The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. VI. Differential tolerance susceptibility in adult spleen as a function of B-cell maturation level.

The maturation level of the B-lymphocyte subpopulations involved in trinitrophenyl (TNP)-specific immunological tolerance in adult mice induced by the injection of trinitrobenzenesulfonic acid (TNBS) was investigated using in vitro antigen-specific and nonspecific polyclonal stimulation. The maturity of the B-cell subsets being studied was defined by the antigen or polyclonal activator which evoked a response. Thus, the thymic independent (TI-1) antigen TNP-lipopolysaccharide (TNP-LPS) and the polyclonal stimulant LPS were used to activate immature, neonatal-type B lymphocytes, whereas mature, adult-type B cells were responsive to the TI-2 antigen, TNP-Ficoll, and the nonspecific activator, purified protein derivative (PPD). Whereas unresponsiveness in TNP-LPS-reactive (immature) B cells 4 d after TNBS treatment was previously shown to be the result of functional deletion, partially reversible receptor blockade was detected in this study early after tolerogen treatment. By the 24-h point, tolerance was irreversible, as assessed by 24-h of antigen-free incubation and cocultivation of tolerant cells with control splenocytes. Tolerance was induced more rapidly in immature, TI-1 B cells than in mature TI-2 B lymphocytes. B lymphocytes reactive to TNP-Ficoll were also less susceptible to receptor blockade. Using LPS as a nonspecific probe for immature B cells, 60% tolerance in high affinity TNP-specific cells was induced within 12 h of TNBS treatment, and complete unresponsiveness by 24 h. In contrast, no significant decrease in response to the mature B-cell activator, PPD, occurred until day 2. Furthermore, the 50% tolerance level was achieved in TNP-specific LPS-reactive B cells by 100 times less tolerogen than required for PPD-responsive cells. Thus, TNBS-induced unresponsiveness in cells reactive to TNP-LPS is initially a result of reversible receptor blockade which leads within 4 d to functional deletion. Immature, TI-1 B lymphocytes, which give polyclonal responses to LPS and antigen-specific responses to TNP-LPS, are rendered tolerant to TNBS more rapidly and at lower tolerogen does than mature, TI-2 mouse B cells which react polyclonally to PPD and specifically to TNP-Ficoll. Moreover, these data show that both the immature and the mature B lymphocyts with these characteristic tolerance susceptibilities and specific and nonspecific immune response patterns are present in the adult mouse spleen.

Antigen-initiated B-lymphocyte differentiation. VIII. Sedimentation velocity and buoyant density characterization of virgin antibody-forming cell progenitors in the adoptive immune response of unprimed CBA mice to 4-hydroxy-3-iodo-5-nitrophenylacetic acid-polymerized bacterial flagellin antigen.

The characteristics of antibody-forming cell (AFC) progenitors lacking previous contact with specific antigen (virgin AFC progenitors) has been studied using sedimentation velocity and buoyant density separation for the investigation of physically distinct B-cell subpopulations. Functional characterization of isolated subsets was made using a quantitative adoptive immune assay for the IgM AFC progenitors responding to the antigen 4-hydroxy-3-iodo-5-nitrophenylacetic acid conjugated polymerized bacterial flagellin. Extensive heterogeneity is present among B lymphocytes, only some subpopulations of which exhibit AFC progenitor function. In the spleen of adult conventional CBA mice, atypically fast sedimenting cells of low buoyant density are active, while typical small B lymphocytes do not appear to be progenitors of IgM AFC. Spleen of adult specific pathogen-free (SPF), germfree, and athymic nude mice give similar results, although a minor population of typical slowly sedimenting dense cells are active in the latter two sources. Adult conventional bone marrow cells are as physically and functionally heterogeneous as splenic B cells, and although a significant proportion of AFC progenitor activity is found among dense, slowly sedimenting cells, most of the activity is among low density, faster sedimenting cells. In contrast to this situation in adult animals, where most of the unprimed AFC progenitors are large, atypical B cells, the spleens of neonatal mice provide a site where virgin AFC progenitors with the physical properties of typical small B lymphocytes are found. While being present in conventional and SPF neonatal spleens, these virgin cells are predominant in 7-day-old germfree mouse spleen. These findings suggest that the newborn virgin B cell is a typical small lymphocyte. However, few cells of this type are found in the adult animal. The unprimed AFC-progenitor population in the adult consists of large, fast sedimenting, low buoyant density, adherent cells, the physical properties of which are characteristic of activated B lymphocytes. It is suggested that these atypical cells are derived from the small newborn virgin B cell by the nonspecific effects of environmental antigenic stimuli.

