T cell responses to human recombinant acetylcholine receptor-alpha subunit in myasthenia gravis and controls.

Antibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction are detectable in most patients with myasthenia gravis (MG) and assumed to participate in the destruction of the AChR, thereby, causing the characteristics signs and symptoms of the disease. The extent and importance of T cell responses to AChR and its subunits in MG are still unsettled. We have now examined T cell reactivities using human recombinant AChR-alpha subunit as antigen. Upon recognition of appropriate antigen in an MHC-class II-restricted fashion, memory T cells secrete interferon-gamma (IFN-gamma). Adopting this principle in an immunospot assay we found that 73% of MG patients had recombinant human AChR-alpha subunit-reactive T cells at a median value of 1 per 56,000 blood mononuclear cells, while only 27% of the MG patients responded to the alpha subunit in a conventional lymphocyte proliferation assay. This compares with even lower numbers of AChR-reactive T cells and 14% positivity in the proliferation assay among control subjects. The T cell responses to the control antigens purified protein derivative and myelin basic protein did not differ between MG and controls, underlining the specificity of an augmented T cell reactivity to AChR-alpha subunit in MG. Alpha Subunit-specific T cell lines and clones propagated from patients with MG and healthy controls yielded a high proportion of alpha subunit-reactive T cells in the IFN-gamma immunospot assay. Their appearance was inhibited by the addition of monoclonal anti-MHC class II antibodies, demonstrating that an MHC-restricted T cell response was measured. Our data underline that the AChR-alpha subunit is a major T cell autoantigen in MG.     

B and T cell involvement in anti-acetylcholine receptor antibody formation in myasthenia gravis.

The in vitro method of antibody production was applied to ascertain the contribution of B and T cells to the formation of anti-acetylcholine receptor (AChR) antibody. The anti-AChR antibody in the culture supernatant was estimated by radioimmunoassay, and the anti-AChR antibody-forming cells from cultures were detected by autoradiography of the antigen-binding cells. Thymic B cells from myasthenia gravis (MG) patients formed antibody when they were cultured with thymic T cells from MG patients and stimulated with AChR antigen. The antibody formation was more vigorous with thymic B cells, which contained more germinal centres. The antibody was also formed from the B and T cell combination of peripheral blood lymphocytes, although the amount was less than that produced by thymic lymphocytes from MG patients. The antibody produced by lymphocytes from MG patients. The antibody produced by lymphocytes from MG patients was suppressed by the addition of T cells from the culture supernatant of normal individuals, but not by autologous or allogeneic T cells from MG patients. The suppression by T cells from normal individuals was abolished when the cells were treated with mitomycin C. These observations indicated that AChR-specific B cells and helper T cells are active, while the suppressor T cells, which are usually present in normal individuals, are defective in MG patients.     

Immunological heterogeneity of autoreactive T lymphocytes against the nicotinic acetylcholine receptor in myasthenic patients

The response of human T lymphocytes against the nicotinic acetylcholine receptor (AChR) was studied in five patients with myasthenia gravis (MG) and in six healthy donors using either native Torpedo AChR or recombinant protein derived from the mammalian AChR α subunit (X4, residues 6–216 of mouse AChR α subunit). The present study demonstrates that (a) AChR-specific T helper cell lines can be generated from MG patients [either from peripheral blood lymphocytes (PBL) or from thymocytes] as well as from PBL of normal controls, (b) lymphocytes from MG patients, but not from controls, recognize the mammalian AChR but not the Torpedo receptor, (c) in humans, the HLA-DR2-associated T cell epitope is probably located in the region of residues 162–216 of the AChR α subunit and (d) there is a considerable heterogeneity of autoreactive T cell responses: (i) Tcell lines from different HLA-type donors have distinct epitope profiles; (ii) the epitope specificity of the PBL-derived T cell line is different from that of the thymocyte-derived line; (iii) the epitope specificities of patient-derived T cell lines are different from those generated from normal controls who share the same HLA phenotype.     

