The anti-lymphoma activities of anti-CD137 monoclonal antibodies are enhanced in FcγRIII−/− mice

Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc–FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR^?/? strains. 2A-treated FcRγ^?/? mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII^?/? animals. These improved anti-tumor responses were associated with increased splenic CD8β T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.     

A similarity in peptide cross-reactivity between alloantigen- and nominal antigen-induced CD8+ T cell responses in vitro

Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (FcγRI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8⁺ T cells after co-culturing. The CD8⁺ T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-γ) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8⁺ T cell responses. Interestingly, the allo- and self-HLA-restricted CD8⁺ T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.     

IgE regulates T helper cell differentiation through FcγRIII mediated dendritic cell cytokine modulation

Asthma and allergy are characterized by dysregulation of inflammatory responses toward Th2 responses and high serum levels of IgE. IgE plays a role in the effector phase by triggering the degranulation of mast cells after antigen-crosslinking but its role in the induction of helper T cell differentiation is unknown. We have previously shown lymphotoxin is required for maintaining physiological levels of serum IgE which minimize spontaneous Th1-mediated airway inflammation, suggesting a physiological role for IgE in the regulation of T helper cell differentiation. We describe the mechanism in which IgE modulates inflammation by regulating dendritic cell cytokine production. Physiological levels of IgE suppress IL-12 production in the spleen and lung, suggesting IgE limits Th1 responses in vivo. IgE directly stimulates dendritic cells through FcγRIII to suppress IL-12 in vitro and influences APC to skew CD4⁺ T cells toward Th2 differentiation. We demonstrate a novel role for IgE in regulating differentiation of adaptive inflammatory responses through direct interaction with FcγRIII on dendritic cells.     

Comparative conformational studies on the tryptic digestion fragments of human immunoglobulins M and G

IgM¹ immunoglobulins were cleaved into Fabμ and (Fc)5μ fragments by tryptic digestion. Comparative circular dichroism studies with the corresponding IgG fragments show that the Fab portions of IgG and IgM proteins have very similar CD spectral features, although the same is not true for their Fc fragments. These studies indicate the presence of higher amount of beta-structured regions in Fcμ than in Fcγ. Also, there are considerable differences in their pH-dependent structural transitions as measured by CD spectral changes. The conformational differences between IgG and IgM immunoglobulins are more pronounced in their Fc portions, which carry out class specific biological functions, rather than in Fab portions, which contain antigen combining sites.     

Macrophage-T cell interactions. IV. A new receptor on T cells: Ia antigens as receptors for Ig-antigen complexes which appear early after immunization.

The Fc-receptor-positive T cells have been shown to bear a new receptor which is involved in the uptake of cytophilic complexes of Ig and antigen which are detected in the serum of mice within 6 hr following immunization. It was shown that the Fc receptor on T cells is not necessary for the uptake of these complexes. This new receptor is labile during culture of the T cells in vitro and disappears together with the Fc receptors. The new receptor is taken up by the T cells from a macrophage supernate. The evidence presented suggests that the receptor for these complexes bears determinants coded by the I region of the major histocompatibility complex but is distinct from conventional Fc receptor in that it does not bind to Fc fragments of Ig.     

Protection against the allergic airway inflammation depends on the modulation of spleen dendritic cell function and induction of regulatory T cells in mice

Background

Allergen-induced imbalance of specific T regulatory (Treg) cells and T helper 2 cells plays a decisive role in the development of immune response against allergens.

Objective

To evaluate effects and potential mechanisms of DNA vaccine containing ovalbumin (OVA) and Fc fusion on allergic airway inflammation.

Methods

Bronchoalveolar lavage (BAL) levels of inflammatory mediators and leukocyte infiltration, expression of CD_11c ⁺CD₈₀ ⁺ and CD_11c ⁺CD₈₆ ⁺ co-stimulatory molecules in spleen dendritic cells (DCs), circulating CD₄ ⁺ and CD₈ ⁺ T cells, Foxp3⁺ in spleen CD₄ ⁺ T cells and spleen CD₄ ⁺ T cells were measured in OVA-sensitized and challenged animals pretreated with pcDNA, OVA-pcDNA, Fc-pcDNA, and OVA-Fc-pcDNA.

Results

OVA-Sensitized and challenged mice developed airway inflammation and Th2 responses, and decreased the proliferation of peripheral CD₄ ⁺and CD₈ ⁺ T cells and the number of spleen Foxp₃ ⁺ Treg. Those changes with increased INF-γ production and reduced OVA-specific IgE production were protected by the pretreatment with OVA-Fc-pcDNA.

Conclusion

DNA vaccine encoding both Fc and OVA showed more effective than DNA vaccine encoding Fc or OVA alone, through the balance of DCs and Treg.     

