Cellular basis of regulation of expression of idiotype. I. T-suppressor cells specific for MOPC 460 idiotype regulate the expression of cells secreting anti-TNP antibodies bearing 460 idiotype.

An idiotype of the dinitrophenyl-binding myeloma protein MOPC 460 was expressed on a small but significant proportion of anti-TNP antibodies which appeared after in vivo or in vitro immunization of BALB/c mice with three T-independent TNP antigens. In vitro experiments show that the depletion of T cells before culture increased significantly the number of plaques secreting anti-TNP antibodies bearing MOPC 460 idiotype (460Id). T cells from BALB/c mice, but not from C.B20 mice, exhibit this suppressor activity. Plate-binding experiments indicate that the suppressive action of the T-lymphocyte population depends on a cell which can bind to MOPC 460 myeloma protein. The possible role of these normally occurring, idiotype-specific T cells on expression of 460Id in the anti-TNP antibody response of BALB/c mice is discussed.

CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3.

Injection of anti-CD3 antibodies causes prompt expression of interleukin (IL)-4, IL-2, and interferon gamma (IFN-gamma) mRNA among spleen cells. The optimal dose of anti-CD3 for such induction was 1.33 microgram/animal; lymphokine mRNA was first observed at 30 min, peaked at 90 min, and was undetectable (for IL-4) or had declined markedly by 4 h. Cells harvested from spleens of mice injected with anti-CD3 90 min earlier secreted IL-4, IL-2, and IFN-gamma without further stimulation. By contrast, in vitro stimulation with anti-CD3 of spleen cell suspensions or splenic fragments from noninjected donors failed to cause prompt production of IL-4 and, even after 24 h of stimulation, the amount of IL-4 produced in such cells was substantially less than that secreted within 1 h by spleen cell suspensions or splenic fragments from mice injected with anti-CD3 90 min earlier. Production of IL-4 by spleen cells from anti-CD3-injected mice was not inhibited by pretreatment with anti-IL-4 antibody or with IFN-gamma or tumor growth factor beta nor enhanced by treatment with IL-4. By contrast, CTLA-4 immunoglobulin (Ig) treatment clearly diminished IL-4 production in response to in vivo anti-CD3, indicating that cellular interactions involving CD28 (or related molecules) were important in stimulation. Cell sorting analysis indicated that the cells that produced IL-4 in response to in vivo injection of anti-CD3 were highly enriched in CD4pos cells with the phenotype leukocyte cell adhesion molecule-1 (LECAM-1)dull, CD44bright, CD45RBdull, NK1.1pos. Indeed, the small population of CD4pos, NK1.1pos cells had the great majority of the IL-4-producing activity of this population. Injection with Staphylococcal enterotoxin B also caused prompt induction of IL-4 mRNA; the cells that were principally responsible for production also had the phenotype of CD4pos, NK1.1pos. These results suggest that possibility that this rare population of T cells may be capable of secreting IL-4 at the outset of immune responses and thus may act to regulate the pattern of priming of naive T cells, by providing a source of IL-4 to favor the development of T cell helper 2-like IL-4-producing cells.

Impaired interleukin 4 signaling in T helper type 1 cells.

Cluster of differentation (CD)4+ T helper cells (Th)1s fail to produce interleukin (IL)-4. Even if restimulated in the presence of IL-4, a condition that induces IL-4-producing capacity in naive CD4+ T cells, Th1s fail to become IL-4 producers. We report that Th1 cells have a major impairment in IL-4 signaling. When compared to both Th2s and naive T cells, they display a striking diminution in phosphorylation of Stat6. They also show reduced phosphorylation of Janus kinase (JAK)-3 and insulin receptor substrate (IRS)-2 when compared to Th2s. Stat6 and JAK-3 are present in equivalent amounts in Th1s and Th2s, but IRS-2 protein levels are much lower in Th1s than in Th2s. Altered sensitivity to IL-4, the major inducer of the Th2 phenotype, may explain the stability of the Th1 state.

The specificity of cellular immune responses in guinea pigs. III. The precision of antigen recognition by T lymphocytes.