Immunological tolerance to a hapten. I. Induction and maintenance of tolerance to trinitrophenyl with trinitrobenzene sulfonic acid.

Treatment of mice with a nonimmunogenic preparation of free reactive hapten, trinitrobenzene sulfonic acid (TNBS), leads to the induction of a state of tolerance to the hapten, 2,4,6-trinitrophenyl (TNP). This is determined by the lack of response to the haptenic moiety in an immunogenic hapten-carrier conjugate (TNP-SRBC) as assayed both by serum antibody titrations and the hemolytic plaque assay. The tolerance produced is specific for the hapten, since the anticarrier responses are essentially unaltered compared with the control values. The unresponsiveness induced by TNBS treatment is a dose-dependent phenomenon, becoming less complete at lower doses of TNBS. The tolerance is of a definite length, both in its induction phase and in the duration of the established unresponsive state. Tolerance can be maintained and extended, and may also be reentered once escape has been initiated.

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. I. Prolongation of rat cardiac and renal allograft survival by the PG27 extract and immunosuppressive synergy in combination therapy with cyclosporine.

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant, Tripterygium wilfordii. We tested PG27 in rat cardiac and renal allotransplantation, and we examined the immunosuppressive interaction with cyclosporine (CsA). METHODS: Brown Norway (BN) rat heart or kidney allografts were transplanted into the abdomen of Lewis rats, which were treated by the intraperitoneal or oral route with PG27, CsA, or both. RESULTS: PG27 administered intraperitoneally to Lewis recipients for 16 days at 10-30 mg/kg/day significantly increased the median survival time of BN heart allografts from 7 to 18-22 days. Oral administration was effective, with cardiac allograft survival prolonged to > 100 days with 52 days of treatment. PG27 at 20-30 mg/kg/day significantly extended the median survival time of BN kidney allograft recipients from 9 to 36.5-77 days, and 30 mg/kg/day for 52 days extended survival beyond 200 days. PG27 combined with CsA significantly enhanced heart and kidney allograft survival, even at doses of CsA ineffective when administered alone. The addition of 5 or 10 mg/kg/day PG27 reduced by 50-75% the CsA dose needed for 100% kidney allograft survival. The combination index was less than 1.0, indicating synergy of PG27 with CsA in prolonging cardiac and renal allograft survival. Furthermore, the PG27/CsA combination exerted a positive influence on renal allograft function. PG490 (triptolide, a constituent of PG27) and PG490-88 (a semisynthetic derivative of PG490) suppressed rejection of cardiac and renal allografts. CONCLUSIONS: The PG27 herbal extract demonstrated immunosuppressive activity by prolonging heart and kidney allograft survival, displaying synergy in the immunosuppressive interaction with CsA, and improving renal allograft function in combination with CsA. PG490 and PG490-88 compounds were also effective.

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. II. Prolongation of hamster-to-rat cardiac xenograft survival by combination therapy with the PG27 extract and cyclosporine.

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant Tripterygium wilfordii, which we investigated alone and in combination with cyclosporine (CsA) in a concordant, hamster-to-rat cardiac xenotransplantation model. METHODS: Golden Syrian hamster hearts were heterotopically transplanted into the abdomen of Lewis rat recipients, which were treated intraperitoneally or orally with PG27, CsA, or both. RESULTS: Combination therapy with 30 mg/kg(day of PG27 and CsA at 10 mg/kg/day successfully suppressed acute hamster-to-rat cardiac xenograft rejection. Treatment with PG27 or CsA alone was ineffective. Among several effective combinations, the best regimen involved PG27 at 30 mg/kg/day and CsA at 5 mg/ kg/day from days 8 to 35 and then CsA at 5 mg/kg/day from days 36 to 100, which produced 100% survival beyond 100 days. CsA suppressed the heterospecific lymphocytotoxic antibody response and inhibited IgG but not IgM xenoantibody production (which led to xenograft rejection), whereas PG27 alone did not prevent antibody production. The PG27/CsA combination blocked the lymphocytotoxic antibody response and IgG and IgM xenoantibody production induced by cardiac xenotransplantation. CONCLUSIONS: PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, as well as completely inhibiting xenoantibody production.

