Cell-depleted Spleen Research Articles

Stroma-dependent development of two dendritic-like cell types with distinct antigen presenting capability

Novel antigen presenting cells (APCs) have been described in the murine spleen. Cells have a distinct CD11c(lo)CD11b(hi)MHC-II(-)CD8α(-) phenotype as highly endocytic dendritic-like cells that cross-present antigen to CD8(+) T cells but fail to activate CD4(+) T cells. These cells are named "L-DCs" because they reflect dendritic cells (DCs) produced in long-term spleen cultures (LTC). Similar cells were produced when bone marrow progenitors were cocultured over the splenic stromal line 5G3. Cocultures continuously produced a majority of L-DCs and a transient population of cells reflecting conventional dendritic cells (cDCs). Both the L-DC and cDC-like subsets cross-present antigen to CD8(+) T cells, inducing their activation and proliferation. However, as MHC-II(-) cells, L-DCs are unable to activate CD4(+) T cells, while MHC-II(+) cDC-like cells present antigen for CD4(+) T cell activation. These results distinguish two APC subsets produced invitro: a transient population of cDC-like cells and L-DCs that are continuously produced, presumably from self-renewing progenitors. These subsets are not developmentally linked via a precursor or progeny relationship. L-DCs and cDC-like cells are also distinct in terms of cytokine expression, with 65 of 84 tested genes displaying greater than a twofold difference by quantitative reverse-transcriptase polymerase chain reaction. Splenic stroma supports production of two APC subsets reflecting different lineage origins.

Induction of Specific Th1 Responses and Suppression of IgE Antibody Formation by Vaccination with Plasmid DNA Encoding Cyn d 1

Background: DNA vaccines encoding allergens have been developed to prevent or to treat specific IgE responses. Objective: To evaluate the potential preventive and therapeutic effect of DNA vaccines encoding Cyn d 1 alone or combined with different adjuvants on specific allergies. Methods: Recombinant plasmid Cyn d 1 (pCyn d 1) was constructed by insertion of Cyn d 1 cDNA into the vector pcDNA3. BALB/c mice were injected with pCyn d 1 alone or plus adjuvants such as bupivacaine, bestatin, liposome, or CpG. Control mice were treated with pcDNA3 or PBS. They were boosted 3 weeks later and then sensitized twice with recombinant Cyn d 1 and alum. Their serum antibody responses and cytokine profiles of spleen cells were studied. Adoptive transfer of spleen cells of pCyn d 1-vaccinated mice was also performed. Results: Vaccination of mice with pCyn d 1 induced Th1 responses characterized by IgG2a responses and spleen cell secretion of interferon-γ. Vaccination with pCyn d 1 not only prevented the induction of specific IgE responses but also suppressed ongoing IgE responses. The mice receiving untreated, CD4+- or CD8+-depleted spleen cells from pCyn d 1-vaccinated mice all had suppression of IgE responses. Conclusion: This study confirms the prophylactic and therapeutic effects of DNA vaccines encoding Bermuda grass pollen allergen Cyn d 1 on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulatory effect of pCyn d 1 on specific IgE responses.

Separation of antileukemic effects from graft-versus-host disease in MHC-haploidentical murine bone marrow transplantation: participation of host immune cells

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. In clinical studies of HLA-mismatched HSCT, strong GVL effects have been reported. In the present study, we addressed the mechanism of the GVL and GVH response using MHC-haploidentical murine bone marrow transplantation (BMT) models. Recipient BDF1 (H-2(b/d)) mice received T cell-depleted bone marrow and spleen cells from B6C3F1 (H-2(b/k)) or C57BL/6 (H-2(b)) mice with or without P815 mastocytoma cells (H-2(d)) after receiving lethal total body irradiation. B6C3F1 --> BDF1 (hetero-to-hetero type) recipients showed more powerful antileukemic effects with less severe GVHD than C57BL/6 --> BDF1 (parent-to-F1 type) recipients. Compared with C57BL/6 --> BDF1 recipients, significantly higher in vitro cytotoxic activity against P815 cells was observed in B6C3F1 --> BDF1 recipients. Significantly lower CXCR3 expression on donor T cells and higher interferon (IFN)-gamma expression were considered to be associated with strong antileukemic effects with less severe GVHD in B6C3F1 --> BDF1 recipients. Furthermore, host immune cells, especially natural killer cells and CD8(+) T cells, were found to contribute remarkably to high IFN-gamma production in B6C3F1 --> BDF1 recipients. Thus, in MHC-haploidentical HSCT, host immune cells may change the balance between GVH and GVL response through IFN-gamma production.

