Biomedical subjects

F D Finkelman

Publications and source records attributed to F D Finkelman.

At least 109 records · Page 6Linked to original sources

PubMed1991

Specificity of the murine IgD receptor on T cells is for N-linked glycans on IgD molecules.

IgD receptors on murine T cells have been reported in this issue [Tamma, S. M. L., Amin, A. R., Finkelman, F. D., Chen, Y.-W., Thorbecke, G. J. & Coico, R. F. (1991) Proc. Natl. Acad. Sci. USA 88, 9233-9237] to bind either the first or third constant region of the heavy-chain of IgD molecules--findings that could not be satisfactorily explained by IgD amino acid sequences. We now find that boiled IgD molecules or low-Mr fragments from protease-digested IgD still inhibit binding of IgD-coated erythrocytes to IgD receptors. This inhibitory activity can be absorbed with the murine IgD-binding lectin from Griffonia simplicifolia 1 (GS-1) immobilized on Sepharose. N-linked glycans, obtained from N-glycanase-treated IgD and purified by binding to GS-1-Sepharose, also inhibit rosette formation of T-helper cells bearing receptors for IgD with IgD- or mutant IgD-coated erythrocytes. Deglycosylated IgD, produced by treatment with N-glycanase, no longer binds to the lectin and fails to inhibit IgD rosetting. Binding of intact IgD to T cells is also competitively inhibited by N-acetylgalactosamine, galactose, N-acetylglucosamine, and neoglycoproteins containing these sugars. These results clearly show that N-linked glycans, present in both the first and third constant regions of the delta heavy-chain domains, are prerequisites for binding of IgD to IgD receptors.

Animals↗

PubMed1991

Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody.

Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy.

Animals↗

PubMed1991

Antibodies to IL-3 and IL-4 suppress helminth-induced intestinal mastocytosis.

Rodents infected with the nematode parasite Nippostrongylus brasiliensis (Nb) develop intestinal mastocytosis, eosinophilia, and elevated serum IgE levels. Although IL-4 and IL-5 are necessary for stimulation of IgE synthesis and eosinophilia, respectively, the cytokines that regulate gut mast cell hyperplasia have not been identified. To address this question, 6- to 8-wk-old BALB/c mice were injected on day 0 and day 7 of Nb infection with a rat anti-mouse IL-4 mAb, and with polyclonal sheep (day 0) and rabbit (day 7) anti-mouse IL-3 IgG antibodies. Additional Nb-infected mice received equal doses of isotype- and species-matched control antibodies. Mice were sacrificed on days 12 or 13 post-infection, and mucosal mast cells (MMC) in sections of the small intestine were enumerated. Nb infection induced a 25- to 40-fold increase in MMC over that observed in uninfected controls. Anti-IL-3 or anti-IL-4 alone suppressed the Nb-induced MMC response by 40 to 50%, whereas both antibodies combined suppressed the MMC response by 85 to 90%. Anti-IL-3 alone had no effect on the serum IgE levels, which were essentially abrogated in the Nb-infected mice treated with anti-IL-4. Blood eosinophilia was not affected by treatment with anti-IL-3 and/or anti-IL-4. These studies demonstrate that IL-3 and IL-4 are physiologically important stimuli of mastocytosis in vivo, and suggest therapeutic interventions that may counteract adverse host responses to allergens as well as to parasites.

Animals↗

PubMed1991

Cryptosporidium infection in an adult mouse model. Independent roles for IFN-gamma and CD4+ T lymphocytes in protective immunity.

