Spontaneous internalization of Class I major histocompatibility complex molecules in T lymphoid cells.

A low proportion of T lymphocytes in normal mouse spleen contains small intracytoplasmic vesicles showing Class I MHC molecules. After stimulation in vitro in a mixed lymphocyte reaction or by addition of Con A, the proportion of T cells with such intracytoplasmic vesicles increases progressively and becomes the majority. Labeling with fluorochrome-conjugated antibodies has shown that the vesicles are formed by internalization of molecules from the plasma membrane. The process is spontaneous and does not require cross-linking by antibodies or other ligands; it is selective inasmuch as other molecules (Thy-1 and T200 antigens) are not included and it is specific since it is not performed by other cells such as B lymphoid cells or fibroblasts. On the whole the process shows similarities with the internalization and recycling of other receptors, such as the receptors for different macromolecules of metabolic or informational significance, as seen in other cells. On the other hand, the specificity of Class I MHC mobilization in T lymphoid cells suggest a role for this process which is related to the immune function of these molecules.

Immunoglobulin spots on the surface of rabbit lymphocytes.

Small and medium lymphocytes from the peripheral blood and lymphoid tissues of the rabbit react in suspension with antibodies directed against different immunoglobulin determinants. Through immunofluorescence, it was possible to show that numerous discrete spots on the surface of the positive lymphocytes carry immunoglobulin molecules. The positive lymphocytes are about one-half of all lymphocytes in the different preparations; thymus lymphocytes are all negative. With antisera specific for rabbit IgM as well as with antisera directed against allotypic determinants specific for IgM or IgG, it was possible to show that about nine-tenths of the immunoglobulin-positive lymphocytes carry IgM molecules on their surface. With antisera directed against a- and b-locus determinants, it was also possible to demonstrate that both heavy and light chains were present in the surface immunoglobulins. Furthermore, in animals which were heterozygous at the a or the b locus, it was found that each lymphocyte had immunoglobulins synthesized under the influence of only one of two alleles. A very small proportion of lymphocytes could be shown to have a specific surface reaction with one antigen (horse ferritin); the proportion of these cells increased very much after immunization.

Blocking and redistribution ("capping") of antigen receptors on T and B lymphocytes by anti-immunoglobulin antibody.

Antigen-binding T and B lymphocytes were studied by combined autoradiography and immunofluorescence; mouse spleen lymphocytes binding the antigens, [(125)I]MSH or [(125)I]TIGAL, were incubated with rhodamine-labeled anti-Ig reagents or with a rhodamine-labeled IgG fraction of anti-theta serum. B cells were identified as Ig+ or theta-, T cells as Ig- or theta+. It was found that: (a) 20% (1-2 mo after priming) to 30% (3.5-4 mo after priming) of the antigen-binding cells were T cells. (b) The range of antigen molecules bound by B and T cells was similar. (c) Binding of antigen to B and T cells was inhibited by polyvalent anti-Ig, anti-micro, or anti-L reagents. Binding to T cells was more readily inhibited than to B cells. Normal rabbit serum, antimouse lymphocyte serum, or anti-theta did not inhibit antigen binding. (d) When Ig at the surface of B cells was induced, by noninhibiting concentrations of anti-Ig reagents, to redistribute into polar caps and the cells subsequently exposed to [(125)I)antigen under noncapping conditions, the [(125)I]antigen silver grains were distributed in caps superimposed on the Ig fluorescent cap. Of crucial importance, antigen was found in cap in the same proportion of T cells as B cells. Significant capping of antigen receptors was not induced in B or T cells with normal rabbit serum or by anti-Ig reagents absorbed with mouse Ig. The main conclusions of this series of experiments using direct visualization of antigen-binding B and T lymphocytes is that T cells have antigen-specific receptors, probably of IgM nature, and that the number of these receptors appears to range in the order of thousands.

A new HLA-linked T cell membrane molecule, related to the beta chain of the clonotypic receptor, is associated with T3.