Liposomal KRN7000 (RGI-2001) Synergizes with Low-Dose Rapamycin in Preventing GvHD by promoting expansion of naturally-Occurring CD4+FOXP3+ Regulatory T Cells.

Abstract 4491 IntroductionRGI-2001 is a liposomal formulation of KRN7000 (a synthetic derivative of a-galactosylceramide). We previously demonstrated that RGI-2001 potently reduces GvHD lethality in a mouse model by inducing host-specific tolerance through the expansion of donor-derived CD4+Foxp3+ Regulatory T cells (Tregs) (ASH 2008 Poster III-582). In the present study, we examined if the combination with low-dose rapamycin (LD-RAPA) may further improve the efficacy of RGI-2001. In addition, the origin of Tregs induced by RGI-2001 was investigated. MethodsA fully MHC mismatched GvHD mouse model using lethally-irradiated Balb/c (H-2d) recipients transplanted with C57BL/6 (H-2b) T cell depleted bone marrow cells with total spleen cells were used. In some experiments, Foxp3-GFP reporter mice (C57BL/6 background) were used as the donor. RGI-2001 (0.01 ug/kg to 1 ug/kg) was given on day 0 via single intravenous administration. LD-RAPA (0.1 mg/kg) was given daily from day 0 to day 14 intraperitoneally. Treg frequency was determined on day 15 post-BMT by flow cytometry either by using an antibody for Foxp3 or by detecting GFP as a surrogate marker for Foxp3. ResultsFirst, the pharmacological dose induction of Tregs by RGI-2001 was investigated. Day 15 post-BMT analysis of mice treated with RGI-2001 demonstrated a dose-dependent increase in the % CD4+Foxp3+ Treg cells in the spleen, lymph nodes, and bone marrow. As shown in the Table, frequencies of CD4+Foxp3+ Tregs in the spleen increased in a dose dependent manner, from 3.0 ± 1.4 % in untreated mice to 14.9 ± 5.8% in 1 ug/kg RGI-2001 treated mice. The total Treg cell counts in the spleen also increased in a dose dependent manner with 1 ug/kg RGI-2001 consisting of 4.2×10e5 Tregs as compared with 0.3×10e5 Tregs in untreated mice. The frequency of CD8+ T cell population decreased in a dose dependent manner, suggesting an inverse relationship with frequencies of Tregs. When LD-RAPA was combined, a robust synergy between the two compounds was noted. The frequency of Treg in mice that received combination treatment was significantly higher than that in each monotherapy group (p = .0005 vs. RGI-2001 only, p< .0001 vs. LD-RAPA only). Absolute Treg counts in the spleen were markedly increased in mice received the combination treatment. In three independent experiments, Tregs in combination treatment group were consistently ∼3-fold higher than that in RGI-2001 monotherapy group and ∼9-fold higher than that in LD-RAPA monotherapy group. In contrast, CD8+ T cell frequencies decreased in mice treated with LD-RAPA containing regimen. Efficacy in survival was also improved in mice that received the combination treatment with 80-100% survival over 100 days.Table 1:Cellular composition of Day 15 post-BMT spleen in mice treated by RGI-2001 with or without LD-RAPA.RGI-2001-0.01 ug/kg0.1 ug/kg1 ug/kg-0.1 ug/kgRAPA----0.1 mg/kg0.1 mg/kg%Foxp3+/CD4+3.0 ± 1.4 (n=15)6.9 ± 3.0 (n=17)11.9 ± 4.1 (n=9)14.9 ± 5.8 (n=11)6.2 ± 3.1 (n=12)19.8 ± 4.1 (n=10)%CD8+/CD3+12.3 ± 1.4 (n=9)11.2 ± 4.0 (n=17)9.8 ± 4.7 (n=14)8.5 ± 4.2% (n=11)7.6 ± 2.3% (n=8)6.7 ± 2.6% (n=8)Next, the origin of the Tregs induced by RGI-2001 was investigated using the Foxp3-GFP reporter mice as the spleen cell donor. While 2.5×10e6 total spleen cells from Foxp3-GFP mice gave rise to 7.2 ± 2.4 % Tregs (n= 7) when treated with RGI-2001 (1 ug/kg), equivalent number of GFP+ Treg-depleted spleen cells gave rise to 1.4 ± 0.3 % Tregs (n=5, p < .0006). Likewise, when mice were treated with the combination of RGI-2001 (1 ug/kg) and LD-RAPA, Treg frequencies in mice that received total spleen cells and GFP+ Treg -depleted spleen cells were 17.8 ± 3.8 % (n=7) and 1.5 ± 0.4 % (n=7), respectively (p < .0001). These results together suggest that the majority of Tregs induced by RGI-2001 with or without LD-RAPA were derived from the pre-existing naturally-occurring Treg population, and the contribution of de-novo Tregs was minimal in the GvHD setting. ConclusionsRGI-2001 prevented GvHD by promoting the expansion of naturally-occurring Tregs. LD-RAPA synergistically enhanced the efficacy of RGI-2001 by significantly increasing Treg expansion while suppressing effector T cells. Disclosures:REGiMMUNE employees (OD, AL, JL, TT), Founder (YI), and consultant (RN). YI and RN are stockholders. Disclosures:No relevant conflicts of interest to declare.