Cryptosporidium is a protozoan parasite that can cause chronic life-threatening diarrhea in immunocompromised persons. Host immune responses are poorly understood, an impediment to development of effective therapy. In mice, normal adult BALB/c animals resist infection whereas chronic symptomatic cryptosporidiosis develops in adult nude mice and in neonatally infected BALB/c mice treated with anti-CD4 mAb. To define further the immune defects that allow mice to be infected with Cryptosporidium, adult BALB/c mice were treated with cytolytic anti-CD4 or anti-CD8 or with neutralizing anti-IFN-gamma or anti-IL-2 mAb. Chronic infection, manifested by continuous shedding of sparse but statistically significant numbers of oocysts, occurred with anti-CD4 +/- anti-CD8 mAb treatment although anti-CD8 mAb treatment alone did not allow infection. Treatment with anti-IFN-gamma mAb greatly enhanced oocyst shedding but infection was self-limited. Treatment with a combination of anti-CD4 and anti-IFN-gamma mAb permitted both chronic infection and shedding of large numbers of oocysts. Furthermore mice treated initially with anti-CD4 mAb showed a substantial increase in oocyst shedding when later treated with anti-IFN-gamma mAb; and mice treated initially with both mAbs showed a decline in oocyst shedding when anti-IFN-gamma mAb was stopped. Anti-IFN-gamma mAb treatment of congenitally athymic adult BALB/c mice led to an approximately a 75-fold increase in oocyst shedding. Treatment of adult BALB/c mice with anti-IL-2 mAb did not permit Cryptosporidium infection. These results suggest that redundant immunologic mechanisms limit Cryptosporidium infection such that both CD4+ cells and IFN-gamma are required to prevent initiation of infection whereas either alone can limit the extent (IFN-gamma) or duration (CD4+ T cells) of infection. They also suggest that production of IFN-gamma by a non-T cell contributes to host immunity.

Analysis of Variance↗

PubMed1991

Purified Fc epsilon R+ bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin.

Non-B, non-T cells from spleen and bone marrow cells produce IL-4 in response to cross-linkage of high affinity receptors for Fc epsilon R or Fc gamma RII, and to treatment with calcium ionophores. Cells bearing high affinity Fc epsilon R constituted 1 to 2% of non-B, non-T cells of spleen and of total bone marrow cells from naive donors. In mice whose immune systems had been polyclonally activated by injection with anti-IgD antibodies or had been infected with Nippostrongylus brasiliensis larvae, the frequency of Fc epsilon R+ cells in splenic non-B, non-T cells was also 1 to 2% but in bone marrow from anti-IgD-injected mice donors the frequency was approximately 5%. Cell sorting experiments revealed that all of the capacity to produce IL-4 in response to immobilized IgE or IgG2a or to ionomycin was found in the Fc epsilon R+ fraction. Among the Fc epsilon R+ spleen cells from naive donors, the frequency of IL-4-producing cells was 1/20 to 1/40 whereas in mice that had been injected with anti-IgD or infected with N. brasiliensis, the frequency of IL-4 producing cells in the Fc epsilon R+ population was approximately 1/5.

Animals↗

PubMed1991

Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice.

Parasitic helminths typically induce components of immediate-type hypersensitivity, including elevated serum IgE, eosinophilia, and mucosal mast cells. These responses are T-cell-dependent and associated with rapid expulsion of parasitic worms from a sensitized host; existing experimental systems have failed to define the precise role of cytokines in these responses. We report here that anti-interleukin 4 or anti-interleukin 4 receptor antibodies block the polyclonal IgE response to a parasitic nematode, Heligmosomoides polygyrus, and abrogate protective immunity to the infection. In contrast, anti-interleukin 5 antibody prevented H. polygyrus-induced eosinophilia but did not prevent protection. These data provide evidence that a specific cytokine affects the physiology and survival of a parasitic nematode in the host.

Animals↗

PubMed1991

Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo.

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.

Animals↗

PubMed1991

IL-4 requirements for the generation of secondary in vivo IgE responses.

IL-4 has been shown to induce B lymphocytes to switch from the expression of membrane IgM to the expression of membrane IgE and to be required for the generation of primary polyclonal and secondary Ag-specific IgE responses in mice. To further define the role of IL-4 in the generation of memory IgE responses, we investigated the ability of a combination of anti-IL-4 and anti-IL-4R mAb to block the generation of secondary IgE responses induced by: 1) a second infection with the nematode parasites Nippostrongylus brasiliensis or Heligmosomoides polygyrus; or 2) injection of anti-IgD antibody-primed mice with anti-IgE antibody. The latter stimulus was designed to induce intrinsic membrane IgE-expressing B cells to differentiate into IgE-secreting cells. Although the IgE responses induced by a second nematode infection were completely inhibited by the combination of anti-IL-4 and anti-IL-4R mAb, anti-IgE antibody-induced IgE responses in anti-IgD primed mice were not inhibited by these antibodies to a large degree. Additional experiments demonstrated that the anti-IgE antibody-induced memory IgE response was dependent on CD4+ T cells but did not involve the low affinity B cell Fc epsilon RII. Taken together, these observations provide evidence that IL-4 is required for virgin B lymphocytes to develop into IgE-expressing cells, but is not required for B cells that express intrinsic membrane IgE to differentiate into IgE-secreting cells in a T-dependent response. Furthermore, these data suggest that secondary IgE responses in the parasite models that we have studied develop from B cells that had not previously switched to the expression of IgE.

