Ras isoforms selectively regulate antigen-specific immune response.

H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1-165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated anti-TNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.

Hepatic Stellate Cells Directly Inhibit B Cells via Programmed Death-Ligand 1.

We demonstrated previously that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1), but it remains unknown whether they exert any effects on B cells, the other component of the adaptive immune system. In this study, we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that HSCs inhibited the upregulation of activation markers on activated B cells, as well as the proliferation of activated B cells and their cytokine/Ig production in vitro, and that pharmaceutically or genetically blocking the interaction of PD-L1 with programmed cell death protein 1 impaired the ability of HSCs to inhibit B cells. To test the newly discovered B cell-inhibitory activity of HSCs in vivo, we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll, a T cell-independent Ag, significantly suppressed Ag-specific IgM and IgG production in vivo, whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion, in addition to inhibiting T cells, mouse HSCs concurrently inhibit B cells via PD-L1. This direct B cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver's immune homeostasis.

Caspase-1 is required for maintenance of marginal zone B cells in pristane-induced lupus.

OBJECTIVE: Caspase-1 is required for nephritis and robust autoantibody development in the pristane model of murine lupus. The objective of this study was to evaluate the immune response and to study the splenic B and T cell populations in wild-type (WT) and caspase-1-/- mice following pristane injection in order to develop an understanding of why absence of caspase-1 is protective in pristane-induced lupus. METHODS: Immunization responses to NP-Ficoll and NP-ovalbumin were assessed in WT and caspase-1-/- mice. In vitro IgM and IgG responses to R848 were measured by ELISA. Serum IgM anti-dsDNA and IL-1ß were also measured by ELISA. B and T cell populations 2 weeks and 6 months following pristane injection were measured by flow cytometry in WT and caspase-1-/- mice. RESULTS: Caspase-1-/- mice generate equivalent IgG responses to NP-Ficoll and NP-ova antigens when compared to wild-type mice. Additionally, they secrete IgM and IgG in response to TLR7 activation. Pristane injected WT and caspase-1-/- mice generate robust IgM anti-dsDNA responses. Caspase-1-/- mice have a significant reduction in marginal zone B cell populations compared to WT 6 months after pristane exposure whereas T cell responses are intact in these mice. CONCLUSIONS: Caspase-1-/- mice have intact immune responses but do not develop an expanded marginal zone B cell population in response to pristane-induced lupus. This may be one explanation for reduced IgG autoantibody production in these mice.

gp49B-mediated negative regulation of antibody production by memory and marginal zone B cells.

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvß3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for peripheral B-cell development and function.

The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.

14-3-3sigma regulates B-cell homeostasis through stabilization of FOXO1.

14-3-3σ regulates cytokinesis and cell cycle arrest induced by DNA damage but its role in the immune system is unknown. Using gene-targeted 14-3-3σ-deficient (i.e., KO) mice, we studied the role of 14-3-3σ in B-cell functions. Total numbers of B cells were reduced by spontaneous apoptosis of peripheral B cells. Upon B-cell antigen receptor engagement in vitro, KO B cells did not proliferate properly or up-regulate CD86. In response to T cell-independent antigens, KO B cells showed poor secretion of antigen-specific IgM. This deficit led to increased lethality of KO mice after vesicular stomatitis virus infection. KO B cells showed elevated total FOXO transcriptional activity but also increased FOXO1 degradation. Coimmunoprecipitation revealed that endogenous 14-3-3σ protein formed a complex with FOXO1 protein. Our results suggest that 14-3-3σ maintains FOXO1 at a consistent level critical for normal B-cell antigen receptor signaling and B-cell survival.

Tolerance induction of IgG+ memory B cells by T cell-independent type II antigens.

Memory B cells generated during a T cell-dependent immune response rapidly respond to a secondary immunization by producing abundant IgG Abs that bind cognate Ag with high affinity. It is currently unclear whether this heightened recall response by memory B cells is due to augmented IgG-BCR signaling, which has only been demonstrated in the context of naive transgenic B cells. To address this question, we examined whether memory B cells can respond in vivo to Ags that stimulate only through BCR, namely T cell-independent type II (TI-II) Ags. In this study, we show that the TI-II Ag (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll cannot elicit the recall response in mice first immunized with the T cell-dependent Ag NP-chicken γ-globulin. Moreover, the NP-Ficoll challenge in vivo as well as in vitro significantly inhibits a subsequent recall response to NP-chicken γ-globulin in a B cell-intrinsic manner. This NP-Ficoll-mediated tolerance is caused by the preferential elimination of IgG(+) memory B cells binding to NP with high affinity. These data indicate that BCR cross-linking with a TI-II Ag does not activate IgG(+) memory B cells, but rather tolerizes them, identifying a terminal checkpoint of memory B cell differentiation that may prevent autoimmunity.

Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens.

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.

Dibenzo[def,p]chrysene (DBC) suppresses antibody formation in spleen cells following oral exposures of mice.

Dibenzo[def,p]chrysene (DBC) is a potent environmental carcinogen in rodents, fish, and human cells examined in culture. There are numerous similarities between the patterns of cytochrome P-450 (P450) activation of DBC and its covalent binding to DNA and proteins with another polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA). Our lab has previously shown that DMBA produces immunosuppression in rodents and human cell systems. Therefore, the purpose of these studies was to examine the immunotoxicity of DBC in a rodent model that was found to be sensitive to the immunosuppressive effects of DMBA. Data showed that DBC had similar potency to DMBA in producing suppression of a T-dependent antibody response (TDAR) and altered spleen cell subsets in a similar manner as DMBA when DMBA was given by gavage for 5 d in corn oil to mice at doses of 1-100 mg/kg total cumulative doses. T-cell-independent antigen (TNP-Ficoll) responses were quantitatively less sensitive to DBC suppression. It was also found that as with DMBA, DBC produced a persistent immunosuppression, which lasted for at least 4 wk following dosing with a novel pill method for self-administration of DBC. In conclusion, DBC appears to possess many of the same characteristics of DMBA in terms of its immunotoxicity.

Influence of molecular shape, conformability, net surface charge, and tissue interaction on transscleral macromolecular diffusion.

The purpose of this study was to investigate the influence of molecular shape, conformability, net surface charge and tissue interaction on transscleral diffusion. Unfixed, porcine sclera was clamped in an Ussing chamber. Fluorophore-labelled neutral albumin, neutral dextran, or neutral ficoll were placed in one hemi-chamber and the rate of transscleral diffusion was measured over 24 h using a spectrophotometer. Experiments were repeated using dextrans and ficoll with positive or negative net surface charges. Fluorescence recovery after photobleaching (FRAP) was undertaken to compare transscleral diffusion with diffusion through a solution. All molecules were 70 kDa. With FRAP, the diffusion coefficient (D) of neutral molecules was highest for albumin, followed by ficoll, then dextran (p < 0.0001). Positive dextrans diffused fastest, followed by negative, then neutral dextrans (p = 0.0004). Neutral ficoll diffused the fastest, followed by positive then negative ficoll (p = 0.5865). For the neutral molecules, transscleral D was highest for albumin, followed by dextran, then ficoll (p < 0.0001). D was highest for negative ficoll, followed by neutral, then positive ficoll (p < 0.0001). By contrast, D was highest for positive dextran, followed by neutral, then negative dextran (p = 0.0021). In conclusion, diffusion in free solution does not predict transscleral diffusion and the molecular-tissue interaction is important. Molecular size, shape, and charge may all markedly influence transscleral diffusion, as may conformability to a lesser degree, but their effects may be diametrically opposed in different molecules, and their influence on diffusion is more complex than previously thought. Each variable cannot be considered in isolation, and the interplay of all these variables needs to be tested, when selecting or designing drugs for transscleral delivery.

Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay.

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.

Basal lamina secreted by MDCK cells has size- and charge-selective properties.

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, respectively, during pressure-driven filtration. Streaming potential measurement indicated the presence of fixed, negative charge in the basal lamina. The sieving coefficient for neutral Ficoll 400 decreased by ∼0.0013 for each 1-Šincrement in solute radius, compared with a decrease of 0.0023 per Šfor the anionic Ficoll 400. In this system, molecular charge played a significant role in determining the sieving characteristics of the membrane, pointing to solute charge as a potential contributor to GBM permselectivity.

Reduced diffusion of charge-modified, conformationally intact anionic Ficoll relative to neutral Ficoll across the rat glomerular filtration barrier in vivo.