Spreading tolerance by converting

Unlike some religious leaders, suppressive T cells need more than just charisma to spread messages of tolerance: the cells rely on a cell surface cytokine complex to convert others, according to Andersson et al. (page 1975). Figure 1 Naive-turned-suppressor cells dampen colitis (left). Regulatory T (T reg) cells suppress other T cells in part via the cytokine TGF-β, which can convert naive CD4+ T cells into suppressor cells. Andersson et al. now find that naive CD4+ T cells turn into suppressor cells only when they bump into T reg cells that have an inactive TGF-β complex on their surface. Proteolysis of the inactive complex, which liberates soluble protein, was required for conversion. Inhibiting the protease or swamping the cells with inactive complex prevented cleavage and thus conversion. The converted cells could suppress gut inflammation when transferred into mice suffering from colitis. Other immune cells also secrete soluble TGF-β, but they do not seem to force conversion—at least in this system. Previous experiments showed that naive T cells cultured with T reg cell–depleted spleen cells, which secrete TGFβ, fail to convert. Non-T reg cells don't express the inactive surface complex, so perhaps its cleavage is needed to trigger other conversion-supporting signals. The latent TGF-β complex was present only on T reg cells that had been stimulated via their T cell receptors. This specificity might allow T reg cells to limit conversion by delivering the TGF-β signal only to T cells bound to the same antigen-presenting cell and thus on the verge of being activated. “We wouldn't want T reg cells to run amok and convert all naive T cells,” says lead author Ethan Shevach. How often T reg cells rely on this mechanism and its impact on the induction of tolerance in vivo are unknown.

Mode-of-Action, Efficacy, and Safety of a Homologous Multi-Epitope Vaccine in a Murine Model for Adjuvant Treatment of Renal Cell Carcinoma

Background and objectiveIn a phase-III trial it was recently shown that an adjuvant renal cell carcinoma (RCC) vaccine (Reniale) reduces the risk of tumour progression following nephrectomy. This clinical trial focused on efficacy and did not investigate end-points relating to mode-of-action of the vaccine. In a murine model we investigated mode-of-action, efficacy, and safety of a homologous RENCA cell-based vaccine. Design, setting, and participantsSix groups with 12 BALB/c mice per group received five vaccinations (lysate of 1×106–1×107 RENCA cells, manufactured with or without prior IFN-γ incubation) at 3-wk intervals before tumour transplantation and one vaccination 14 d afterwards. Controls (12 mice) received only solvent. All mice were sacrificed 21 d after tumour transplantation. MeasurementsAnimal welfare, tumour growth, number of metastases, and the presence of cytotoxic T-lymphocytes as determined by a 51chromium-release assay. Adoptive immune transfer experiments (vaccination of nine mice with the RENCA vaccine or saline and transfer of serum, spleen cells, and CD4 and/or CD8 depleted spleen cells into five recipient mice each) were carried out to demonstrate involvement of different immune mechanisms. ResultsAll controls developed a renal tumour, compared to 7/72 animals (9.7%) in the vaccine groups. The mean number of lung metastases was 100 (range 3–750) in controls and 4 (range 0–196) in the vaccine groups, respectively. Tumour uptake and number of metastases were not related to the vaccine dose. The 51chromium-release assay confirmed a significant tumour-specific cytolytic activity and marginally increased NK activity of splenocytes from vaccinated mice against RENCA cells compared to controls. Adoptive immune transfer experiments showed that the antitumoural effective immune mechanisms are cell-based. ConclusionsWe could demonstrate the mode-of-action, efficacy, and safety of a homologous tumour vaccine in a RENCA model. These findings support the positive results from a phase-III trial with Reniale.

