B-1a transitional cells are phenotypically distinct and are lacking in mice deficient in IκBNS.

B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93(+)IgM(+)CD5(+)) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93(+)IgM(+)CD5(-) cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS.

Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety. OBJECTIVE: To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice. METHODS: We transplanted Was(-/-) mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was(-/-) cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum. RESULTS: Here we show that WAS protein(+) B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies. CONCLUSION: WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector.

Regulation of T-cell-independent and T-cell-dependent antibody production by circadian rhythm and melatonin.

Melatonin is a hormone that has immunomodulatory activity and is believed to influence the production of antibodies in mammals. The aim of the present study was to investigate the effect of suppressed melatonin synthesis on the antibody production. BALB/c mice were immunized with T-cell-dependent (TD) and T-cell-independent (TI) antigens and kept under (i) normal lighting, (ii) constant exposure to light, (iii) exposed to light and treated daily with melatonin. It was revealed that melatonin modulated TD and TI antibody production. Suppressed melatonin synthesis increased the amount of IgM, IgG1, IgG2b and IgG3 antibodies after immunization with TI antigen. The level of TD antibodies IgM, IgG2a, IgG2b and IgG3 also increased, however, the antigen-specific antibodies of IgG1 isotype significantly decreased in mice exposed to light. Daily melatonin treatment brought the antibody level back to normal. The antibody concentration in the sera of mice kept at normal lighting was significantly higher when the immunizations were performed in the evening. The action of melatonin on B cells via MT2 receptor was shown in vitro and in vivo.

Nasal cholera toxin elicits IL-5 and IL-5 receptor alpha-chain expressing B-1a B cells for innate mucosal IgA antibody responses.

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.

Antibody structural variation in rainbow trout fluids.

Rainbow trout (Oncorhynchus mykiss) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) and the redox structure of induced anti-TNP antibodies from the serum, mucus, egg and ovarian fluid was examined. In conducting these studies it was determined that all TNP-specific antibody from each source possessed the mAb-specific H chain (1-14) epitopes, which facilitated the direct structural analysis of the induced antibodies. A protocol was developed which ensured complete adsorption of all specific anti-TNP antibody from each fluid. Together these protocols permitted the unbiased compositional analysis of all redox forms of the anti-TNP antibodies from each source. All antibodies, regardless of source, possessed the same molecular mass, characteristic of the trout tetramer (800 kDa). It was found that specific antibody titers were significantly higher in male than female trout, while the degree of disulfide polymerization was relatively invariant in male antibodies, while being highly variable in female antibodies. Within the females, no distinctively different redox ratios were between antibodies isolated from sera, ovarian fluid or eggs: however, mucus antibodies possessed a unique redox structure consisting of halfmeric constituents that were not observed in antibodies from other fluids.

Antibody repertoire development in fetal and neonatal piglets. IX. Three pathogen-associated molecular patterns act synergistically to allow germfree piglets to respond to type 2 thymus-independent and thymus-dependent antigens.

Newborn piglets maintained germfree (GF) cannot respond to either thymus-dependent (TD) or type 2 thymus-independent Ags (TI-2) unless colonized with bacteria. We show here that pathogen-associated molecular patterns (PAMPs), including muramyl dipeptide (MDP), LPS, and a B-class CpG oligonucleotide (CpG-B), can substitute for gut flora in the induction of neonatal immunoresponsiveness. These PAMPs alone or in combination had little effect on serum IgG and IgA levels, but CpG-B and CpG-B + MDP elevated total IgM levels 3- to 7-fold above that seen in colonized controls after booster immunization. Although only CpG-B could alone stimulate immunoresponsiveness, co-administration of LPS or MDP resulted in a 5-fold increase in the IgG response to both immunogens. Co-administered MDP did not promote secondary IgG responses to either Ag but instead pronounced secondary IgM responses to the epitopes of both immunogens. LPS co-administered with CpG-B may promote class switch recombination or cause differentiation of previously switched cells that become responsive after exposure to CpG-B. Primary and secondary IgG responses equally recognized the epitopes of the TI-2 and TD immunogens, whereas IgM responses favored the TI-2 epitope. Because PAMPs alone can result in Abs to 2,4,6-triitrophenyl and FLU without immunization, it suggests they alone cause differentiation of B cells of the preimmune repertoire. The finding that both bacterial PAMPs and colonization are capable of stimulating Ab responses in both immunized and nonimmunized piglets suggests that PAMPs derived from host flora may play a major role in awakening adaptive immunity in neonates.