Myasthenia Gravis and its Animal Model: T Cell Receptor Expression in an Antibody Mediated Autoimmune Disease

Myasthenia gravis (MG) is a prototypic antibody-mediated autoimmune disease. Since the primary target antigen of the autoimmune response is known and a well-characterized animal model is available, MG is often considered an excellent situation for the application of novel specific immunotherapies, many of which are directed at T lymphocytes. CD4⁺ helper T cells are required for the development of the animal model, experimental autoimmune MG (EAMG). Even though the target antigen, acetylcholine receptor (AChR) is immunologically complex, the T cell response to AChR in mice is dominated by recognition of a single peptide by about 50% of the T cells. These T cells, in turn, utilize a restricted set of TCR gene elements and conserved CDR3 regions. While specific therapy directed at the immunodominant T cells is capable of reducing the magnitude of the anti-AChR response, considerable flexibility is apparent and reveals the ability of additional T cells to provide the requisite B cell help. In human MG patients, AChR-specific T cells have been identified but in many studies the frequencies were surprisingly low. In a very few cases, AChR-specific T cells have been cloned from MG patients. Analysis reveals heterogeneity in epitope recognition and MHC restriction. Little information on TCR structure is available. Our own studies using antigen-specific as well as non-specific methods for examining clonal T cell expansions in MG have led to an alternative hypothesis concerning T-B collaboration in MG.     

Experimental autoimmune myasthenia gravis induction in B cell-deficient mice

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG). Autoantibody-induced functional loss of nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane is an important pathogenic feature of both MG and EAMG. To evaluate the extent at which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (muMT) and wild- type C57BL/6 mice with AChR and complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha146-162 were intact in muMT mice as in wild-type mice. Similar amounts of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in muMT and wild-type mice. However, muMT mice had no detectable anti-AChR antibodies and remained completely free from clinical EAMG. We conclude that B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming.      

An immunodominant site of acetylcholine receptor in experimental myasthenia mapped with T lymphocyte clones and synthetic peptides

Myasthenia gravis (MG) is an autoimmune disease of man caused by antibodies directed against the acetylcholine receptor (AChR). In the experimental model of MG in mice, murine experimental autoimmune myasthenia gravis (EAMG), an anti-AChR immune response is induced by immunization with Torpedo AChR, and anti-AChR antibodies. AChR-sensitized T cells, and neuromuscular dysfunction result. The production of antibodies to AChR is thymus-dependent. In order to define the epitopes of the AChR identified by AChR-specific T cells, we generated T cell populations and T cell hybridoma clones and tested their reactivity to synthetic uniform-sized overlapping peptides representing the entire extracellular portion of the alpha-chain of the AChR. The predominant reactivity of the T cell clones and the parent lines was to a peptide corresponding to residues 146-162 of Torpedo AChR. This data is consistent with a highly limited recognition of AChR determinants in murine EAMG by AChR-specific T cells.     

Myasthenia gravis and its animal model: T cell receptor expression in an antibody mediated autoimmune disease

Myasthenia gravis (MG) is a prototypic antibody-mediated autoimmune disease. Since the primary target antigen of the autoimmune response is known and a well-characterized animal model is available, MG is often considered an excellent situation for the application of novel specific immunotherapies, many of which are directed at T lymphocytes. CD4+ helper T cells are required for the development of the animal model, experimental autoimmune MG (EAMG). Even though the target antigen, acetylcholine receptor (AChR) is immunologically complex, the T cell response to AChR in mice is dominated by recognition of a single peptide by about 50% of the T cells. These T cells, in turn, utilize a restricted set of TCR gene elements and conserved CDR3 regions. While specific therapy directed at the immunodominant T cells is capable of reducing the magnitude of the anti-AChR response, considerable flexibility is apparent and reveals the ability of additional T cells to provide the requisite B cell help. In human MG patients, AChR-specific T cells have been identified but in many studies the frequencies were surprisingly low. In a very few cases, AChR-specific T cells have been cloned from MG patients. Analysis reveals heterogeneity in epitope recognition and MHC restriction. Little information on TCR structure is available. Our own studies using antigen-specific as well as non-specific methods for examining clonal T cell expansions in MG have led to an alternative hypothesis concerning T-B collaboration in MG.     