Prevention of graft-versus-host disease by a novel immunosuppressant, PG490-88, through inhibition of alloreactive T cell expansion.

BACKGROUND: PG490-88 is a water soluble, semisynthetic derivative of a novel compound PG490 (triptolide) purified from the Chinese herb Tripterygium Wilfordii Hook F. METHODS: PG490-88 was administrated into recipient mice in a model (B10.D2-->BALB/c) of lethal graft-versus-host disease (GVHD) to study the effects of PG490-88 on GVHD and on the various steps involved in the pathological course of GVHD. RESULTS: Injection of PG490-88 i.p. at a dose of 0.535 mg/kg/day for the first 3 weeks after transplantation protected all the recipients from developing GVHD up to 100 days after transplantation. PG490-88 inhibited in vivo both CD4+Vbeta3+ and CD8+Vbeta3+ T cell (alloreactive T cells in this model) expansion in the spleen by 64.09 and 34.02%, respectively, at the time when Vbeta3+ cell expansion was in the logarithmic phase (day 3 after transplantation). Intracellular cytokine staining without further in vitro activation demonstrated 47.42% inhibition of IL-2 production among CD4+ spleen cells in PG490-88-treated mice as compared to GVHD control on day 3 after transplantation. In contrast, CD25 (alpha chain of interleukin-2 receptor) expression did not differ. CONCLUSIONS: PG490-88 is highly effective in prevention of murine GVHD. The immunosuppressive effect of PG490-88 is mediated by inhibition of alloreactive T cell expansion through interleukin-2 production.

Suppression of graft-versus-host disease by succinyl acetone in a rat allogeneic bone marrow transplantation model.

The efficacy of succinyl acetone (SA, 4,6-dioxoheptanoic acid) was explored in the allogeneic rat bone marrow transplant model of graft-vs.-host disease. Lethally irradiated Wistar Furth rats receiving Fischer 344 allogeneic bone marrow and spleen cells developed severe GVHD, resulting in mortality at 25-45 days posttransplant. Treatment for 14 days with 250 mg/kg/day of SA by Alzet osmotic pumps implanted subcutaneously 3 days before cell transfer prevented GVHD and produced long-term survivors that were allogeneic hematopoietic chimeras. SA doses below 250 mg/kg/day and treatment for less than 14 days were less efficacious. Initiation of SA therapy could be effectively delayed up to 7 days after BMT. Pharmacokinetic studies with i.v. bolus administration in normal CD rats revealed a plasma mean residence time that increased with dose and a systemic clearance that decreased with dose. Three dose-dependent half lives were apparent (ca. 7-18 min, 0.8-3 hr, and 12 hr). The s.c. bioavailability was ca. 82%. Relatively constant plasma SA levels were obtained with s.c. Alzet osmotic pumps, indicating no change in clearance with continuous exposure. Allogeneic BMT exerted no major influence upon SA clearance. These studies show that SA is a robust therapeutic agent that suppressed GVHD in the allogeneic rat BMT model under a variety of circumstances.

Pharmacologic, toxicologic, and marrow transplantation studies in dogs given succinyl acetone.