Rapid induction of splenic and peritoneal B-1a cells in adult mice by thymus-independent type-2 antigen.

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.

Scurfin (FoxP3) controls T-dependent immune responses in vivo through regulation of CD4+ T cell effector function.

Scurfin, the protein product of the FoxP3 gene, is a forkhead-family transcription factor that negatively regulates T cell function. Mice carrying a loss-of-function mutation in FoxP3 (scurfy mice) present with fatal autoimmune-like disease caused by hyperresponsive CD4(+) T cells. Mice that overexpress scurfin (FoxP3 Tg mice) possess fewer mature T cells with reduced functional capabilities compared with normal littermate control mice. We analyzed the ability of CD4(+) T cells and B cells from FoxP3 Tg mice to respond to a T-dependent Ag and found that immunized FoxP3 Tg mice displayed reduced total and Ag-specific serum Ig and disorganized splenic architecture. However, when cultured in vitro, FoxP3 Tg B cells responded normally, suggesting that the poor Ab response was a result of defective T cell help in vivo. When challenged, CD4(+) T cells from FoxP3 Tg mice display reduced up-regulation of CD40 ligand and fewer IFN-gamma-producing cells. Overall, these findings show that overexpression of scurfin reduces T cell responses in vivo such that CD4(+) T cells cannot provide help to B cells during a T cell-dependent Ab response.

Architectural defects in the spleens of Nkx2-3-deficient mice are intrinsic and associated with defects in both B cell maturation and T cell-dependent immune responses.

Mice lacking the homeodomain transcription factor Nkx2-3 are either asplenic or develop a spleen of significantly reduced size with poorly organized white pulp. In this report, we analyze the effect of this mutation on B lymphocyte development and differentiation. Follicular dendritic cells in spleen, but not lymph node, of Nkx2-3(-/-) mice fail to express a developmental Ag (follicular dendritic cell-M2) and show an abnormal association with B cells, despite essentially normal expression of several chemokine genes. Bone marrow reconstitution studies show the splenic disorganization and absence of marginal zone B cells to be of stromal rather than hemopoietic origin. Furthermore, Nkx2-3(-/-) mice show an excess of conventional B cells in mesenteric lymph node and peritoneal cavity, whereas transitional B cells are rare in spleen but overrepresented in bone marrow. Finally, immunization of Nkx2-3(-/-) mice with a T cell-dependent Ag elicits clusters of germinal center B cells, although these fail to develop to the same extent as in controls and there is no evidence of affinity maturation in serum Ab. Similarly, Ab-forming cells fail to aggregate into foci early in the response. Collectively, these data indicate a substantial role for Nkx2-3 in the correct association of lymphocytes and splenic stromal elements that is independent of chemokine expression.

IKK beta is required for peripheral B cell survival and proliferation.

NF-kappaB activity in mammalian cells is regulated through the IkappaB kinase (IKK) complex, consisting of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma). Targeted deletion of Ikkbeta results in early embryonic lethality, thus complicating the examination of IKKbeta function in adult tissues. Here we describe the role of IKKbeta in B lymphocytes made possible by generation of a mouse strain that expresses a conditional Ikkbeta allele. We find that the loss of IKKbeta results in a dramatic reduction in all peripheral B cell subsets due to associated defects in cell survival. IKKbeta-deficient B cells are also impaired in mitogenic responses to LPS, anti-CD40, and anti-IgM, indicating a general defect in the ability to activate the canonical NF-kappaB signaling pathway. These findings are consistent with a failure to mount effective Ab responses to T cell-dependent and independent Ags. Thus, IKKbeta provides a requisite role in B cell activation and maintenance and thus is a key determinant of humoral immunity.

The Yaa mutation promoting murine lupus causes defective development of marginal zone B cells.

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.

B cell developmental requirement for the G alpha i2 gene.

Null mutation of the Galphai2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that Galphai2 is required for the development of IL-10-producing B cells. Galphai2(-/-) mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2(-/-) mice with Galphai2(-/-) bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the Galphai2(-/-) genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of Galphai2(-/-) mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, Galphai2(-/-) mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for Galphai2 in MZ and B-1 B cell development. Disorders of this Galphai2-dependent process in B cell development may represent a mechanism for IBD susceptibility.