T lymphocytes from guinea pigs immunized with 2,4-dinitrophenyl (DNP) derivatives of mycobacteria respond to a variety of DNP conjugates. Preincubation of such cells with a given DNP conjugate under conditions which lead to the inactivation of responding cells causes a loss of the response to that conjugate, but has little effect on the response to DNP coupled to unrelated carriers. Thus, the responses of such cells to a variety of DNP conjugates can best be explained by the presence of a mixture of highly specific cells each responding to a different antigenic dterminant rather than by the presence of T cells with specificity limited to the hapten itself. Furthermore, the activity of T cells from DNP-mycobacteria-primed donors could not be blocked by a variety of nonstimulatory DNP conjugates. This suggests that while such T cells clearly recognize DNP with great precision, the receptor does not contain a very high affinity site for the hapten. A possible model for such a T-cell receptor is discussed.

Synergistic genetic defect in B-lymphocyte function. I. Defective responses to B-cell stimulants and their genetic basis.

CBA/N female mice, which express an X-linked defect in B-lymphocyte function, were mated with C3H/HeJ male mice, which are unresponsive to lipopolysaccharide (LPS). The resulting F1 hybrid females were mated to C3H/HeJ males. Approximately one-half of the backcross (BC.1) males obtained from this mating expressed a more profound immunologic defect than either of the parental strains. Spleen cells from these mice were unresponsive to a series of B-cell mitogens including LPS prepared from Escherichia coli K235 and from E. coli 0111:B4, lipoprotein mitogen from E. coli, and Nocardia water-soluble mitogen (NWSM). They failed to give in vitro antibody responses to the thymus-independent type 2 (TI-2) antigen trinophenylated Ficoll and most were unresponsive to the TI-1 antigens trinitrophenylated Brucella abortus, trinitrophenylated LPS, and trinitrophenylated NWSM. This synergistic defect in B-lymphocyte function depended on the presence of the CBA/N xid gene but the critical gene(s) from the C3H strain was not the defective Lps gene (Lpsd). These mice should provide a valuable tool for the elucidation of B-lymphocyte ontogeny, heterogeneity, and function.

A study of the passive transfer of delayed hypersensitivity to DNP-poly-L-lysine and DNP-GL in responder and nonresponder guinea pigs.

A study of the passive transfer of delayed hypersensitivity to DNP-poly-L-lysine and to DNP-GL was performed in Hartley guinea pigs. Delayed hypersensitivity to DNP-PLL and DNP-GL could be transferred successfully only by means of sensitized cells from genetic responder guinea pigs and in most cases, only into those guinea pigs genetically capable of responding to PLL. The inability to transfer delayed hypersensitivity to DNP-PLL or DNP-GL to genetic nonresponder guinea pigs is not the result of the early destruction of the transferred cells by an incompatible host, since it was shown that delayed hypersensitivity to ovalbumin could be successfully transferred from guinea pigs with the PLL gene into genetic nonresponder animals. The requirement of active participation of specific and genetically controlled host mechanisms in the successful passive transfer of delayed sensitivity to DNP-PLL and DNP-GL has been demonstrated.

Stimulus-response in the mixed lymphocyte reaction.

Mixed lymphocyte reactions occur when mouse spleen cell populations depleted of thymus-derived (T) lymphocytes are cultured with allogeneic target cells inactivated by mitomycin C or X irradiation, and when F(1) hybrid responder cells are cultured with inactivated parental target cells. These responses might be interpreted as indicating that T lymphocytes are not required for responsiveness and that F(1) lymphocytes recognize parental alloantigens. Data reported here indicate that the more likely explanation for these surprising results is that inactivated target cells recognize the "responding" cells and this recognition leads to the response observed.

Receptors on immunocompetent cells. IV. Direct measurement of avidity of cell receptors and cooperative binding of multivalent ligands.

The interaction of antigen with specific, cell-associated receptors was measured in thermodynamic terms. The binding of (125)I-labeled 2,4-dinitrophenyl guinea pig albumin (DNP(16)GPA-(125)I) to lymphocytes from guinea pigs immunized to DNP(16)GPA is a temperature-dependent, reversible process. Measurement of association and dissociation rates of antigen-receptor complexes permits calculation of antigen-cell binding constants. These may also be calculated by equilibrium-binding techniques. Although differences in the constants calculated in these two ways exist, a clear increase in avidity of cell receptor for antigen occurs in the course of the immune response. This change in receptor avidity provides evidence that the time-dependent change in affinity of serum antibody (maturation) indeed has a cellular basis. The magnitude of the equilibrium constant is, in part, due to binding of more than one DNP group per molecule of antigen. Thus, multivalent ligands bind more effectively to cell receptors than univalent or paucivalent ligands when measured by the number of antigen molecules bound, the dissociation rate of antigen-receptor complexes, and in the relative capacity to inhibit a standard multivalent ligand (DNP(16)GPA-(125)I) from binding.