The glomerular filtration barrier (GFB) is commonly conceived as a negatively charged sieve to proteins. Recent studies, however, indicate that glomerular charge effects are small for anionic, carboxymethylated (CM) dextran vs. neutral dextran. Furthermore, two studies assessing the glomerular sieving coefficients (θ) for negative CM-Ficoll vs. native Ficoll have demonstrated an increased glomerular permeability for CM-Ficoll (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Physiol 291: F1083-F1089, 2006; Guimarães M, Nikolovski J, Pratt L, Greive K, Comper W. Am Physiol Renal Physiol 285: F1118-F1124, 2003.). The CM-Ficoll used, however, showed a larger Stokes-Einstein radius (a(e)) than neutral Ficoll, and it was proposed that the introduction of negative charges in the Ficoll molecule had made it more flexible and permeable. Recently, a negative FITC-labeled CM-Ficoll (CMI-Ficoll) was produced with a conformation identical to that of neutral FITC-Ficoll. Using these probes, we determined their θ:s in anesthetized Wistar rats (259 ± 2.5 g). After blood access had been achieved, the left ureter was cannulated for urine sampling. Either polysaccharide was infused (iv) together with a filtration marker, and urine and plasma were collected. Assessment of θ FITC-Ficoll was achieved by high-performance size-exclusion chromatography (HPSEC). CMI-Ficoll and native Ficoll had identical elugrams on the HPSEC. Diffusion of anionic Ficoll was significantly reduced compared with that of neutral Ficoll across the GFB for molecules of a(e) ∼20-35 Å, while there were no charge effects for Ficoll of a(e) = 35-80 Å. The data are consistent with a charge effect present in "small pores," but not in "large pores," of the GFB and mimicked those obtained for anionic membranes in vitro for the same probes.

Transient and sustained increases in glomerular permeability following ANP infusion in rats.

The present study was performed to investigate the effects of systemic atrial natriuretic peptide (ANP) infusion on the glomerular permeability to macromolecules in rats. In anesthetized Wistar rats (250-280 g), the left urether was cannulated for urine collection while simultaneously blood access was achieved. Rats were continuously infused intravenously with ANP [30 ng·kg(-1)·min(-1) (Lo-ANP; n=8) or 800 ng·kg(-1)·min(-1) (Hi-ANP; n=10)] or 0.9% NaCl (SHAM; n=16), respectively, and with polydisperse FITC-Ficoll-70/400 (molecular radius 13-90 Å) and 51Cr-EDTA for 2 h. Plasma and urine samples were taken at 5, 15, 30, 60, and 120 min of ANP infusion and analyzed by high-performance size-exclusion chromatography (HPLC) for determination of glomerular sieving coefficients (θ) for Ficoll. GFR was also assessed (51Cr-EDTA). In Hi-ANP, there was a rapid (within 5 min), but bimodal, increase in glomerular permeability. θ to high-molecular-weight Ficoll thus reached a maximum at 15 min, after which θ returned to near control at 30 min, to again increase moderately at 60 and 120 min. In Lo-ANP, there was also a rapid, reversible increase in glomerular θ, returning to near control at 30 min, followed by just a tendency of a sustained increase in permeability, but with a significant increase in "large-pore" radius. In conclusion, in Hi-ANP there was a rapid increase in glomerular permeability, with an early, partly reversible permeability peak, followed by a (moderate) sustained increase in permeability. In Lo-ANP animals, only the initial permeability peak was evident. In both Lo-ANP and Hi-ANP, the glomerular sieving pattern observed was found to mainly reflect an increase in the number and radius of large pores in the glomerular filter.

The murine equivalent of the A181E TACI mutation associated with common variable immunodeficiency severely impairs B-cell function.

TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NF kappaB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI(-/-) background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.

The C104R mutant impairs the function of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) through haploinsufficiency.

BACKGROUND: TNFRSF13B, which encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), is mutated in 10% of patients with common variable immunodeficiency. One of the 2 most common TACI mutations in common variable immunodeficiency, C104R, abolishes ligand binding and is found predominantly in the heterozygous state. The murine TACI mutant C76R is the equivalent of the human TACI mutant C104R. OBJECTIVE: We sought to define the consequence of the C76R mutation on TACI function in mice that express both wild-type TACI and the murine C76R mutant. METHODS: Transgenic mice that express murine TACI C76R, the counterpart of human TACI C104R, on the TACI(+/-) B6/129 background (C76R/TACI(+/-) mice) were constructed. Serum immunoglobulins and antibody responses to the type II T-independent antigen trinitrophenylated (TNP)-Ficoll were determined by means of ELISA. B-cell proliferation in response to a proliferation-inducing ligand was determined based on tritiated thymidine incorporation into DNA. IgG1 secretion by B cells in response to a proliferation-inducing ligand plus IL-4 was determined by means of ELISA. RESULTS: C76R/TACI(+/-) mice had significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll compared with TACI(+/+) B6/129 control animals, and their B cells were impaired in their capacity to proliferate and secrete IgG1 in response to TACI ligation. Unexpectedly, TACI(+/-) mice had similarly impaired B-cell function as C76R/TACI(+/-) littermates. Impaired TACI function caused by haploinsufficiency was confirmed in TACI(+/-) mice on the C57BL/6 background. CONCLUSION: These results suggest that the human TACI mutant C104R might impair TACI function in heterozygotes through haploinsufficiency.