HLA-DQ6 transgenic mice resistance to experimental autoimmune myasthenia gravis is linked to reduced acetylcholine receptor-specific IFN-gamma, IL-2 and IL-10 production.

To comprehend the reduced susceptibility of HLA-DQ6 transgenic mice in comparison with HLA-DQ8 mice, to experimental autoimmune myasthenia gravis (EAMG), we immunized them with acetylcholine receptor (AChR) and examined in vitro, the proliferative and cytokine responses to AChR. When immunized with AChR and examined for AChR-specific lymphocyte responses to AChR, EAMG-resistant DQ6 mice exhibited significantly reduced in vitro lymphoproliferative and cytokine responses to AChR, compared to DQ8 mice. The differences in susceptibility were not linked to a difference in peptide recognition by AChR-specific lymphocytes. AChR T cell epitope mapping showed that both DQ6 and DQ8 responded to the same epitopes, although to varying degrees. Resistance of DQ6 transgenic mice to EAMG was linked to a dramatic suppression of AChR-specific IFN-gamma, IL-2 and IL-10 productions by AChR-primed lymph node cells.     

T cell receptor transgenic mice recognizing the immunodominant epitope of the Torpedo californica acetylcholine receptor

Myasthenia gravis (MG) is an autoimmune disease caused by T cell-dependent antibody-mediated reduction of acetylcholine receptors (AChR) at the neuromuscular junction. Immunization of animals with Torpedo californica AChR (TAChR) results in an experimental model of MG. We used the variable regions of alpha and beta T cell receptor (TCR) genes recognizing an immunodominant peptide containing amino acids 146-162 from the alpha subunit of TAChR presented in the context of I-A(b) to generate TCR-transgenic mice. We found that the transgenic TCR was strongly positively selected and that transgenic T cells proliferated robustly to the immunodominant peptide and TAChR. Unexpectedly, there was a variable paucity of B cells in the blood and spleen from transgenic mice, which averaged about 16% of peripheral blood lymphocytes, compared to 55% in wild-type B6 mice. Unselected transgenic mice immunized with TAChR exhibited weak anti-TAChR antibody responses. However, transgenic mice selected to have relatively higher B cell numbers produced anti-TAChR titers equal to B6 mice and a predominance of Th1-induced antibody isotypes were observed in certain experiments. The incidence and severity of clinical disease was variable following immunizations. These mice should be useful for studying the pathogenesis and treatment of MG.     

'Split tolerance' induction by intrathymic injection of acetylcholine receptor in a rat model of autoimmune myasthenia gravis; implications for the design of specific immunotherapies.

Experimental autoimmune myasthenia gravis (EAMG) in the Lewis rat, induced by a single injection of acetylcholine receptor (AChR) protein, is a model used to study human myasthenia gravis (MG). The production of anti-AChR antibodies in the animal model and human MG is T cell-dependent, and AChR-specific T cells have been considered as a potential target for specific immunotherapy. Intrathymic injection of antigens induces antigen-specific tolerance in several T cell-mediated autoimmune models. We examined the effect of intrathymic injection of AChR on T cell responses and the production of antibodies to AChR in EAMG rats. Primed lymph node cells from rats receiving intrathymic injection of AChR exhibited reduced proliferation to AChR with marked suppression of interferon-gamma (IFN-gamma) secretion in the antigen-stimulated culture, compared with those of rats injected with PBS. However, neither anti-Narke AChR nor anti-rat AChR antibody production was suppressed or enhanced in intrathymically AChR-injected animals compared with that of animals injected intrathymically with PBS or perithymically with AChR. This 'split tolerance' may be attributable to the suppression of type-1 T helper cells (Th1). Our results suggest that the suppression of Th1 function alone may not be sufficient for the prevention of antibody-mediated autoimmune diseases.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.      