Receptors on immunocompetent cells. V. Cellular correlates of the "maturation" of the immune response.

During the course of the immune response to dinitrophenylated guinea pig albumin (DNP-GPA), a striking and parallel increase in avidity for epsilon-DNP-L-lysine occurs in the receptors on antigen-binding lymphocytes, antibody secreted by individual plaque-forming cells, and serum antibody molecules. A detailed analysis of the avidity distribution of antibody produced by plaque-forming cells indicates that this "immunologic maturation" is primarily due to a preservation of the high avidity subpopulation and a striking loss in the low avidity population rather than to sequential appearance of these cells. Moreover, the demonstration of the increased avidity of receptors of antigen-binding lymphocytes, which appear to be precursors of antibody-synthesizing cells, strongly suggests that the antigen-driven selectional process operates primarily on this cell type.

Alloantiserum-induced inhibition of immune response gene product function. I. Cellular distribution of target antigens.

It has been previously shown that alloantisera prepared by reciprocal immunization of strain 2 and strain 13 guinea pigs specifically block the activation of T lymphocytes from immune guinea pigs by antigens, the response to which is controlled by Ir genes. In this report we have examined the effect of absorption of the 13 anti-2 serum with different populations of lymphoid cells. It is unlikely that the inhibitory activity of the anti-2 serum on the proliferation of (2 x 13)F(1) lymphocytes to a DNP derivative of a copolymer of L-glutamic and L-lysine (DNP-GL) is due to the presence of antibodies specific for the unique antigenic determinants (idiotypes) of clonally distributed T-lymphocyte receptors. Thus, cells obtained from a normal animal and a DNP-GL immune animal were equivalent in their absorptive capacity. Populations of T lymphocytes were ineffective in absorbing either the cytotoxic or inhibitory activity of the anti-2 serum, while L(2)C leukemia cells, a malignant B-cell population, were most efficient in absorbing both activities. Thus, the antigen(s) against which the cytotoxic and inhibitory activities are directed are present to a greater extent on B lymphocytes than on T lymphocytes. However, these results do not allow us to definitively determine whether the inhibitory activity of the alloantisera is due to antibodies specific for Ir gene products or antibodies specific for linked antigens in the MHC. We also examined the effect of a number of anti-immunoglobulin reagents which had specificity for the heavy and/or light chains of guinea pig immunoglobulin on the in vitro lymphocyte proliferative response to antigen. Under conditions in which we were able to completely and specifically suppress the response of (2 x 13)F(1) lymphocytes to DNP-GL with anti-2 serum, the anti-immunoglobulin reagents were devoid of inhibitory effect on the response of these same F(1) cells to DNP-GL, a copolymer of L-glutamic and L-tyrosine (GT), or purified protein derivative of tuberculin (PPD). These results strongly suggest that conventional serum-type immunoglobulin is not important in antigen recognition by the T cells involved in the DNA synthetic response.

Alloantiserum-induced inhibition of immune response gene product function. II. Genetic analysis of target antigens.

It has been previously demonstrated that alloantisera can specifically block the activation of T lymphocytes by antigens, the response to which is linked to the presence of histocompatibility (H) types against which the alloantisera are directed. Thus, strain 13 anti-2 serum can inhibit the activation of (2 x 13)F(1) T lymphocytes by a DNP derivative of a copolymer of L-glutamic acid and L-lysine (DNP-GL), an antigen the response to which is controlled by a 2-linked Ir gene. It was proposed that alloantisera can inhibit T-lymphocyte antigen recognition through interference with the activity of immune response (Ir) gene products. In order to further study whether the inhibitory antibodies within the alloantisera are directed against H antigens or against the products of the Ir genes, we have examined whether the anti-2 serum can inhibit the function of an Ir gene (the L-glutamic acid and L-alanine [GA] gene), which is normally linked to strain 2 H genes when this gene occurs in an outbred animal lacking strain 2 H genes. In the majority of cases, the anti-2 serum was capable of inhibiting the in vitro proliferative response to GA of T cells derived from animals that were GA(+)2(+), but the serum had little if any effect on the GA response of T cells from GA(+)2(-) animals. Furthermore, an antiserum prepared in strain 13 animals against the lymphoid cells of a GA(+)2(-) outbred animal was devoid of inhibitory activity on the GA response of cells from a (2 x 13)F(1), while an antiserum prepared in strain 13 animals against the lymphoid cells of a GA(+)2(+) outbred animal was capable of specifically inhibiting the response to GA. It thus appears that the inhibition of the GA response by the anti-2 serum is primarily mediated via antibodies directed toward strain 2 H antigens rather than antibodies specific for the product of the GA Ir gene. The mechanism of alloantiserum induced suppression of Ir gene function would then be by steric interference with the Ir gene product on the cell surface, rather than by direct binding to it. This conclusion implies that the products of both the H genes and the Ir genes are physically related on the cell surface. The implications of such a relationship in terms of the fluid-mosaic model of the lymphocyte surface are discussed.