Animals↗

PubMed1991

Receptor-mediated elimination of phosphocholine-specific B cells in x-linked immune-deficient mice.

The combined expression of the M167 mu/kappa anstiphosphocholine (PC) transgenes with the x-linked immunodeficiency gene, xid, results in an almost total failure to develop B cells in the peripheral lymphoid organs of such mice. Although there is no significant difference between the normal transgene positive (TG+) female offspring and the immunodeficient TG+ xid males with respect to the number of B220+ pre-B cells and IgM+B220+B cells that develop in their bone marrow, the hemizygous xid males have 85% fewer B cells in their spleens than the phenotypically normal heterozygous F1 females. In xid M167-mu-transgenic mice, PC-specific B cells also fail to develop in the spleen; however, numerous B cells bearing the mua+VH1(+)-transgene product associated with endogenous kappa L chains that do not give rise PC-specific antibodies are present. In the phenotypically normal TG+ (B6.CBA/N x mu 243-4)F1 female mice, PC-specific B cells represent almost 10% of the total B cell population, and these B cells express an M167-Id that has been produced by association of the VH1 transgene product with an endogenous V kappa 24L chain. B cells expressing the normally dominant T15-Id are not detectable in the spleens of these M167 mu-transgenic mice. Furthermore, M167-Id+ B cells are present at a fivefold lower level in the bone marrow of mu-TG+ normal mice than in their spleens. These data suggest that the PC-specific B cells that develop in TG+ xid mice are either clonally deleted via some "IgR-directed" mechanism or they fail to receive the appropriate signals to exit the bone marrow or to enter the peripheral lymphoid tissues. This hypothesis is supported by the finding that TNP-specific B cells develop normally and do not undergo clonal deletion in xid mice carrying the Sp6 mu/kappa anti-TNP transgenes.

Animals↗

PubMed1991

Kinetics of interleukin-4 induction and interferon-gamma inhibition of IgE secretion by Epstein-Barr virus-infected human peripheral blood B cells.

Interleukin-4 (IL-4) acts directly on purified human peripheral blood B cells cultured in the presence of Epstein-Barr virus (EBV) to induce IgE secretion and to enhance the secretion of IgG and IgM. Interferon-gamma (IFN-gamma) inhibits IgE secretion in this system, without affecting the secretion of the other Ig isotypes. To identify the time period during which EBV-infected B cells can be induced by IL-4 to secrete IgE, we have studied the effects of delayed addition of IL-4, or the termination of IL-4 stimulation by wash out or by neutralization with anti-IL-4 antibodies, on the induction of an IgE response. To induce a maximal IgE response, IL-4 had to be added to cultures of B cells plus EBV no later than 2 days after the initiation of culture, and had to remain present through the tenth day of culture. These two time points correspond to the initiation of detectable DNA synthesis (Days 3 to 4) and the earliest detectable Ig secretion (Days 10 to 12) by EBV-stimulated B cells. No IgE response was induced if the period during which EBV-stimulated B cells were cultured with IL-4 was less than 4 days, or if IL-4 were added later than the tenth day of culture, regardless of how long the culture was continued beyond that time. In contrast, IL-4 considerably enhanced IgG and IgM secretion and B cell CD23 expression, even if it was added after the tenth day of culture. IFN-gamma strongly inhibited the IgE response of B cells cultured with IL-4 plus EBV if added within 6 days of the initiation of culture, but had little effect on the generation of IgM or IgG responses made by these cells, regardless of the time of addition. Neither IL-4 nor IFN-gamma affected ongoing IgE secretion by an established, IgE-secreting, EBV-transformed cell line. These observations suggest that: (i) IL-4 first becomes able to induce EBV-activated B cells to secrete IgE as these cells begin to synthesize DNA, must stimulate B cells for at least 4 days to induce IgE secretion, and loses its ability to induce IgE secretion as these cells differentiate into Ig-secreting cells; (ii) the ability of IFN-gamma to suppress an IgE response is limited to this same time period; and (iii) IL-4 enhancement of CD23 expression and IgM and IgG secretion are independent of IL-4 induction of an IgE response.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1991

Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils.

Splenic and bone marrow cells from normal mice, and from mice that have been polyclonally activated by injection of anti-IgD antibody, contain cells that produce interleukin 4 (IL-4) in response to crosslinkage of Fc epsilon receptors (Fc epsilon R) or Fc gamma R or to ionomycin. Isolated Fc epsilon R+ cells have recently been shown to contain all of the IL-4-producing capacity of the nonlymphoid compartment of spleen and bone marrow. Here, purified Fc epsilon R+ cells are shown to be enriched in cells that contain histamine and express alcian blue-positive cytoplasmic granules. By electron microscopy, the vast majority of cytoplasmic granule-containing cells are basophils; they constitute approximately 25% and approximately 50%, respectively, of Fc epsilon R+ spleen and bone marrow cells from anti-IgD-injected mice. The Fc epsilon R- populations contain cells that form colonies typical of mast cells. The Fc epsilon R+ populations also contain cells that, upon culture with IL-3, form colonies of alcian blue-positive cells, but (in contrast to colonies derived from Fc epsilon R- populations) the colonies are small, and all the cells die within 2-3 weeks. The Fc epsilon R+ cells synthesize histamine during a 60-hr culture with IL-3, while the Fc epsilon R- cells do not. These results indicate that IL-4-producing Fc epsilon R+ cells are highly enriched in basophils.

Animals↗

PubMed1991

Lack of Fc epsilon RII expression by murine B cells after in vivo immunization is directly associated with Ig secretion and not Ig isotype switching.

The "low affinity" Fc receptor for IgE (Fc epsilon RII) has been reported to be absent from normal murine and human B cells that express a membrane (m)Ig isotype other than mIgM or mIgD in vivo. This would suggest that Fc epsilon RII expression is specifically lost after in vivo Ig isotype switching. We demonstrate that during a murine immune response to the bacterium Brucella abortus, to goat anti-mouse IgD (G alpha M delta) antibody, or to infection with the nematode parasites Nippostrongylus brasilienis or Heligmosomoides polygyrus, Fc epsilon RII expression is low or absent on virtually all B cells secreting IgM, IgG1, IgG2a, and IgE. However, up to 50% of B cells that express mIgG1 after G alpha M delta injection continue to express Fc epsilon RII. These mIgG1 + Fc epsilon RII+ cells secrete little, if any, IgG1 when placed in vitro, in contrast to their mIgG1 + Fc epsilon RII- counterparts. The mIgG1 + Fc epsilon RII+ cells may be a transitional cell population, because they undergo substantial loss of Fc epsilon RII in culture, unlike mIgM+ Fc epsilon RII+ cells, which maintain constant levels of Fc epsilon RII throughout a comparable culture period. Thus, low or absent expression of Fc epsilon RII after immunization in vivo is directly associated with B cell differentiation to Ig production in the presence or absence of Ig isotype switching. However, all post-switched B cells may eventually lack Fc epsilon RII expression, independently of their differentiative state.

Animals↗

PubMed1991

Generation and cloning of stable human IgE-secreting cells that have rearranged the C epsilon gene.