Targeting the antigen-binding site of HLA-restricting alleles in treatment of autoimmune disease

In autoimmune disease, production of disease-causing auto-antibodies (Abs) depends on autoreactive T cells that recognize the epitopes of the pathogenic antigen in the context of MHC class II molecules. It is possible that selective inhibition of an antigen-presenting function of disease-associated MHC alleles could lead to suppression of the disease. Myasthenia gravis (MG) is a disabling neuromuscular disease in which autoimmune responses against acetylcholine receptor (AChR), especially against the alpha chain of AChR, cause a postsynaptic defect. HLA linkage of MG has been thus far best detailed for DQB1. Recently, we have shown that certain DQ haplotypes are associated with presentation of AChR alpha-chain peptides in MG. In a mouse model for MG, which can be induced in disease-susceptible C57BL/6 (B6, H-2b) mice by injection with Torpedo AChR, region 62-76 of I-Ab beta chain is involved in the disease mechanism. Monoclonal Abs (mAbs) against synthetic peptide I-Abetab62-76, which localizes at the rim of the antigen-binding site of I-Ab, inhibited in vitro proliferation of disease-associated T cells. Passive transfer of these mAbs as well as vaccination with this peptide strongly suppressed occurrence of clinical MG in B6 mice. In both cases, Ab and T-cell responses against AChR, especially those related to disease pathogenesis, also decreased. mAbs against peptides from the ridge of the antigen-binding region of the correlate DQB1 sequences inhibited in vitro the proliferation of AChR-specific T cells from MG patients. The results indicated that the function of disease-associated MHC alleles may be blocked by directly and selectively targeting the antigen-presenting region on these MHC molecules. The strategy could provide an effective means for immunointervention in other autoimmune and allergic responses.     

Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies

In the autoimmune disease myasthenia gravis (MG), antibodies against the muscle nicotinic acetylcholine receptor (AChR) cause loss of functional AChR in the neuromuscular junction. To isolate AChR-specific human antibody fragments (Fab), a phage-display library was constructed from an MG patient's thymic B lymphocytes. The first Fab isolated had a low affinity for human AChR, but two sequential antibody chain shufflings using the MG donor heavy and light chain gene repertoires resulted in isolating two new Fab with an approximately 30-fold higher binding ability. The selected Fab contained extensively mutated heavy and light chains and probably represent intraclonal variants of a common progenitor having diverged in vivo by somatic hypermutation. Interestingly, the isolated Fab bound to an extracellular highly immunogenic region located either on an alpha-subunit site affected by the gamma/epsilon-subunits or on the interface between alpha- and gamma/epsilon-subunits. This region is not the previously described "main immunogenic region" (MIR), although it seems to be close to it, as one improved Fab and an anti-MIR mAb competed for AChR binding with distinctly different subpopulations of MG sera. Furthermore, this Fab protected surface AChR in cell cultures against MG autoantibody-induced antigenic modulation, suggesting a potential therapeutic use in MG, especially in combination with a human anti-MIR Fab.     

Pathogenesis of myasthenia gravis

 Various studies over the last 25 years in Man and animal models have revealed many steps in the pathogenesis of myasthenia gravis (MG) which is now considered the classical organ specific, autoantibody mediated and T cell dependent human autoimmune disease. Though not a disease entity, MG is associated with pathological alterations of the thymus in about 80% of cases. These are described here with reference to distinct models of autoimmunization against the acetylcholine receptor (AChR). In MG with thymitis, intrathymic production of AChR-specific autoantibodies is the result of a classical antigen-driven immune reaction that occurs completely inside the thymus and probably involves AChR on myoid cells as the triggering (myasthenogenic) antigen. Genetic factors contribute essentially to the pathogenesis of this form of MG. In thymoma-associated MG genetic factors are probably of marginal significance. Neither intratumour autoantibody production nor T cell activation seem to occur and the AChR is not the myasthenogenic antigen. Instead, abnormal neurofilaments that share epitopes with the AChR and other autoantigen targets in paraneoplastic MG are expressed in thymomas and may trigger autoantigen-specific, non-tolerogenic T cell selection by molecular mimicry. These data support the hypothesis that initial steps in the pathogenesis of most MG cases take place within abnormal thymic microenvironments, be they inflammatory or neoplastic. Where these initial steps occur in MG cases without thymic pathology is not known. Likewise, the factors involved in the initial triggering of MG remain enigmatic in all MG subtypes. Received: 16 September 1996 / Accepted: 28 October 1996     