Dissociating GVT from GVHD in Murine BMT Models through TNFα Dependent CD4+ T Cell Mediated GVHD and IFNγ Dependent CD8+ T Cell Mediated Anti-Tumor Effects.

Tumor relapse and acute graft-versus-host disease (GVHD) remain the major obstacles limiting the efficacy of allogeneic bone marrow transplantation (allo-BMT). However, there has been a strong correlation between the beneficial graft-vs.-tumor effects (GVT) and the deleterious GVHD. Recent studies have provided novel evidence for the divergent roles of the cytokines IL-17 and IFNγ in the establishment and maintenance of inflammatory disease. Because proinflammatory responses are greatly involved in the pathogenesis of acute GVHD, we examined the role of CD8+ T cell-derived IFNγ and CD4+ T cell-derived TNFα mediated cytokine response in the development of GVHD/GVT in two different murine BMT/GVHD models as well a delayed bortezomib-induced GVHD dependent gut toxicity model that we have shown to be associated with increased serum levels of IL-1, IL-6 and TNFα (Blood, 2005). We found that removal of CD4+ but not CD8+ T cells from donor graft markedly lowered post-transplant serum TNFα but not IFNγ levels. This correlated with significantly decreased acute GVHD as well as delayed bortezomib-induced GVHD dependent gut toxicity. The use of TNFα knockout mice as donors revealed that the delayed bortezomib exacerbated acute GVHD-dependent mortality was dependent on CD4+ T cell-derived TNFα. In addition, we found that donor CD4+ IL-17+ cells were significantly increased in the GVHD target organs (gut and liver) but not the spleen of acute GVHD mice. Donor CD4+ IL-17+ cells in the gut were further increased in delayed bortezomib-induced GVHD dependent gut toxicity model. Thus, removal of CD4+ T cells from the graft significantly altered post-transplant serum cytokine patterns resulting in reduced acute GVHD lethality as well as delayed bortezomib-induced GVHD dependent mortality. To determine the effect of bortezomib treatment on CD8+ T cell mediated GVT, A20 lymphoma bearing BALB/c recipients were given FvB cellular grafts. No significant protective anti-tumor effects of the BMT alone with or without post-transplant bortezomib therapy were observed in these advanced-tumor-bearing recipients. When the tumor-bearing mice received allogeneic BMT with donor splenocytes that were depleted of CD4+ T cells, marked and significant increases in survival were observed, indicating that significant GVT effects had been obtained in the absence of donor CD4+ T cells. Survival was further improved with the addition of prolonged post-transplant bortezomib therapy, indicating synergistic anti-tumor responses had been obtained with CD4+ T cell depleted spleen cells and bortezomib treatment (P < 0.05). The use of IFNγ deficient mice, as donors of CD8+ enriched T cells, revealed that the GVT activity and bortezomib-enhanced GVT responses in this model was dependent on donor CD8+ T cell-derived IFNγ. These data suggest that selection for a CD8+ T cell mediated IFNγ but not CD4+ T cell mediated TNFα cytokine response may be a novel therapeutic target for the separating GVT from acute GVHD.