An antisense oligonucleotide complementary to a sequence in I gamma 2b increases gamma 2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion.

An antisense phosphorothioate (S)-oligonucleotide to a sequence in the intervening (I) region of the gamma 2b immunoglobulin (Ig) heavy chain gene inhibits Ig secretion by B cells stimulated with lipopolysaccharide (LPS) or LPS plus interleukin 4. It is also a striking stimulant of DNA synthesis by resting B cells. The antisense S-oligonucleotide causes a 10-20-fold increase in the expression of the gamma 2b germline transcript. Among mutants of the antisense S-oligonucleotide, some show all the effects whereas others are inactive. A similar hierarchy exists in the quantitative biological activities of mutant S-oligonucleotides and in their capacity to hybridize to the sense oligonucleotide, strongly suggesting that an I gamma 2b sequence in the RNA transcript or in the noncoding strand of the DNA is the target of the antisense S-oligonucleotide. The possible relationship of the overexpression of the germline gamma 2b transcript to the biological functions of the I gamma 2b antisense S-oligonucleotide is discussed.

Hapten carrier relationships in the DNP-PLL foreign albumin complex system: induction of tolerance and stimulation of cells in vitro.

Genetic nonresponder guinea pigs made tolerant to BSA and then immunized with DNP-PLL.BSA failed to make anti-DNP-PLL antibodies. Thus, tolerance to a carrier protein renders animals unresponsive to the hapten which it bears. The addition in vitro of DNP-PLL or DNP-GL to lymph node cell cultures derived from genetic responder animals immunized with these materials led to a significant stimulation of (3)H-thymidine incorporation into DNA. However, the addition of DNP-PLL or DNP-GL to lymph node cell cultures from nonresponder animals immunized with these materials failed to produce any stimulation of DNA synthesis. Furthermore, the addition of DNP-PLL to lymph node cell cultures from nonresponder animals immunized with DNP-PLL.BSA or DNP-PLL.OVA also failed to stimulate cell proliferation in spite of the fact that the lymph node cells of these animals were producing anti-DNP-PLL antibodies. The above facts suggest that the function of the PLL gene product is to act at an early crucial step in the immune mechanism to form an antigen-inducer complex. The specificity of this early step may be of a simple order and different than that of the antibody which is later produced in the immune response.

The behavior of hapten-poly-L-lysine conjugates as complete antigens in genetic responder and as haptens in nonresponder guinea pigs.

30 to 40% of Hartley strain guinea pigs have previously been demonstrated to possess a dominant autosomal gene which enables them to recognize the antigenicity of hapten-poly-L-lysine conjugates as expressed by the development of both antihapten antibodies and delayed hypersensitivity to the immunizing antigen. In the present study, it was shown that PLL alone was weakly antigenic in such genetic responder animals. Immunization with DNP-PLL electrostatically combined with foreign albumins elicits the production of anti-DNP antibodies in all Hartley strain guinea pigs, although the percentage of animals demonstrating a delayed response to DNP-PLL and therefore considered genetic responders remains 30 to 40%. Immunization with nonantigenic polyanions combined with DNP-PLL does not produce such an effect. Some degree of PLL specificity of purified anti-DNP antibodies produced by genetic nonresponder animals by immunization with DNP-PLL combined with foreign albumins was demonstrated by means of fluorescence quenching.

Studies on the effect of the carrier molecule on antihapten antibody synthesis. I. Effect of carrier on the nature of the antibody synthesized.

1. Fluorescence quenching has been calibrated, by comparison with equilibrium dialysis, for measurement of antibody-hapten association constants with guinea pig antibody to the 2,4-dinitrophenyl (DNP) determinant. The maximum quenching with all sites occupied which gave best agreement with equilibrium dialysis was found to be 100%. 2. The carrier to which the DNP-hapten is coupled influences significantly the amount, class (gamma(1) or gamma(2)), and affinity of the antibody of DNP specificity made by individual guinea pigs. 3. The affinities of gamma(1)- and gamma(2)-antibodies from individual guinea pigs are generally very similar. 4. Precipitin curves with gamma(1)-antibodies show more marked inhibition of precipitation in antigen excess than do precipitin curves with the less charged gamma(2)-antibodies, indicating the importance of nonspecific forces in the precipitation reactions.

