paraproteins seemed also to bear private idiotypes on their H and L chains.

Neutralizing monoclonal antibody specific for Naja nigricollis toxin alpha: preparation, characterization, and localization of the antigenic binding site.

One homogeneous population of high-affinity monoclonal antibodies (KD = 0.35 nM) specific for Naja nigricollis toxin alpha has been produced. It neutralizes the biological activity of the toxin under both the vivo and in vitro conditions. The molecular zone of the toxin to which the antibody binds has been precisely defined on the basis of cross-reaction experiments using five derivative of toxin alpha monomodified at a single amino group and two naturally occurring homologous toxins. The epitope is located at the base of the first beta-sheet loop of the toxin, involving the two positive charges at the N-terminal position and lysine-15 proline-18, and probably threonine-16. It is shown that this region is topographically distinct from the "toxic" site of toxin alpha. Several possibilities are offered to explain the mechanisms(s) of specific neutralization.

Enhancement of Lewis lung carcinoma growth by sera or spleen cells from tumour-bearing mice.

Lewis lung carcinoma(3LL) cells grafted in syngeneic adult C57BL/6 mice produced local tumours associated with lung metastasis in 78% of recipients. Adult thymectomized, lethally irradiated, bone marrow cell-reconstituted animals (B mice) were more resistant since this tumour grew in only 46% of recipients and especially the weight of lung metastasis was almost 8 times less than in normal animals. Sera from tumour-bearing mice transferred into B mice enhanced tumour incidence and weight of metastasis to the level observed in normal mice. In vitro cultured 3LL cells displayed an intense mitogenic activity as measured by 3H-thymidine incorporation. Addition into these cultures of serum from tumour-bearing animals did not alter this activity. Addition of normal spleen cells in a ratio 40/1 reduced this mitogenic activity to a half or a third. Spleen cells from tumour-bearing mice, either normal or serum-enhanced B mice, mixed in vitro with 3LL cells, produced a 4-fold increase of mitogenic activity of the latter. These results indicate that the lymphoid system may contribute to the growth and spreading of 3LL tumours.

Non-specific inhibitory processes of immunological and mitogenic cellular responses.

The suppressive effects of four agents on several types of in vitro immune response and on in vitro responses to T and B mitogens were studied comparatively in spleen cells from C57B1/6 mice, previously injected with each of these agents. It was found that the in vitro PFC response to sheep erythrocytes was the most constantly and extensively inhibited: from 56% after treatment with Con A to 85% after treatment with HSA; LPS and C. parvum also provoked a strong inhibition (74-78%). Inhibition of MLC was constant but less effective, ranging from 36% (LPS) to 57% (C. parvum); HSA and Con A depressed it by about 50%. The CML reaction was substantially inhibited by C. parvum (50%), moderately by Con A and LPS (respectively, 23 and 28%) and slightly by HSA (15%). The inhibition of mitogenic response to PHA and LPS varied widely with the agent used: the PHA response was strongly inhibited by Con A (84%) to a lesser extent by C. parvum (54%) and even less by LPS (27%), whereas HSA did not affect it. LPS reactivity was well inhibited by C. parvum (57%), moderately by Con A (25%), slightly by HSA and not at all by LPS. Most of these agents produced a slight but significant prolongation of skin graft survival time. In vitro experiments using mixtures of spleen cells from treated and normal animals showed that these inhibitory effects were mediated by suppressive cells that developed as a result of the treatment used. The degree of inhibition observed in the mixed cultures satisfactorily paralleled the direct inhibition observed in cells from treated animals. The more consistently suppressive agents seemed to be C. parvum and Con A, the effects of the other two (HSA and LPS) on the cellular responses studied were less regular.

Three M-components IgA lambda + IgG kappa n + IgG kappa h in one patient (DA): lack of shared idiotypic determinants between IgA and IgG, and the presence of an unusual kappa h chain of 30,000 M.W.

Two apparently homogeneous electrophoretic bands were found in the serum of a patient (DA) with multiple myeloma. These M-components were identified as IgA-lambda and IgG-kappa paraproteins bearing different idiotypic determinants. Further analysis of the L chains showed that the lambda-chain was homogeneous but the kappa-chain could be separated by SDS-polyacrylamide gel electrophoresis into two different bands. Both of them were associated with gamma-chains but one (termed kappa n) had normal m.w. (24,500) whereas the other (termed kappa h) was larger (m.w. 30,000). Sugar content of the two DA IgG, as determined by anthrone reaction, was similar in DA IgG kappa n (0.73%) and in DA IgG kappa h (1.1%), clearly demonstrating that the difference in m.w. was not due to a large sugar chain. Furthermore, the peptide map of the kappa h chain included nine peptides absent in those of four other control kappa-chains. Sequence analysis showed that the first 25 N-terminal amino acids of the kappa n differed from those of the kappa h chain at positions 4, 5, 15, 18, and 21. Thus the two kappa-chains had different framework regions.