A novel immunosuppressant, succinyl acetone (4,6-dioxoheptanoic acid), was studied in dogs. Results with bolus intravenous injections at doses ranging from 50 to 1600 mg/kg showed dose-dependent alpha and beta half-lives, ranging from 30 to 80 min and 7 to 27 hr, respectively. Results suggested that continuous i.v. infusion was necessary to maintain constant plasma levels. Four dogs were given 9.2 Gy total-body irradiation and autologous marrow transplants along with continuous i.v. infusion of succinyl acetone at 50, 100, 200, or 400 mg/kg/day for 21 days, and all four had rapid, sustained hematopoietic engraftment. However, two of the four dogs receiving 200 and 400 mg succinyl acetone/kg/day, respectively, developed bilateral hind-limb ataxia, with histologically confirmed cerebellar lesions in the dog given the higher dose, thus establishing a potential dose-limiting neurotoxicity. Prevention of graft-versus-host disease was studied in recipients of allogeneic marrow. Dogs were given 9.2 Gy TBI, followed by hematopoietic grafts from unrelated DLA-nonidentical or DLA-haploidentical littermate dogs. Succinyl acetone was given as continuous infusion for 21 days after transplant at doses of 100-300 mg/kg/day. Starting succinyl acetone on the day of marrow infusion in four dogs failed to prevent rapid onset of acute GVHD, and dogs survived no longer than controls. Starting succinyl acetone 3 days before transplant delayed the onset of acute GVHD and prolonged survival significantly compared with that of dogs not given postgrafting immunosuppression (P = 0.008); survival was comparable to that in previously reported dogs given either methotrexate or cyclosporine as postgrafting immunosuppression (P = 0.88 and 0.99, respectively). Seven of the sixteen allogeneic recipients developed evidence of neurotoxicity during succinyl-acetone infusion. Neurological dysfunctions were manifested by hind-limb ataxia and posterior paresis. In conclusion, succinyl acetone significantly delayed the onset of GVHD and prolonged survival of DLA-nonidentical marrow graft recipients but did not induce graft-host tolerance and was associated with dose-limiting neurotoxicity.

MRx102, a triptolide derivative, has potent antileukemic activity in vitro and in a murine model of AML.

Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC(50) values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4-11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34(+) progenitor, and more importantly, CD34(+)CD38(-) stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

Induction of hapten-specific immunological tolerance and immunity in B lymphocytes. VII. Correlation between trinitrobenzenesulfonic acid administration, serum trinitrophenyl content, and level of tolerance.

The induction of immunological tolerance with trinitrobenzenesulfonic acid (TNBS) was studied by a comparison of the concentration of trinitrophenyl (TNP) in the serum of tolerant mice (TolS) and the degree of unresponsiveness induced as the dose and time of tolerogen injection were varied. The concentration of TNP in TolS was greater with a larger dose of TNBS, as expected, and decreased with time after tolerogen injection in a biphasic manner. The rapid initial decline followed on Day 10 by a more gradual decrease in TNP concentration suggests that there were two classes of TNP conjugates produced by TNBS injection. The serum TNP concentration appeared to correlate to the in vivo response of TNBS-treated mice to thymic-dependent and thymic-independent antigenic challenge while little correlation was evident with the in vitro response.

The induction of hapten-specific immunologic tolerance and immunity in B lymphocytes. I. The effect of delayed immunization on the adoptive response to TNP-LPS.

An improved adoptive transfer procedure has been developed for the thymus-independent conjugate TNP-LPS. This technique involving delayed immunization results in increased sensivity and makes possible studies of various cell types, the response of which is normally very low or which are difficult to manipulate experimentally in situ. The standard adoptive immune response of adult spleen cells to TNP-LPS was low in comparison to the primary AFC response of intact mice. Adult bone marrow cells gave a 30-fold lower and neonatal spleen cells an 8-fold lower adoptive response than an equivalent number of adult spleen cells. If the administration of antigen was delayed past the normal time of immunization (1 hr after cell transfer), the resulting AFC response of adult spleen, adult bone marrow, and neonatal cells was enhanced. The peak response occurred with a delay interval of 4 days, and the magnitude decreased with greater delay. Part of the reason for the decline was an acceleration in the attainment of a peak response in mice given antigen past the 4-day optimum. The enhancement of the adult splenic response was not restricted to the antigen TNP-LPS, and was a transferrable phenomenon which did not require antigen to persist past the 4-day optimum delay period. The ineffectiveness of host pre-irradiation indicated that host recovery alone was not involved. Although the results are more compatible with B cell proliferation causing the enhancement, a combination of effects may be involved.

Antigen-initiated B lymphocyte differentiation: non-specific stimulation changes the physical properties of virgin AFC-progenitors in neonatal mouse spleen.