The 38 kD molecule is noncovalently associated with beta 2 microglobulin (beta 2m)-free HLA heavy chain-like molecule, and thus forms a second heterodimer distinct from the clonotypic alpha/beta T cell receptor expressed by the same clone of leukemia cells. This second heterodimer (38 kD/HLA) is variably expressed and appears to be associated with the T3 molecule. We suggest, therefore, that it has a functional role in T cell activation.

Induction of an antibody response in cultures of human peripheral blood lymphocytes.

A culture system is descirbed which provides adequate conditions for in vitro immunization of humand peripheral blood lymphocytes to heterologous erythrocytes. Making use of this method we could obtain, with a number of different donors, an antibody response which peaked at about day 8 of culture with 30-300 plaque-forming cells (PFC) per 10(6) input lymphocytes. However, in a number of experiments poor or negative results were obtained, even with donors that had previously given good response. This variability in the results was shown not to be due to a too low number of precursor cells present in the blood and could be overcome by treating the cells, before initiation of the culture, with a factor produced by mouse T cells educated to sheep erythrocytes (SRBC). Under these conditions a PFC responce was obtained which peaked at about day 8 and which in some experiments could be as high as 20,000 PFC per 10(6) input lymphocytes. Paralleling the increase in PFC was an increase in cell number. The cells recovered from the treated cultures were at all times more numerous than in the nontreated cultures. The height of both the proliferative and antibody-producing responses varied from experiment to experiment, a higher proliferative response, accompanying a higher PFC response. Although the mechanisms that are at the basis of the antibody response in vitro described in this paper still need to be clarified, this system may become a useful tool in studying the immune response in man.

Immunoglobulin D as a lymphocyte receptor.

A large proportion of the human peripheral blood lymphocytes of adults and newborns having IgD were found also to have IgM on their membranes and vice versa. A few lymphocytes had one of these classes only. IgD and IgM could be capped independently on the same cell. The possibility that IgD was acquired by a cytophilic process was excluded by the finding that IgD-bearing cells were of one light chain type only, and by the direct demonstration of reappearance of IgD on the lymphocyte membrane during incubation in an IgD-free culture medium. On the basis of these findings, it is proposed that IgD functions as a lymphocyte antigen receptor.

Specific binding of alloantigens to T cells activated in the mixed lymphocyte reaction.

Immunoglobulin (Ig) is present on a large fraction of T cells from unfractionated lymphocytes activated by in vitro stimulation with H-2-incompatible cells (mixed lymphocyte reaction [MLR]). Removal of bursa equivalent-derived (B) cells from the responder cell population before mixed culture, by filtration through nylon wool columns, reduces the percentage of Ig-bearing responder T blasts to background levels. Thus, Ig on the T blast is probably of B cell origin. A large fraction of T blasts activated against the stimulator cells. This staining occurs with "early" and hyperimmune alloantisera, including the 7S fraction of the latter. B-depleted responder cells were activated against a mixture of two different stimulator cells and the resulting T blasts stained with different concentrations of sera directed either against one or both stimulator cells. We obtained results which strongly suggest that most or all responder T blasts stain with only one antistimulator serum. When antisera directed against different segments of the H-2 complex of the stimulator cells were used, it seemed that most responder T cells only bound antibody directed against a single segment. We propose that T cells activated in MLR carry stimulator alloantigens on their surface, and that this is due to specific antigen binding, not requiring the presence of B-cell-derived antibody. These histocompatibility antigen-binding T blasts can be detected by appropriate antistimulator alloantibodies.

Physiology of IgD. IV. Enhancement of antibody production in mice bearing IgD-secreting plasmacytomas.

Immune responses to trinitrophenylated hemocyanin (TNP-KLH), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-BA) were examined in BALB/c mice bearing subcutaneous transplants of TEPC-1017 and TEPC-1033, the two known IgD-secreting BALB/c plasmacytomas. Both primary and secondary 19S and 7S splenic plaque-forming cell (PFC) responses in spleen to intravenously injected TNP-KLH were enhanced three to fivefold. Primary responses to TNP-Ficoll were 1.5-2 times higher than in control mice (particularly the 7S PFC response). Primary responses to TNP-BA were enhanced by TEPC-1017 but suppressed by TEPC-1033, while secondary responses to TNP-BA were enhanced three to sevenfold by both tumors. Intraperitoneal injections of ascites fluid from mice bearing TEPC-1017 or TEPC-1033, or of IgD isolated from such ascites fluid, caused a similar enhancement of the primary response to TNP-KLH, as did the tumor itself, particularly when injected approximately 1 wk before antigen injection. IgD-containing ascites fluid had no effect on the response of athymic (nu/nu) BALB/c mice to TNP-KLH. These findings suggest the existence of an IgD-responsive immunoregulatory T cell.