Studies on the effect of the carrier molecule on antihapten antibody synthesis. II. Carrier specificity of anti-2,4-dinitrophenyl-poly-l-lysine antibodies.

Equilibrium measurements of interactions of anti-DNP antibodies, prepared using DNP-PLL and several DNP-proteins for immunization, with DNP(0.6)-PLL(240) and with the univalent hapten, epsilon-DNP-L-lysine, were made utilizing the technique of fluorescence quenching. Carrier specificity of anti-DNP-PLL antibodies was demonstrated by a higher average intrinsic association constant (K(0)) of anti-DNP-PLL antibodies with DNP(0.6)-PLL(240) than with epsilon-DNP-L-lysine. The free energy contribution of the PLL carrier to the interaction of intact anti-DNP-PLL antibodies with DNP(0.6)-PLL(240) was from -0.8 to -2.1 kcal/mole. On the other hand, intact anti-DNP-protein antibodies displayed a lower energy of interaction with DNP(0.6)-PLL(240) than with epsilon-DNP-L-lysine of up to +2.4 kcal/mole. Fab' fragments of both anti-DNP-PLL and anti-DNP-BGG antibodies have K(0)'s with epsilon-DNP-L-lysine identical to the K(0)'s of the intact anti-DNP antibodies from which they were prepared. However, K(0) of interaction of Fab' fragments with DNP(0.6)-PLL(240) (a large proportion of the conjugated PLL molecules in this preparation bear more than one DNP group) is considerably lower than that of the intact antibody. Thus a cooperative effect in the binding of bivalent antibody and bivalent (or greater) antigen exists and is of the order of -1.2 to -2.0 kcal/mole of IgG antibody. Although the direct contribution of the carrier to the interaction of Fab' fragment of anti-DNP-PLL and DNP(0.6)-PLL(240) is -0.4 kcal/mole, the energy of carrier specificity, based upon consideration of cooperative effects and of repulsion of anti-DNP-protein antibodies for portions of the DNP-PLL determinants, is of the order of -3 kcal/mole (approximately 30% of total binding energy).

Histocompatibility-linked immune response gene function in guinea pigs. Specific inhibition of antigen-induced lymphocyte proliferation by alloantisera.

A number of autosomal dominant immune response (IR) genes have been identified in both mice and guinea pigs. These IR genes have been shown to be linked to the major histocompatibility antigens of the species and to be functionally expressed primarily in T lymphocytes. In order to more fully understand the relationship between IR genes, histocompatibility antigens, and immune recognition, the effect of specific alloantisera on lymphocyte stimulation induced by antigens under control of IR genes was examined. Using lymphocytes from strain 2 or strain 13 animals, the in vitro proliferative responses both to antigens which are known to be under genetic control (DNP-GL in strain 2 guinea pigs and GT in strain 13 guinea pigs) and to an antigen which is not known to be under genetic control (PPD) were inhibited to a similar degree and to a much greater extent than the response to phytohemagglutinin. However, when cells from F(1) (2 x 13) animals are used, the alloantisera markedly inhibit only the response which is linked to the histocompatibility antigens against which the serum is directed. Thus, the anti-2 serum inhibited the response to DNP-GL but not to GT; the anti-13 serum inhibited the response to GT but did not affect DNP-GL response. The inhibitory activity of the alloantisera could not be removed by absorption with gamma globulin of the opposite strain. It can be concluded from these observations that immune response genes produce a cell surface-associated product and that this product plays a role in the mechanism of antigen recognition by the T lymphocyte. The mechanisms by which alloantisera block this process of antigenic recognition is not resolved nor is the relationship between the IR gene product and the antigen-binding receptor of the T lymphocyte. The approach described here offers a powerful tool for the resolution of these problems.

T-lymphocyte-enriched murine peritoneal exudate cells. II. Genetic control of antigen-induced T-lymphocyte proliferation.