Although the secretion of Ig isotypes other than IgM is generally accompanied by a DNA rearrangement that deletes C mu (and the other IgCH genes located between VDJ and the expressed CH gene), a system has recently been described that generates a high frequency of IgE-secreting cells that have failed to delete IgCH genes or to rearrange their C epsilon genes. These cells, derived from EBV-transformed human PBMC, secrete IgM and IgD as well as IgE. To determine whether the absence of C epsilon rearrangement and CH gene deletion is a general phenomenon for human IgE-secreting cells, we have characterized IgE-secreting cells that are generated by culturing purified human B cells with EBV plus IL-4 in the presence of irradiated human PBMC. In contrast to the earlier observation, we have not been able to detect any cells that demonstrate cytoplasmic staining for IgE and concurrently stain for a second Ig isotype. Stable IgE-secreting cell lines and clones produced by this method have rearranged one of their C epsilon genes and have deleted both C mu genes. These observations demonstrate that the generation of human IgE-secreting cells can involve the same gene rearrangement and deletional mechanisms that lead to the generation of cells that secrete other isotypes.

B-Lymphocytes↗

PubMed1991

Dextran-conjugated anti-Ig antibodies as a model for T cell-independent type 2 antigen-mediated stimulation of Ig secretion in vitro. I. Lymphokine dependence.

We have previously demonstrated that dextran-conjugated anti-IgD antibodies (anti-delta-dex) stimulate high levels of B cell proliferation at concentrations that are 1000-fold lower than that required by unconjugated anti-Ig. We now show that anti-delta-dex may provide a suitable model to study Ig secretion stimulated by soluble T cell-independent type 2 Ag exemplified by TNP-Ficoll. Thus, both TNP-Ficoll and anti-delta-dex stimulate low to undetectable levels of Ig secretion when cultured with resting B cells. Addition of IL-5 or IL-2 stimulated enhanced anti-TNP responses in the presence of TNP-Ficoll, or induced polyclonal Ig secretion in the presence of anti-delta-dex. Both TNP-Ficoll and anti-delta-dex conjugates stimulated Ig production by Percoll-separated low density (partially activated) B cells in the absence of added lymphokines. These findings point to the similarities in the activation requirements of TNP-Ficoll and anti-delta-dex and suggest that dextran-anti-Ig conjugates, which can induce B cell activation irrespective of Ag specificity, may provide a useful model for studying various parameters that characterize the responses to soluble TI type 2 Ag.

Animals↗

PubMed1991

Polyclonal activation of the murine immune system by an antibody to IgD. XI. Contribution of membrane IgD cross-linking to the generation of an in vivo polyclonal antibody response.

The injection of mice with a foreign, polyclonal antibody to IgD sequentially induces: 1) activation of B cells by cross-linking of their cell membrane (m) IgD; 2) B cell processing and presentation of the bound anti-IgD antibody to T cells; 3) activation of these T cells; and 4) T-dependent stimulation of B cell differentiation into IgG1 secreting cells. To determine whether the cross-linking of B cell membrane IgD and/or the resulting B cell activation that follows contribute to the generation of the polyclonal IgG1 response, we examined the abilities of three sets of anti-delta mAb or mAb fragments to stimulate polyclonal IgG1 production. Within each set mAb were matched for species and Ig isotypic determinants, but differed in avidity for IgD or in ability to cross-link IgD. In addition, experiments were performed to determine whether the anti-delta mAb had to be foreign to the immunized mouse to stimulate an IgG1 response. Results of these experiments indicate that: 1) recognition of the injected anti-delta antibody as foreign is required for the induction of a polyclonal IgG1 response; 2) the cross-linking of B cell membrane Ig, which directly activates B cells, can contribute considerably to the generation of in vivo IgG1 production; and 3) that even relatively weak cross-linking of membrane Ig by ligands that bind it with low avidity can make this contribution.

Animals↗

PubMed1991

Increased frequency of interleukin 4-producing T cells as a result of polyclonal priming. Use of a single-cell assay to detect interleukin 4-producing cells.