Immunological properties of Fc receptor on lymphocytes. 1. Functional differences between Fc receptor-positive and negative lymphocytes in humoral immune responses.

Using an EA rosetting system, it was observed that Fc receptors (FcR) were present on the surface of T cells as well as B cells, and that functional differences existed between FcR-positive (FcR⁺) and FcR-negative (FcR^?) cells in both T and B cells in in vivo humoral immune responses. Approximately 15% of splenic T cells obtained by nylon wool passage are FcR⁺. The number of surface immunoglobulinbearing cells as detected by immunofluorescent staining accounted for less than 10% of these FcR⁺ cells. FcR⁺ and FcR^? T+B-cell populations obtained from spleens contain 60 and 20% of surface immunoglobulin-positive cells, respectively. In the adoptive primary response in which horse RBC and dinitrophenyl-conjugated dextran (DNP-DE) were used as T-dependent and T-independent antigens, respectively, the majority of precursor B cells were FcR^?. In the secondary response using hapten-primed B cells and carrier-primed T cells, the majority of memory B cells for a haptenic determinant were also FcR^?. Furthermore, the majority of functional cells exerting helper activity in the same hapten-carrier system are FcR^? cells, and FcR⁺ T cells collaborate much less effectively with either memory B cells or helper FcR^? T cells.     

Sedimentation Coefficient and Fragility under Hydrodynamic Shear as Measures of Molecular Weight of the DNA of Phage T5

T5 DNA molecules resemble fragments of T2 DNA of molecular weight 84 × 10⁶ with respect to sedimentation coefficient and susceptibility to breakage under hydrodynamic shear. The sedimentation coefficient falls by the same factor when either T2 or T5 DNA is broken at its characteristic critical shear rate. At a given high rate of shear, both DNA's are broken into fragments exhibiting the same sedimentation coefficient. It follows that 84 × 10⁶ is a proper estimate of the molecular weight of T5 DNA, and that particles of phage T5, like those of T2, contain a single DNA molecule.     

Th1 to Th2 immune deviation facilitates,but does not cause,islet allograft tolerance in mice

It has been reported that Th1 to Th2 immune deviation effectively promotes peripheral tolerance in situations involving a limited T cell clone size, such as T cell-dependent autoimmunity and transplantation across minor, but not major, histocompatibility barriers. In this study, we tested the hypothesis that while Th1 to Th2 immune deviation fails to induce tolerance in the MHC-mismatched islet allograft model, it may promote a state that is permissive for tolerance induction. Here, we report that anti-IL-12 did not prevent acute rejection of islet allografts when administered alone. In conjunction with CTLA4/Fc, however, anti-IL-12 greatly facilitated long-term engraftment in three MHC-mismatched strain combinations. Similarly, while non-cytolytic IL-4/Fc, a long-lasting form of IL-4, did not prevent acute graft rejection when administered alone, a low, but not a high, dose of IL-4/Fc synergized with CTLA4/Fc in inducing significant levels of islet allograft tolerance. Moreover, by using a skin allograft adoptive transfer model, we show that these effects induced by anti-IL-12 and IL-4/Fc treatment were associated with an enhancement of the suppressive properties of CD4⁺CD25⁺ regulatory T cells. Thus, anti-IL-12 and low-dose IL-4/Fc facilitate, but do not cause, islet allograft tolerance in mice by increasing the immunosuppressive potency of CD4⁺CD25⁺ regulatory T cells.     

Induction of human B cell differentiation by Fc region activators. II. Stimulation of IL-6 production

Fc region fragments derived from the enzymatic cleavage of human IgG have been shown to induce human peripheral blood-derived B cells to differentiate into Ig secreting cells (ISC). The synthetic peptide p23, corresponding to residues 335 to 357 in the Fc region of human IgG1, represents a region of the molecule responsible for stimulation of ISC formation. Fc region-induced ISC formation requires at least two signals; one supplied by Fc region activators and one supplied by a T cell-derived factor(s). In this report we show that the coculture of human PBMC with pFc' or p23, results in the release of factor(s) that resemble IL-6 in its pattern of biologic activity. This conclusion is based on the observations that supernatants from Fc region-stimulated PBMC cultures contained increased levels of elements that scored as positive in two assays for IL-6: the B9.9 hybridoma growth and the CESS cell differentiation assays. Moreover, RNA from Fc region-stimulation PBMC contained increased levels of IL-6 cDNA-hybridizable elements. Finally, it was observed that rabbit anti-IL-6 inhibited the ability of supernatants derived from Fc region-stimulated PBMC cultures to induce B9.9 cell proliferation as well as p23-induced ISC formation in intact PBMC cultures. Fc region fragments induce both monocytes and T cells to produce IL-6. Taken together, these results indicate that IL-6 is produced in Fc region-stimulated PBMC cultures and is involved in B cell activation by these activators.     

Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer

 Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL_17-1A-VH_17-1A-VH_CD3-VL_CD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (⁵¹Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8⁺ as well as CD4⁺ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8⁺ cells induced high ⁵¹Cr release within 4 h while CD4⁺ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto. Accepted: 14 October 1997     

An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

Intranasal Administration of Antibody-Bound Respiratory Syncytial Virus Particles Efficiently Primes Virus-Specific Immune Responses in Mice

Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn^−/− mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4⁺ T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8⁺ T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.     

Helicobacter pylori pediatric infection changes FcεRI expression in dendritic cells and Treg profile in vivo and in vitro

H. pylori infection shows an inverse relationship with allergies. Dendritic cells regulate mucosal immune responses including the induction of T regulatory cells which are fundamental in Helicobacter pylori-induced dampening of allergies. In this respect expression of high-affinity IgE receptor (FcεRI) has been associated with a regulatory dendritic cell profile. Therefore we aimed to evaluate possible mechanisms by which H. pylori infection might modify atopy in pediatric patients. Here we show that H. pylori-infected children exhibited both increased expression of FcεRI on peripheral myeloid and plasmacytoid dendritic cells and higher levels of Foxp3 and Latency Associated Peptide on T regulatory cells. Moreover, exposure to H. pylori drove increased FcεRI expression and IL-10 secretion by both pediatric H. pylori-exposed monocyte derived dendritic cells and T cells. Finally, we show a positive correlation between expression of FcεRI in circulating myeloid DCs and total Treg cells, suggesting that in children, H. pylori infection may have a modulating role in atopy, mediated by both altered surface expression of FcεRI on children's DC and an increased T regulatory cell profile.     

The effects of corticosteroids on immunoregulation in sarcoidosis.

The effects of in vivo hydrocortisone administration on the kinetics and functional capabilities of cells involved in the immune response in sarcoidosis were examined. Untreated sarcoidosis patients have a decrease in the absolute numbers of circulating T lymphocytes (P < 0.05). However, with regard to the proportions of T lymphocyte subpopulations, there is an increase in the relative proportions of IgG Fc receptor positive T cells (T_G) (P < 0.01), which have suppressor capabilities in certain in vitro systems of mitogen-induced antibody production, and a relative decrease in IgM Fc receptor positive T lymphocytes (T^M) which have helper effects in this system (P < 0.05). Additionally, sarcoidosis patients have circulating “suppressor” monocytes capable of suppressing anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses by pokeweed mitogen (PWM)-stimulated lymphocytes. The in vitro removal of this cell abrogated this depressed response (P < 0.01). Intravenous administration of hydrocortisone produced a transient absolute T lymphocytopenia (P < 0.01) accompanied by a relative increase in T^G cells (P < 0.01) and a relative decrease in T_M cells (P < 0.02). Four hours after hydrocortisone therapy, at the point of maximal hydrocortisone-induced monocytopenia (P < 0.01), the suppressed ability of sarcoidosis lymphocytes to synthesize and secrete in vitro anti-SRBC antibody after polyclonal activation was corrected (P < 0.01), and PFC responses comparable to those seen in untreated normal subjects were obtained. These studies demonstrate that corticosteroid administration has profound effects on certain in vitro demonstrable immunoregulatory abnormalities in sarcoidosis.     

FcRγ Controls the Fas-Dependent Regulatory Function of Lymphoproliferative Double Negative T Cells

Patients with autoimmune lymphoproliferative syndrome (ALPS) and lymphoproliferation (LPR) mice are deficient in Fas, and accumulate large numbers of αβ-TCR⁺, CD4⁻, CD8⁻ double negative (DN) T cells. The function of these DN T cells remains largely unknown. The common γ subunit of the activating Fc receptors, FcRγ, plays an important role in mediating innate immune responses. We have shown previously that a significant proportion of DN T cells express FcRγ, and that this molecule is required for TCR transgenic DN T cells to suppress allogeneic immune responses. Whether FcRγ plays a critical role in LPR DN T cell-mediated suppression of immune responses to auto and allo-antigens is not known. Here, we demonstrated that FcRγ⁺, but not FcRγ⁻ LPR DN T cells could suppress Fas⁺ CD4⁺ and CD8⁺ T cell proliferation in vitro and attenuated CD4⁺ T cell-mediated graft-versus host disease. Although FcRγ expression did not allow LPR DN T cells to inhibit the expansion of Fas-deficient cells within the LPR context, adoptive transfer of FcRγ⁺, but not FcRγ⁻, DN T cells inhibited lymphoproliferation in generalized lymphoproliferative disease (GLD) mice. Furthermore, FcRγ acted in a cell-intrinsic fashion to limit DN T cell accumulation by increasing the rate of apoptosis in proliferated cells. These results indicate that FcRγ can confer Fas-dependent regulatory properties on LPR DN T cells, and suggest that FcRγ may be a novel marker for functional DN Tregs.     