IgM-producing tumors in the BALB-c mouse: a model for B-cell maturation.

Five adjuvant induced BALB/c tumors producing IgM-McPc 1748, W 3469, TEPC 183, McPc 774, and Y 5781-were characterized morphologically by electron microscopy, analysis of the distribution of surface-bound and intracytoplasmic IgM using immunofluorescence, and by biochemical study of IgM synthesis, turnover, and secretion. The cells of different tumors appear to represent different stages in B-cell maturation when compared to normal, lipopolysaccharide-stimulated B cells. Thus, McPc 1748 tumor cells resemble 10-25-h stimulated normal B cells, 3469 cells resemble 20-35-h stimulated B cells, TEPC 183 cells resemble 45-65-h stimulated B cells, Y 5781 cells resemble 80-110-h stimulated B cells, and McPc 774 cells resemble 100-130-h stimulated B cells.

Physiology of IgD. I. Compensatory phenomena in B lymphocyte activation in mice treated with anti-IgD antibodies.

The role of delta-positive cells in the immune response was studied by comparing the effects of treatment with allotype-specific IgD hybridoma antibody on homozygous BALB/c or SJL/J and heterozygous (BALB x SJL)F1 mice. Homozygous mice, injected from birth with the relevant anti-delta antibody, made primary or secondary immune responses to intravenously injected trinitrophenyl (TNP)-Brucella abortus, TNP-Ficoll, and TNP-keyhole limpet hemocyanin, which did not differ significantly from those of control mice, despite the fact that IgD+ cells were depleted and Ig+ cells were markedly reduced in the spleens of treated mice. Responses in nodes draining a local injection of TNP-Brucella abortus were, however, significantly suppressed. Heterozygous mice, injected from birth with either anti-Ig-5a or anti-Ig-5b, showed a marked reduction in the number cells producing IgG antibody of linked allotype specificity in the secondary response to intravenously injected sheep erythrocytes. A corresponding decrease in the amount of serum IgG2a of that allotype specificity was also noted. However, in agreement with the results obtained in homozygotes, heterozygotes injected simultaneously with anti-IgD directed against each of the allotypes made normal, if not enhanced, plaque-forming cell responses of both allotype specificities. Similarly, serum IgG2a levels were normal in all but one mouse treated in this fashion. These results indicate that IgD+ cells are not essential for an immune response in vivo. Although the delta-positive cell is used preferentially under normal conditions, it appears that an alternative mechanism exists by which, in the absence of these cells, the animal is able to make a normal immune response.

Role of CD8+ T cells in murine experimental allergic encephalomyelitis.

The course of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis, is affected by immunoregulatory T lymphocytes. When animals are immunized with encephalitogenic peptide of myelin basic protein and recover from the first episode of EAE, they become resistant to a second induction of this disease. Animals depleted of CD8+ T cells by antibody-mediated clearance were used to examine the role of CD8+ T cells in EAE. These cells were found to be major participants in the resistance to a second induction of EAE but were not essential for spontaneous recovery from the first episode of the disease.

Intracellular accumulation of T-cell receptor complex molecules in a human T-cell line.

This work was aimed at understanding the mechanisms of T-lymphocyte function by studying the cellular distribution and traffic of molecules of the T-cell receptor complex. The accumulation of specific molecules in intracytoplasmic vesicles is related to the activation of T lymphocytes. Some of these molecules include acid hydrolases, the transferrin receptor, and class I antigens of the major histocompatibility complex. Molecules of the T-cell receptor complex have now also been found in intracytoplasmic vesicles in a human T-cell line derived from a lymphoblastic leukemia. Such vesicles were tightly associated with the cytoplasmic microtubule network. One functional aspect of this association is a cellular pathway by which vesicles traveling to and from the cell surface converge in an area of the cells that is rich in processing enzymes.