Differential requirements for expression of CD80/86 and CD40 on B cells for T-dependent antibody responses in vivo.

The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.

Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens.

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.

The absence of Tssc6, a member of the tetraspanin superfamily, does not affect lymphoid development but enhances in vitro T-cell proliferative responses.

The tetraspanins are a family of integral membrane proteins with four transmembrane domains. These molecules form multimolecular networks on the surfaces of many different cell types. Gene-targeting studies have revealed a role for tetraspanins in B- and T-lymphocyte function. We have isolated and deleted a novel tetraspanin, Tssc6, which is expressed exclusively in hematopoietic and lymphoid organs. Using a gene-trapping strategy, we generated an embryonic stem (ES) cell line with an insertion in the Tssc6 locus. Mice were derived from these ES cells and, using RNase protection and reverse transcription-PCR, we demonstrated that the insertion resulted in a null mutation of the Tssc6 allele. Mice homozygous for the gene trap insertion (Tssc6(gt/gt) mice) were viable and fertile, with normal steady-state hematopoiesis. Furthermore, responses to hemolysis and granulocyte colony-stimulating factor-induced granulopoiesis were equivalent to those of wild-type mice. Lymphoid development was normal in Tssc6(gt/gt) mice. Whereas Tssc6(gt/gt) B cells responded normally to lipopolysaccharide, anti-CD40, and anti-immunoglobulin M stimulation, Tssc6(gt/gt) T cells showed enhanced responses to concanavalin A, anti-CD3, and anti-CD28. This increased proliferation by Tssc6-deleted T lymphocytes was due to increased interleukin 2 production following T-cell receptor stimulation. These results demonstrate that Tssc6 is not required for normal development of the hematopoietic system but may play a role in the negative regulation of peripheral T-lymphocyte proliferation.

Low-level hypermutation in T cell-independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers.

Exceptionally germinal center formation can be induced without T cell help by polysaccharide-based antigens, but these germinal centers involute by massive B cell apoptosis at the time centrocyte selection starts. This study investigates whether B cells in germinal centers induced by the T cell-independent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to Ficoll undergo hypermutation in their immunoglobulin V region genes. Positive controls are provided by comparing germinal centers at the same stage of development in carrier-primed mice immunized with a T cell-dependent antigen: NP protein conjugate. False positive results from background germinal centers and false negatives from non-B cells in germinal centers were avoided by transferring B cells with a transgenic B cell receptor into congenic controls not carrying the transgene. By 4 d after immunization, hypermutation was well advanced in the T cell-dependent germinal centers. By contrast, the mutation rate for T cell-independent germinal centers was low, but significantly higher than in NP-specific B cells from nonimmunized transgenic mice. Interestingly, a similar rate of mutation was seen in extrafollicular plasma cells at this stage. It is concluded that efficient activation of hypermutation depends on interaction with T cells, but some hypermutation may be induced without such signals, even outside germinal centers.

Accessory cell defect in unresponsiveness of neonates and aged to polysaccharide vaccines.

T independent antigens elicit antibody responses in the absence of carrier specific T helper cells but require signals from accessory cells (macrophages and dendritic cells) or specific cytokines. They are further subdivided into TI-1 and TI-2 categories based on the ability of TI-1 but not TI-2 antigens to elicit immune responses from neonates. Most bacterial polysaccharides including the pneumococcal polysaccharide vaccines belong to the TI-2 class. It is hypothesized that defects in accessory cell function play a critical role in the failure of neonates to respond to such TI-2 antigens. Immune responses to these TI-2 stimuli are also reduced in the aged, also due to a quantitative deficiency in accessory cells. Agents that can stimulate accessory cell function may provide an alternative strategy to improve the immunogenicity of the polysaccharide vaccines in the neonates and the aged.

Carrier-induced, hapten-specific suppression: a problem of antigen presentation?