While the virgin AFC-progenitors for an adoptive immune response in neonatal germ-free CBA mouse spleen are small, dense cells, the equivalent cells in the adult are a larger, lighter density population. The effects of injections of unrelated antigens on the physical properties of the AFC-progenitors in neonatal spleen were investigated to test the postulate that the physically distinct "virgin" AFC-progenitors in the adult arose by a process of non-specific activation. Spleen cells from 7-day-old germ-free CBA mice were separated by sedimentation at unit gravity or by density on continuous albumin gradients, and the fractions were tested for NIP-specific AFC-progenitor activity using an adoptive immune assay which gave a direct linear measure of B cell activity. If the donor neonatal animals were injected one day previously with POL or PPD, the NIP-specific AFC-progenitor activity shifted from the typical small, dense lymphocytes to larger, lighter cells. The physical properties of these stimulated AFC-progenitors resembled those of IgM AFC-progenitors in normal adult mice. These results experimentally confirm the theory that environmental stimuli induce a non-specific "activation" of a particular subset of "virgin" B cells.

Selective immunomodulation by the antineoplastic agent mitoxantrone. I. Suppression of B lymphocyte function.

Novantrone mitoxantrone, an antineoplastic agent with antiproliferative properties, is under investigation as an immunomodulating agent. The impact of mitoxantrone treatment on B lymphocyte reactivity is presented here. Administered i.p. in H2O at a dose of 0.5 mg/kg, daily for 14 days, mitoxantrone abrogated both the in vivo antibody response (to ovalbumin) and the in vitro plaque-forming cell (PFC) response (to SRC). In addition to the effects on thymus-dependent reactivity, PFC responses to the thymus-independent antigens TNP-LPS and TNP-Ficoll were also inhibited when tested in vivo or in vitro. B cells were identified as a target for the suppressive activity of mitoxantrone by using T cell-replacing factor to reconstitute the in vitro anti-SRC PFC response of a T lymphocyte-depleted spleen cell preparation. LPS-induced B cell mitogenesis was largely inhibited by mitoxantrone treatment. However, depletion of Sephadex G-10-adherent cells significantly restored the proliferative response. Flow cytometric analysis revealed a dramatic decrease in splenic B lymphocyte content. Therefore, mitoxantrone exerted a potent suppressive influence on the humoral immune system through a direct reduction in B cell number augmented by macrophage-mediated inhibition of B cell proliferation.

Selective immunomodulation by the antineoplastic agent mitoxantrone. II. Nonspecific adherent suppressor cells derived from mitoxantrone-treated mice.

Mitoxantrone exerts a potent suppressive influence upon humoral immune responses. The B cell is a likely target for this inhibitory effect, and we have reported evidence supporting this possibility. The impact of mitoxantrone upon T lymphocyte reactivity was assessed as a second mode of action of this novel antineoplastic drug. TH and TS lymphocyte induction were tested in the in vitro anti-sheep erythrocyte response, and a surprising differential effect of mitoxantrone was observed. Helper activity was abrogated and suppressor function was enhanced. In apparent disagreement with this result, mitoxantrone inhibited the in vivo induction of TS cells using trinitrophenylated spleen cells. Macrophages were investigated as potential mediators of these effects upon immunoregulatory function. Replacement of macrophages in mitoxantrone-treated spleen cell preparations by normal adherent cells allowed the induction and complete expression of TH lymphocyte function. Conversely, replacement of mitoxantrone-treated macrophages with normal adherent cells before induction of TS cells failed to generate TS cell function. Thus, TH cells were resistant and TS cells were completely susceptible to mitoxantrone. Furthermore, supplementation of normal TH cell cultures with splenic macrophages from mitoxantrone-treated mice inhibited the induction of helper function. Production of the lymphokines IL 2 and TRF in mitoxantrone-treated mice was normal. This is consistent with the retention of functional TH cells in drug-treated spleens. Macrophages in the spleens of mitoxantrone-treated mice were responsible for the abrogated helper function and the enhanced suppressor activity. Although TS cell induction was directly inhibited by the drug, the effect upon TH cell function was secondary to the action of mitoxantrone-induced suppressor macrophages. Mitogen-stimulated lymphokine production was normal. Thus, mitoxantrone is a selective immunomodulator. The macrophage-mediated suppression of TH cell induction and humoral immunity investigated in spleens from mitoxantrone-treated mice is an intriguing finding that may have significant implications for immunotherapy.

Antigen-initiated B-lymphocyte differentiation. VII. Quantification of AFC progenitor levels in adoptive and culture responses to NIP-POL antigen.