A limiting dilution assay capable of detecting interleukin 4 (IL4) production by a single cell has been developed. This assay is based on the stimulation of T cells, in the presence of IL2 (5 U/ml), with anti-CD3 antibody bound to the surface of Terasaki wells. Cells of the IL4-selective indicator line, CT.4S, are added 24-36 h later and IL4 production is determined based on their survival 24-48 h thereafter. A frequency of 0.98 was obtained for IL4 production by T cells of the D10.G4 cell line. T cells from naive donors capable of producing IL4 in response to anti-CD3 plus IL2 were quite rare, with a frequency in four experiments ranging between 0.0003 and 0.0018. Treatment of mice with polyclonal activators known to increase the IL 4-producing capacity of T cells when assayed in bulk culture caused striking increases in the frequency of IL4-producing cells. Similarly, culturing cells in vitro with anti-CD3, IL2 and IL4 for 5 days caused a marked increase in the frequency of cells capable of producing IL4, to 0.031.IL4 production by individual T cells is dependent upon IL2. Thus, in naive T cell populations, the frequency of IL4-producing cells in response to stimulation with anti-CD3 in the absence of IL2 was below the limit of detection. T cells from primed donors showed a striking inhibition in the frequency of IL4-producing cells in response to anti-CD3 when IL2 was not present. The availability of a simple assay to measure the frequency of cells capable of producing IL4 should have substantial utility in allowing the evaluation of conditions that regulate IL4 production in vivo and in vitro.

Animals↗

PubMed1991

Allostimulatory analysis of a newly-defined and widely-distributed Mls superantigen.

We previously noted that Mlsa,c C58/J responder cells proliferated unexpectedly to H-2k-compatible Mlsa or Mlsc prototypic stimulator cells in a primary mixed lymphocyte reaction. The present investigation was performed to evaluate whether the response of C58/J T cells to these H-2- and Mls-compatible stimulator cells could functionally identify a newly-defined member of the Mls superantigen family through its allostimulatory ability. We observed that C58/J responder cells also proliferated when cultured with H-2-compatible prototypic Mls(null), Mlsb (nonstimulatory), or Mlsa,c splenic stimulator cells. The widely distributed nature of the non-MHC ligand recognized by C58/J T cells is indicated by the finding that 11 of 12 H-2k inbred mouse strains clearly expressed this specificity. A gradient of stimulatory capacity from low to high across this newly-defined non-MHC difference was detected with splenocytes from these different inbred mouse strains. The Mls(a,c) genetic composition of C58/J was confirmed by the observation that crossing C58/J with parental B10.BR (responsive to both Mlsa and Mlsc determinants) generated F1 progeny that were unresponsive to H-2k-compatible Mlsa, Mlsc, or Mls(a,c) stimulator cells. Like prototypic Mlsa and Mlsc, the non-MHC specificity recognized by C58/J responder cells, termed Mlsf, was particularly sensitive to radiation (versus mitomycin C) treatment of the stimulator cells, was greatly augmented after anti-IgD activation of splenic stimulator cells, was blocked with anti-MHC class II antibody, and was effectively presented by phenotypically normal female but not B cell-defective xid+ male CBA/N F1 stimulator cells.

Animals↗

PubMed1991

Intracellular routing rather than cross-linking or rate of internalization determines the potency of immunotoxins directed against different epitopes of sIgD on murine B cells.

Several variables influence the potency of an immunotoxin (IT) prepared with a monoclonal antibody (mAb) and ricin A chain (IT-A). These include the affinity of the mAb, the nature and density of the target antigen (Ag), the epitope on the target Ag bound by the mAb, the type of cell target, and the rate of endocytosis and route of internalization of the bound IT-A. In a previous report, we demonstrated that anti-delta mAbs directed against epitopes which are putatively more proximal to the plasma membrane make more effective IT-As than those directed against epitopes that are putatively more distal from the plasma membrane. It is known that the latter mAbs cross-link sIgD less effectively than the former. Therefore, in the present study, we determined whether the differential cytotoxicity of IT-As directed against these epitopes is related to their ability to cross-link their specific surface antigen (sIgD). We further determined whether they were internalized at different rates by normal B cells. Our results show that neither cross-linking nor rate of internalization account for the different potencies of anti-Fc vs anti-Fd IT-As. However, when these IT-As were used in the presence of the lysosomotropic agent chloroquine, the less potent IT-A became 100-fold more potent and was as cytotoxic as the effective anti-Fc IT-A. Taken together with the results of other studies, these findings further support the hypothesis that the epitope specificity of a given mAb may be an important factor in determining the intracellular routing of an IT-A after internalization.

Animals↗