Monomeric (7S) IgM found in the serum of rheumatoid arthritis patients share idiotypes with pentameric (19S) monoclonal rheumatoid factors.

Serum from some seropositive (RF+) rheumatoid arthritis (RA) patients contains relatively high concentrations of monomeric (7S) IgM molecules. Seven S IgM molecules fail to bind the Fc portion of IgG, unlike 19S IgM RFs that bind aggregated IgG in classical RF assays. Some pentameric IgM RFs are marked by crossreactive idiotypes (RCRI) defined by prototypic monoclonal RFs. In previous studies, we observed that a proportion of pokeweed mitogen (PWM) induced plasma cells from RA patients' blood lymphocytes express the major RCRI as assayed by indirect immunofluorescence with polyclonal anti-RCRI antibodies. In this study, 7S IgM obtained from three different RF+ RA patients inhibits specific anti-RCRI intracytoplasmic staining of PWM induced RF+ RA-derived plasma cells. These 7S molecules also block polyclonal anti-RCRI antibodies from reacting with red blood cells bearing 7S IgM molecules from RF+ patients with RA or Waldenstrom's macroglobulinemia. We conclude that some 7S IgM molecules in the serum of RF+ RA patients are marked by the major RCRI idiotype and are related to 19S monoclonal and polyclonal RFs.

Cellular localization of rheumatoid factor idiotypes.

the stimulation of lymphocytes from rheumatoid arthritis patients with pokeweed mitogen produces a large number of plasma cells that express the dominant cross-reactive idiotype previously found on monoclonal IgM anti-gamma-globulins from patients with mixed cryoglobulinemia. Similar experiments with the cells of normal individuals show a much lower percentage of these cells with a lower intensity of staining with the fluorescent reagents utilized. Efforts to demonstrate rheumatoid factor in the same cells by fluorescent staining with aggregated gammaglobulin were entirely unsuccessful. This also proved to be the case for pokeweed mitogen-stimulated cells from the mixed cryoglobulinemic patients with large amounts of rheumatoid factor in the serum, despite high percentages of cells expressing the cross-reactive idiotype and also the individual idiotype. On the other hand, native plasma cells from synovial tissue of rheumatoid arthritis patients showed some cells with both the cross-reactive idiotype and aggregate staining. The exact reason for the failure to demonstrate rheumatoid factor by aggregate staining in pokeweed mitogen-stimulated cultures remains to be determined despite considerable effort to resolve the problem. The most likely possibility is that these plasma cells are relatively immature and have not accumulated polymeric IgM in their cytoplasm to the degree seen in synovial tissue plasma cells. The monomeric forms are readily recognized by the antiidiotypic antibodies and these reagents appear to be of particular value for cellular studies of this type.

Spontaneous in vitro differentiation of antigen-specific lymphocytes from a patient with immunoglobulin M gammopathy.

Recently we have identified two monoclonal immunoglobulin M (IgM) proteins that bind Klebsiella polysaccharides. The lymphocytes of one of these patients (M.A.Y.) were available for study. A substantial proportion of the B lymphocytes isolated from this patient's peripheral blood also bound Klebsiella polysaccharides with a pattern of specificity identical to that of the monoclonal IgM, and reacted with an anti-idiotypic antiserum directed against this IgM. Stripping the surface immunoglobulin from these lymphocytes eliminated this reactivity. Although no plasma cells were detected in the freshly isolated peripheral blood lymphocytes of this patient, plasma cells binding Klebsiella polysaccharide appeared after 7 d of in vitro culture. This occurred regardless of whether the cultures were supplemented with autologous plasma, normal human plasma, or fetal calf serum. Pokeweed mitogen neither stimulated nor inhibited the in vitro differentiation of the monoclonal B lymphocytes into plasma cells. This differentiation was, however, abrogated by F(ab')2 fragments of anti-human IgM and by anti-idiotypic antibodies, as well as by the Klebsiella polysaccharide with which the monoclonal IgM reacted.

Characteristics of the cytosol-synthesized proteins generating class-I-bound peptides.