The recent introduction of a reliable, T-lymphocyte proliferation assay, which utilizes thioglycollate-induced, nylon wool column-passed, peritoneal exudate lymphocytes from immune mice (PETLES), allowed us to investigate the genetic control of murine immune responses at the T-lymphocyte level. Examination of the blast cells generated in this population 5 days after stimulation with antigen, revealed that 85% of the cells bore the Thy 1 antigen on their surface, whereas only 5% bore immunoglobulin. Thus, the assay can be considered to measure almost exclusively T-lymphocyte function. This assay was used to examine the T-lymphocyte proliferative responses to seven different antigens: poly(Glu60Ala30Tyr10), poly(Glu58Lys38Tyr4), poly-(Tyr,Glu)-poly-D,L-Ala--poly-Lys, poly-(Phe,Glu)-poly-D,L-Ala--poly-Lys, staphylococcal nuclease, lactate dehydrogenase H4, and the BALB/c IgA myeloma protein, TEPC-15. PETLES from a large number of different inbred mouse strains, including H-2 congenic resistant lines and H-2 recombinants, were studied. The strains could be classified as high responders, low responders, or nonresponders to a particular antigen as judged by the magnitude of the T-lymphocyte proliferative response. In every case but one this classification corresponded to the responder status given the strain based on its ability to mount an in vivo antibody response to the same antigen. For two of the antigens, poly-(Tyr,Glu)-poly-D,L-Ala--poly-Lys and TEPC-15, the immune response genes controlling the T-lymphocyte proliferative response were mapped to the K region or I-A subregion of the major histocompatibility complex, as had previously been shown for the control of the antibody responses to these antigens. This tight linkage of the two phenotypic responses very strongly suggests that the same immune response gene controls the expression of both the proliferative and antibody responses. Since there is essentially no contribution from B lymphocytes in the T-lymphocyte proliferation assay, it seems reasonable to conclude that none of the seven immune response genes studied are expressed solely in B lymphocytes.

Antigen presentation in the murine T-lymphocyte proliferative response. I. Requirement for genetic identity at the major histocompatibility complex.

A method is described for stimulating proliferation in primed populations of murine T lymphocytes using antigen bound to mitomycin-C-treated spleen cells. This form of antigen presentation appears to be an active process because heat-killed spleen cells are ineffective, and because genetic similarity at the major histocompatibility complex (MHC) between the responder T cells and the presenting spleen cells is required for effective interactions. At all times examined, from day 3 to day 6 of the proliferative response, syngeneic spleen cells presented antigen better to peritoneal exudate T-lymphocyte-enriched cells (PETLES) than semisyngeneic F(1) spleen cells, which in turn could present antigen better than totally allogeneic spleen cells. Spleen cell mixing experiments demonstrated that these genetic restrictions were not the result of suppression by the ongoing mixed lymphocyte reactions (MLR) in the allogeneic and F(1) cases. Furthermore, incompatibility at the Mls locus generated a strong MLR but failed to prevent antigen presentation if the spleen cells and PETLES were compatible. Genetic mapping studies demonstrated that compatibility at only the I-A subregion of the MHC was sufficient for effective presentation of the antigen, dinitrophenylated ovalbumin. Compatibility at only the K region, or the K and D regions was not sufficient. These results support the concept that functional activation of primed, proliferating T lymphocytes requires the participation of gene products coded for by the I region of the MHC. This conclusion is consistent with a growing body of evidence which suggests that most T cells recognize antigen in association with MHC gene products.

X-linked B-lymphocyte defect in CBA/N mice. III. Abnormal development of B-lymphocyte populations defined by their density of surface immunoglobulin.

CBA/N mice have an X-linked defect in B-lymphocyte function characterized by a failure to respond to certain thymus-independent antigens. When studied by rapid flow microfluorometry, adult CBA/N splenic B lymphocytes labeled with either fluorescein-conjugated (Fl) anti-Ig or Fl anti-mu had fluorescence profiles which were considerably different from those of B lymphocytes derived from normal mice. By studying progeny of crosses of CBA/N and normal mice, it was shown that the abnormal fluorescence profiles of CBA/N B cells were determined by an X-linked gene. The fluorescence profile of adult CBA/N splenic B lymphocytes labeled with anti-mu were very similar to the patterns of neonatal normal and of neonatal CBA/N splenic B lymphocytes suggesting that the defect of CBA/N mice is due to a failure in the development of a mature B-lymphocyte population. The fluorescence profiles of adult CBA/N splenic B lymphocytes labeled with Fl anti-Ig also had immature characteristics in that the frequency of cells with large amounts of surface immunoglobulin was increased in comparison to that of normal strains and the population of cells with low-to-intermediate density of total surface immunoglobulin, which appear characteristic of normal adult splenic B lymphocytes, was markedly diminished.