Delivery of an miR155 inhibitor by anti‐CD20 single‐chain antibody into B cells reduces the acetylcholine receptor‐specific autoantibodies and ameliorates experimental autoimmune myasthenia gravis

MicroRNA‐155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155‐deficient B cells generated reduced germinal centre responses and failed to produce high‐affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up‐regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up‐regulated in torpedo acetylcholine receptor (T‐AChR)‐stimulated B cells. We used an inhibitor of miR155 conjugated to anti‐CD20 single‐chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell‐activating factor (BAFF)‐R‐related signalling pathway and reduced the translocation of nuclear factor (NF)‐κB into the nucleus. Additionally, AChR‐specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.     

Spontaneous production of anti-IFN-alpha and anti-IL-12 autoantibodies by thymoma cells from myasthenia gravis patients suggests autoimmunization in the tumor

Myasthenia gravis (MG) is mediated by autoantibodies to the acetylcholine receptor (AChR), expressed in muscle and rare thymic myoid cells. Most early-onset cases show thymic lymph node-type infiltrates, including pre-activated plasma cells spontaneously producing anti-AChR antibodies. Since these are not evident in the associated thymomas found in another 10% of MG patients, AChR-specific B cells must be autosensitized elsewhere. Unexpectedly, at diagnosis, >70% of MG/thymoma patients also have high-titer neutralizing autoantibodies to IFN-alpha, and >50% to IL-12; moreover, titers increase strikingly if the thymomas recur, indicating a closer tumor relationship than for anti-AChR. To investigate this, we have measured autoantibody production by cells cultured from thymomas, any available thymic remnants and blood, with or without the B cell stimulant pokeweed mitogen (PWM). To check autoantibody specificity and clonal origins, we isolated Fabs from two combinatorial libraries from producer thymus/thymoma cells. Surprisingly, thymoma cells spontaneously produced antibodies to IFN-alpha and/or IL-12 in >40% of seropositive cases, showing typical plasma cell behavior, whereas they produced anti-AChR only after PWM stimulation. We isolated 15 combinatorial Fabs to IFN-alpha (versus only one to AChR). Their strong binding in radio-immunoprecipitation and Western blots implies high affinities. The four Fabs tested neutralized anti-viral actions of IFN-alpha. The diverse V genes clearly showed ongoing antigen-driven selection. These results imply pre-activation in situ by native IFN-alpha/IL-12 expressed within a 'dangerous' tumor microenvironment. With these molecules, it should be easier to identify provoking cell type(s) that may give novel additional clues to autoimmunization against T-cell epitopes from the more complex AChR.     

Epstein-Barr virus-transformed B cells can present acetylcholine receptor to autologous autoreactive T cells

Epstein-Barr virus- (EBV-)transformed B cells were obtained from a patient with myasthenia gravis, who was also the donor of a T cell line specific for acetylcholine receptor (AChR). After three months of culture, the EBV-transformed B cells could effectively present native membrane-bound AChR to autologous AChR-specific T cells.     