The proteins synthesized in the cytosol are several thousand, and the number of peptides potentially able to be bound by class I molecules they can generate is therefore huge. On the other hand, the actual number of peptide-class I complexes required for CTL activation is around 200. We focused on the peptides bound by B27 molecules and by the whole class I. By comparing our results with analogous data from other laboratories, we found that 31 peptides matched protein sequences in data bases; in four cases, two peptides are derived from the same protein. The finding of four pairs of identical samples in a sampling of 31 peptides from a pool of unknown magnitude suggests that this pool is quite small. We have estimated the size of this pool by combinatorial analysis and by computer simulation, and we have found a most probable distribution of about 100 to the number of self-proteins that can actually generate peptides bound by class I molecules.

Do anergic T cells induce suppressor T lymphocytes through idiotypic interactions?

Suppression by T cells and T cell anergy have been implied, at different periods of immunological research, as the main agents of peripheral down regulation of the immune response. This article discusses the possibility that anergic T cells, with the participation of appropriate co-stimulatory molecules on their membranes, stimulate CD8 cells with an alpha/beta TCR specific for peptides of the TCR of the anergic cell itself processed and presented by class I MHC. The non-anergic (orthoergic) members of the same clone, if activated, process and present their TCR in the same way, but, lacking the co-stimulatory molecule, are unable to stimulate the anti-idiotype CD8 cells. On the other hand the orthoergic, but not the anergic, cells can be induced into death (possibly by apoptosis) by the specific CD8 lymphocytes or, alternatively, can be pushed into the anergic pool by the same CD8 suppressors, thus contributing to the generation of a TCR-restricted circuit in which suppression is dominant. This simple immunosuppressive circuit can adequately explain some recent experiments on the course of experimental allergic encephalomyelitis. It is to be stressed that many elements of the proposal are hypothetical. They are, however, open to experimental study.

The major rheumatoid factor cross-reactive idiotype in rheumatic disease.

The major rheumatoid factor cross-reactive idiotype (RCRI), a tertiary structure formed by both light and heavy chains, is found on 60% of all monoclonal IgM kappa RFs. To determine if the RCRI is expressed in patients with rheumatic disease, we used polyclonal rabbit anti-idiotypic antibodies to detect RCRI in sera and in pokeweed mitogen cultures of blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). We detected increased expression of RCRI+, plasma cells in PWM cultures, and in sera from these patients. We have determined that some 7S IgM molecules from RF+RA patients are RCRI+, and can bind IgG in a sensitive RF ELISA. We have also observed that the CD5+ B cell subset, which is responsible for autoantibody production, generates RCRI+ antibodies. We review these data and discuss the relationship of the idiotypic network of interacting antibodies with rheumatic disease.

Anti-immunoglobulins and their idiotypes: are they part of the immune network?

The anti-gamma globulins represent a very heterogeneous group of proteins with widely different specificities that might be considered a portion of the immune network. The hypothesis is presented that at least some of these proteins have other specificities, with the anti-gamma globulin reactivity being a secondary property. Anti-idiotypic antibodies are possible candidates. This is based on the low binding affinity for gamma globulin of many of these proteins and the results of CRI and sequence studies. All the proteins of the major CRI group have VKIIIb light chains, and these have a dominant but not total influence on this CRI. This has been evident from studies with rabbit antibodies and recently also with monoclonal hybridoma antibodies. Sequence studies have also demonstrated the similarity in light chains that, however, are not greater than with other VKIIIb chains; the heavy chains show little similarity except possibly in the J segment. The possibility is discussed that antibodies with secondary anti-gamma globulin binding properties might have selective advantages.

Comparative study of membrane and intracytoplasmic immunoglobulin classes in human lymphoid cells.

Immunofluorescent and "in vitro" biosynthetic techniques have been used to study the connections between the maturation process of human lymphocytes that leads to the appearance of actively secreting cells and a possible switchover from IgD to IgM production, both in homogeneous CLL cell populations and in heterogeneous cell suspensions from tonsils. The results obtained are compatible with the hypothesis that the switchover from IgD to IgM productions can be a clonal maturation phenomenon.