Prior immunity against a carrier protein has been shown to modulate the serologic response to injected haptens attached to the same carrier. In particular, a carrier/hapten-carrier immunization protocol induces marked suppression for IgG2a anti-hapten Ab production but does not interfere with anti-carrier Ab responses. Although the phenomenon of epitopic suppression has been amply demonstrated, the mechanism underlying the suppression remains unknown. The selective deficiency in IgG2a secretion suggests that IFN-gamma-producing Th1 cells are not properly activated. We and others have shown that the nature of the APCs present during the first encounter with the Ag influences the development of selected Th populations in vivo; dendritic cells (DCs) seem to be required for the induction of primary, Th1-type responses. Since carrier priming induces the clonal expansion of specific B cells that appear to efficiently capture the Ag, we hypothesized that the hapten-carrier conjugate may be presented by B cells in preimmunized animals. Therefore, we immunized mice to the conjugate by injecting syngeneic DCs pulsed in vitro with the Ag. Our data show that an injection of DCs and IL-12 prevents epitopic suppression, suggesting that it may result from defective Ag presentation.

Acute and long-term effects of stressors on pulmonary immune functions.

To study the effects of different types or intensities of stressors on immune reactivity in the lungs, we studied the ex vivo production of nitric oxide (NO) and IL-1beta by alveolar macrophages (AM) after short exposure of rats to restraint stress or inescapable electric footshocks. Exposure to electric footshocks of various intensities resulted in an intensity-dependent decrease in NO production whereas the IL-1beta production by AM had increased. The secretory activity was similarly affected by restraint stress. When the time course of electric footshocks on secretory functions of AM was studied, it was found that the effects on NO and IL-1beta production by AM were normalized 3 days after the stress induction, but reappeared when cells were isolated 1 to 2 wk after stress exposure. Analysis of the effects of electric footshocks of various intensities on antibody production 10 days after the stress session and subsequent lung immunization with trinitrophenyl conjugated keyhole limpet hemocyanin (TNP-KLH), showed a footshock intensity-dependent response. Although exposure to stress induced an increase in plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT), hormone levels did not differ between the various stress-exposed groups. This suggests that the observed stress effects on pulmonary immune functions were not mediated by ACTH or CORT but point to a direct involvement of the autonomic nervous system.

Repeated high dose oral exposure or continuous subcutaneous infusion of 2-methoxyacetic acid does not suppress humoral immunity in the mouse.

2-Methoxyethanol (ME) has been shown to be immunosuppressive in rats but not mice, with oxidation of ME to 2-methoxyacetic acid (MAA) being a prerequisite for immunosuppression. MAA is more rapidly cleared by mice than rats, consequently this study was designed to determine if increasing the bioavailability of MAA in mice might play a role in this species difference. Female B6C3F1 mice were given MAA by oral multiple daily high doses or by continuous subcutaneous infusion via mini-osmotic pumps. Humoral immunity was evaluated in MAA-exposed mice using the plaque-forming cell (PFC) response to either sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). Female F344 rats were also used to compare the effects of multiple daily MAA exposure on these humoral immune responses. Rats and mice were dosed orally twice a day for 4 days by gavage with MAA at dosages ranging from 40-320 mg/kg/day and 240-1920 mg/kg/day, respectively. All animals were immunized on the first day of dosing and body and lymphoid organ weights and PFC responses to SRBC or TNP-LPS were evaluated 4 days later. While body weights in rats were unaffected, thymus weights were reduced at all dosages of MAA and spleen weights were reduced at 160 or 320 mg/kg/day. PFC responses to SRBC and TNP-LPS were suppressed in rats at dosages of 160 and 320 mg/kg/day. In contrast, thymus weights of mice were reduced only at 960 mg/kg/day or greater, with no effect on spleen or body weights. Furthermore, neither the PFC response to SRBC nor the response to TNP-LPS was suppressed in mice exposed to any oral dosage of MAA. In the continuous infusion study, mice were subcutaneously implanted with mini-osmotic pumps containing MAA which was delivered at 840 mg/kg/day over a 7-day period. Continuous exposure to MAA via mini-osmotic pumps did not suppress the PFC response to either SRBC or TNP-LPS, but rather significantly enhanced the response to TNP-LPS. These results indicate that mice are insensitive to MAA even at the high dosages given as a bolus or continuously over 1 week. The data further support earlier work, which suggested that the observed difference between rats and mice for MAA-induced immunosuppression appears to be unrelated to the availability of MAA to target lymphoid tissue in these rodent species.