Anti-Idiotypic T Cells in Early Stages of Myasthenia Gravis: Increase in the Number and Prevalence Correlated to Clinical Improvement in Patients

An idiotypic network involving T and B cells bearing complementary structures has been suggested to be important for the regulation of immune response in healthy and disease situations. A previous study by the authors has demonstrated the presence of a relatively higher concentration of anti-idiotypic antibodies than of idiotypic antibodies in early myasthenia gravis (MG), suggesting that the development of an anti-idiotypic immunity is important in early MG. The present study was conducted to examine the cellular components of the idiotypic network in the same situation. T and B cells reactive to acetylcholine receptor (AChR) or to a disease-related idiotype and to an anti-idiotype were analysed in seven patients with early MG at various times after the start of the disease. The results show that a significant increase in the number of idiotype-reactive interferon-γ-secreting T cells and a shift from AChR-reactive to idiotype- and/or anti-idiotype-reactive T cells in the patients at 6 month follow-up were noted. Such changes seem to correlate to a clinical improvement in the patients. The enhanced anti-idiotypic T-cell response and the clinical improvement in the patients may speak in favour of a role for the anti-idiotypic immunity in controlling the autoimmune response in MG, i.e., down-regulating autoantibody-producing B cells and idiotypic (AChR-specific) T cells. Thus, an immune intervention towards the enhancement of the anti-idiotypic immunity in patients might be a rewarding approach. Further studies with regard to cell interactions and immune regulations in the network are warranted.     

How Subtle Differences in MHC Class II Affect the Severity of Experimental Myasthenia Gravis

Myasthenia gravis is an autoimmune disorder characterized by muscle weakness, due to an antibody-mediated deficit of acetylcholine receptors (AChRs) at neuromuscular junctions. We analyzed the factors that determine the severity of experimental myasthenia gravis (EAMG) induced by immunization withTorpedoAChR, in two congenic strains of mice—B6 mice, which are highly susceptible to EAMG; and bm12 mice, which are relatively resistant, and differ only in a change of three amino acids in MHC Class II. We prepared large numbers of AChR-specific T cell hybridomas from each strain and characterized their epitope specificities and T cell receptor (TCR) gene usage: Half the B6 hybridomas responded to a single AChR peptide (α 146–162), and their TCR genes encoded restricted Vα and Vβ chains and CDR3 motifs. bm12 hybridomas had different epitope specificities and different, less restricted TCR genes. APCs were able to present AChR or AChR-derived peptides virtually exclusively to hybridomas of their own strain. Levels of antibodies toTorpedoand autoantibodies to mouse AChR were higher in B6 mice, and were biased toward the IgG2b isotype. We conclude that the “better fit” of MHC II, peptide, and TCR in the B6 mice enhanced cognate interactions of APCs with T cells, and T cells with B cells, resulting in a more abundant and pathogenic AChR antibody response, and thus more severe EAMG.     

Human peripheral blood leukocytes transplanted on CB17 scid-scid mice are transferred to their offspring.

Severe combined immunodeficient (scid) mice are deficient in functional T cells and B cells. Hence, scid mice reconstituted with human peripheral blood leukocytes (scid-huPBL) provide an excellent model for analysis of the human immune response under in vivo conditions. We have investigated this model further by analyzing human immune responses in the progeny of scid-huPBL (termed scid-humo). We find markedly elevated levels of human immunoglobulins (Ig) in the serum of scid-humo for more than 12 weeks indicating materno-fetal transfer of human B lymphocytes. Consistent with this finding we obtained evidence for the existence of human lymphocytes in scid-humo. Murine Ig levels in scid-humo were also elevated and surface Ig-expressing cells (probably B cells) were demonstrable. In this respect scid-humo resembled "leaky" scid. In contrast to "leaky" scid, scid-humo accepted transfer of human blood leukocytes. Not only leukocytes from autologous but also those from heterologous donors were accepted. Human Ig levels in scid-humo increased more rapidly as compared to normal scid mice. Thus, despite these increased B cell activities in scid-humo, transferred human leukocytes were not affected indicating that materno-fetal transfer of human cells had caused tolerization or conditioning. This is in contrast to scid mice in which elevated Ig levels correlate with increased failure rates of reconstitution with human blood leukocytes. We propose that scid-humo provide an improved model for studying the human immune responses in an in vivo setting.