Acute hyperglycemia induces rapid, reversible increases in glomerular permeability in nondiabetic rats.

This study was performed to investigate the impact of acute hyperglycemia (HG) on the permeability of the normal glomerular filtration barrier in vivo. In anesthetized Wistar rats (250-280 g), the left ureter was catheterized for urine collection, while simultaneously blood access was achieved. Rats received an intravenous (iv) infusion of either 1) hypertonic glucose to maintain blood glucose at 20-25 mM (G; n = 8); 2) hypertonic glucose as in 1) and a RhoA-kinase inhibitor (Y-27632; Rho-G; n = 8); 3) 20% mannitol (MANN; n = 7) or 4) hypertonic (12%) NaCl to maintain plasma crystalloid osmotic pressure (pi(cry)) at approximately 320-325 mosmol/l (NaCl; n = 8) or 5) physiological saline (SHAM; n = 8). FITC-Ficoll 70/400 was infused iv for at least 20 min before termination of the experiments, and plasma and urine were collected to determine the glomerular sieving coefficients (theta) for polydisperse Ficoll (molecular radius 15-80 A) by high-performance size-exclusion chromatography. In G there was a marked increase in for Ficoll(55-80A) at 20 min, which was completely reversible within 60 min and abrogated by a Rho-kinase (ROCK) inhibitor, while glomerular permeability remained unchanged in MANN and NaCl. In conclusion, acute HG caused rapid, reversible increases in for large Ficolls, not related to the concomitant hyperosmolarity, but sensitive to ROCK inhibition. The changes observed were consistent with the formation of an increased number of large pores in the glomerular filter. The sensitivity of the permeability changes to ROCK inhibition strongly indicates that the cytoskeleton of the cells in the glomerular barrier may be involved in these alterations.

Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

X-linked B-lymphocyte immune defect in CBA/N mice. II. Studies of the mechanisms underlying the immune defect.

The mechanisms underlying the X-linked thymus-independent (B) lymphocyte functional defect in the CBA/N (CN) mice and their F1 progeny were studied. Immune defective mice were unable to respond to the T-independent antigen 2,4-dinitrophenyl-lysyl-derivative of Ficoll (DNP-lys-Ficoll) but were able to form antibody against the highly cross-reactive hapten (trinitrophenyl) when it was coupled to an erythrocyte carrier. Immune defective CN X DBA/2N (DN) F1 male mice, which do not normally respond to T-independent antigens, were able to respond to both polyribosinic-polyribocytidylic acid and DNP-lys-Ficoll after the administration of CN X DN F1 female spleen cells even if these cells had been depleted of T lymphocytes. In addition, it was shown that the inability of the CN mice and their F1 progeny to respond to T-independent antigens was not due to an intrinsic abnormality of their microenvironment or the suppressive actions of a T lymphocyte. Our data present evidence that the X-linked defect in the CN mice is due to an intrinsic defect in B-lymphocyte development.

Prolonged survival in vivo of unprimed B cells responsive to a T-independent antigen.

Despite earlier evidence to the contrary, it has recently been claimed that most B lymphocytes, including lymph node (LN) and thoracic duct B cells, are short-lived cells of recent marrow origin. To seek direct information on this question, we transferred unprimed LN or thoracic duct B cells from normal mice to xid mice, i.e., mice unresponsive to the T-independent antigen, trinitrophenyl (TNP)-Ficoll. At varying periods after B cell transfer the recipients were challenged with TNP-Ficoll; anti-TNP plaque-forming cells were assayed in the spleen 6 d later. The results showed that the B cell recipients retained responsiveness to TNP-Ficoll for at least 3 mo after transfer. Responsiveness increased within the first 3 wk but then remained relatively constant. These findings imply that, at least for TNP-Ficoll-reactive cells, B cells residing in LN and thoracic duct lymph are not short-lived cells of recent marrow. Indeed, the data suggest that once the pool of recirculating B cells is fully formed in adult mice, further input of newly formed cells from the marrow into the recirculating pool is very limited.