Identification of key genes and pathways associated with different immune statuses of hepatitis B virus infection

We aimed to identify key genes and pathways associated with different immune statuses of hepatitis B virus (HBV) infection. The gene expression and DNA methylation profiles were analysed in different immune statuses of HBV infection. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified, followed by their functional and integrative analyses. The differential expression of IgG Fc receptors (FcγRs) in chronic HBV‐infected patients and immune cells during different stages of HBV infection was investigated. Toll‐like receptor (TLR) signalling pathway (including TLR6) and leucocyte transendothelial migration pathway (including integrin subunit beta 1) were enriched during acute infection. Key DEGs, such as FcγR Ib and FcγR Ia, and interferon‐alpha inducible protein 27 showed correlation with alanine aminotransferase levels, and they were differentially expressed between acute and immune‐tolerant phases and between immune‐tolerant and immune‐clearance phases. The integrative analysis of DNA methylation profile showed that lowly methylated and highly expressed genes, including cytotoxic T lymphocyte‐associated protein 4 and mitogen‐activated protein kinase 3 were enriched in T cell receptor signalling pathway during acute infection. Highly methylated and lowly expressed genes, such as Ras association domain family member 1 and cyclin‐dependent kinase inhibitor 2A were identified in chronic infection. Furthermore, differentially expressed FcγR Ia, FcγR IIa and FcγR IIb, CD3^?CD56⁺CD16⁺ natural killer cells and CD14^highCD16⁺ monocytes were identified between immune‐tolerant and immune‐clearance phases by experimental validation. The above genes and pathways may be used to distinguish different immune statuses of HBV infection.     

Effects of prolonged treatment with cyanoketone on the zona fasciculata of rat adrenal cortex

Summary We have examined IgG Fc receptor (FcR) activity of human and rabbit arachnoid granulations and leptomeninges using antibody (IgG)-coated erythrocytes (EIgG), covalently crosslinked IgG dimers, trimers and oligomers, immune complexes, aggregated Fc fragments and a monoclonal anti-human neutrophil Fc receptor antibody, 3G8. EIgG bound specifically to cells of the leptomeninges and arachnoid granulations; uncoated erythrocytes, F(ab) ₂-coated, or IgM-coated erythrocytes failed to bind. The specificity of this interaction was demonstrated by inhibition studies. Monomeric IgG and Fc fragments blocked EIgG adherence, whereas bovine serum albumin (BSA), Fab fragments of IgG and the monoclonal anti-neutrophil FcR antibody failed to inhibit EIgG adherence. Monomeric IgG inhibited FcR function in a dose-dependent fashion; maximal inhibition was achieved at 1.7 × 10^-5M IgG, indicating a relatively low avidity receptor. Oligomers of IgG inhibited EIgG adherence more effectively and inhibition was directly related to oligomer size. Additionally, these tissues were positive for specific and non-specific esterases. These studies suggest that the CSF pathway from the perivascular spaces to the arachnoid granulations plays a protective role in the clearance of IgG and IgG immune complexes in infections and immune-mediated disorders.Work partially supported by PHS Grant #Ca 38055, National Cancer InstituteWork primarily supported by the Veterans Administration Merit Program     

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.     

Immunosuppression by cobra factor: distribution, antigen-induced blast transformation and trapping of lymphocytes during in vivo complement depletion.

In vivo administration of cobra factor (CoF), the C3-activating protein of cobra venom, suppresses thymus-dependent antibody production. In a study of possible mechanisms for this effect binding of CoF to murine spleen cells in vitro was not detected, nor was there any effect on C3 or Fc receptors. The numbers of spleen cells bearing C3 receptors, Fc receptors, θ antigen or surface immunoglobulin were not altered by in vivo complement depletion of mice with CoF. The distribution and antigen-induced trapping of transferred ⁵¹Cr-labelled syngeneic spleen cells were unaffected by treatment of either donors or recipients with CoF. Furthermore, the antigen-induced generation, trapping and specific retention of immunospecific blast cells were normal in CoF-treated mice, despite profound suppression in these animals of IgG antibody production. The majority of these blast cells 3 days after immunisation were T cells, suggesting that complement depletion interferes with the process of T-dependent antibody production at a later stage than the activation of T cells by antigen.