Quantitative studies on B cells require a direct assay for antibody-forming cell (AFC) progenitor function, in which the number of AFC produced bears a simple, linear arithmetic relationship to the number of progenitors present. This might be expected under conditions where helper T-cell and accessory cell requirements are by-passed, or provided in excess. This possibility has been tested using as antigen the hapten NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid) on the carrier POL (polymerized bacterial flagellin), in adoptive transfer of normal and nude mouse spleen cells to irradiated recipients, and in cell culture. Primary and secondary IgM responses to this antigen are "T cell-independent'. The secondard IgG response is T cell-dependent but this function can be provided by 'carrier-primed' irradiated recipients. However in no case did the cell dose response curve show a linear, arithmetic relationship between cells transferred or cultured, and AFC produced. If less than 10 X 10(6) cells were adoptively transferred or cultured, a sigmoid curve was obtained, approximately linear with a slope of around 1-6 on a log-log scale. In adoptive transfer, a plateau was then seen above 10 X 10(6) cells, followed by a second sharp rise beginning around 15 X 10(6) cells. Addition of irradiated spleen cells as 'fillers' to maintain cell numbers constant produced a linear (arithmetic scale) dose response curve for the primary IgM responses, both adoptive and in culture. Lipopolysaccharide injection of recipients also produced linear regions in the adoptive transfer system. These techniques provide more direct, quantitative assay systems for the primary IgM responses to this antigen. However, arithmetic linear cell dose response curves were still not obtained for the secondary IgG responses, using irradiated filler cells.

Antifungal activity of recombinant human macrophage colony-stimulating factor in models of acute and chronic candidiasis in the rat.

Models of acute and chronic candidiasis were developed in Fischer 344 rats to evaluate the therapeutic efficacy of recombinant human macrophage colony-stimulating factor (rhM-CSF) alone and in combination with the antifungal agent fluconazole. In the acute model, rats were challenged by intravenous injection with 2 x 10(6) Candida albicans, approximately 4 times the LD50. Daily subcutaneous (sc) bolus injections of rhM-CSF for 10 days plus a single sc bolus dose of 0.3 mg/kg of fluconazole improved the median survival time from 5 days (32% survival) with fluconazole alone to > 30 days (88% survival) in the rhM-CSF- and fluconazole-treated rats. In the chronic model, daily sc bolus injections of rhM-CSF for 10 days plus a single sc bolus dose of 1.0 mg/kg of fluconazole decreased the median titer of C. albicans cultured from the kidneys by 10-fold at 15 and 30 days after infection. These studies showed that rhM-CSF treatment improved the therapeutic outcome in both the acute and chronic rat model of candidiasis when used with fluconazole, a standard fungistatic agent.

Antigen-initiated B lymphocyte differentiation. IX. Characterization of memory AFC progenitors by buoyant density and sedimentation velocity separation.

The characteristics of memory B cell antibody-forming cell (AFC) progenitors from long-term hapten-primed CBA mice were investigated by using sedimentation velocity and buoyant density separation to isolate physically distinct B cell sub-sets. The isolated fractions were assayed by the adoptive immune response to NIP-POL antigen, under conditions where neither T cells nor other accessory cells were limiting the IgM or IgG AFC responses. The results were compared to previous studies on the IgM AFC-progenitors of unprimed adult mice. Splenic IgM and IgG memory AFC-progenitor activity was largely found among the typical B cells of slow to medium sedimentation rate, in contrast to the fastre sedimenting IgM AFC-progenitor activity of unprimed animals. Splenic IgM and IgG memory AFC-progenitor activity was found among the medium to light density cells, and so resembled by this parameter the IgM AFC-progenitor activity in unprimed animals. Thoracic duct lymphocytes from hapten-primed mice also exhibited memory IgM and IgG AFC-progenitor activity in the slow-medium sedimentation range. However, in contrast to spleen, the IgM and IgG memory AFC-progenitor activity in lymph was found among very dense B cells. Two physically distinct sub-populations of memory B cells have thus been identified, namely: i) small, medium-light density, presumably tissue-resident B lymphocytes found in spleen; and ii) small, dense, presumably recirculating B lymphocytes found in lymph. Both physical forms include IgM and IgG progenitors. Both forms are distinct from the larger, medium-light density "virgin" AFC-progenitors in the spleen of unprimed adult mice.