Both CD4+and CD8+T Cells Can Mediate Vaccine‐Induced Protection againstCoccidioides immitisInfection in Mice

To determine which lymphocytes are required for vaccine-induced immunity to coccidioidomycosis, we used a temperature-sensitive mutant of Coccidioides immitis to immunize mice lacking subsets of lymphocytes or specific cytokines and infected the mice 4 weeks later with virulent C. immitis. After 2 weeks, we determined the number of fungi in their lungs and spleens. Vaccine-induced immunity required alpha beta T lymphocytes. beta -2 microglobulin knockout (KO) mice were protected by immunization, and we transferred protection using CD4+ T cells from immunized mice. However, vaccination also protected CD4+ KO mice, which suggests that CD8+ T cells played a role in vaccine-induced immunity, even though they were not required. We adaptively transferred protection using spleen cells from immunized CD4+ KO mice to nonimmune B6 mice, but CD8+ -depleted spleen cells did not protect against infection. Recipients of spleen cells from immunized CD4+ KO mice had 6 times more tumor necrosis factor (TNF)- alpha mRNA in their lungs than did mice that received nonimmune spleen cells, and TNF receptor-1 KO mice were not fully protected by immunization. These results show that both CD4+ and CD8+ T cells can protect against coccidioidomycosis and that TNF- alpha is a necessary component of the acquired immune response.

A New Strategy for Treatment of Malignant Tumor: Intra-Bone Marrow–Bone Marrow Transplantation Plus CD4− Donor Lymphocyte Infusion

Donor lymphocyte infusion (DLI) is clinically used for the treatment of malignant tumors. We have found recently that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to treat various autoimmune diseases, even when radiation doses are reduced. In addition, recently we have found that IBM-BMT can prevent not only graft failure but also graft-versus-host disease (GvHD). Based on these findings, we attempted to prevent and treat the progression of a tumor (Meth-A cell line: BALB/c-derived fibrosarcoma) by DLI plus IBM-BMT. When the tumors had grown to approximately 10 x 10 mm, the tumor-bearing BALB/c (H-2(d)) mice were irradiated with 5 Gy, and whole spleen cells from C57BL/6J (B6) (H-2(b)) mice (as DLI) were then intravenously injected into the BALB/c mice. Simultaneously, bone marrow cells (BMCs) from B6 mice were injected directly into the bone marrow cavity of the BALB/c mice (IBM-BMT). The tumors decreased in size, but the mice died of GvHD. However, when CD4(+) T-cell-depleted spleen cells were used for DLI, the recipients showed only mild GvHD and survived longer, due to the slow growth of the tumor. In contrast, when CD8(+) T-cell-depleted spleen cells were used for DLI, the recipients showed more severe GvHD than those injected with whole spleen cells. These results suggest that IBM-BMT plus DLI (the depletion or reduction of a certain cell population like CD4(+) T cells) could be helpful to suppress both GvHD and tumor growth.

CD4+CD25+ regulatory cells in acquired MHC tolerance.

Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4+ cells. By analogy, regulatory CD4+ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4+ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4+-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8+ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-gamma ELISPOT, and intracellular FACS analysis to study CD8+ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4+CD25+ cells that suppress the development of anti-donor CD8+ T-cell responses in vitro. We conclude that regulatory CD4+CD25+ cells initiate and/or maintain tolerance by preventing the development of CD8+ T-cell alloreactivity.

Induction of proliferation in resting B-cells by a factor released by activated mouse spleen cells.

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.

Interleukin 12 Breaks Ultraviolet Light Induced Immunosuppression by Affecting CD8+ Rather Than CD4+T Cells

Recent studies showed that injection of interleukin (IL)-12 prevents ultraviolet (UV) light mediated suppression of contact hypersensitivity and breaks UV-induced hapten specific tolerance. UV-mediated suppression can be adoptively transferred by injecting splenocytes from UV-irradiated mice; however, suppression is not transferable when donor mice are treated with IL-12 after UV-irradiation. This study was performed to elucidate the mechanisms by which IL-12 counteracts this immunosuppression. To characterize the cells transferring suppression, depletion studies were performed revealing that UV-induced suppression is transferred via CD8+ T cells. To investigate whether IL-12 counteracts UV-induced suppression by either inhibiting the development of CD8+ suppressor T cells or inducing CD4+ effector T cells, splenocytes from mice, which were IL-12 treated and sensitized through UV-exposed skin, were depleted from CD4+ T cells and transferred into naive mice that were subsequently sensitized. Whereas transfer of splenocytes from UV-irradiated mice inhibited sensitization of recipients, no inhibition was observed after transfer of splenocytes from UV-exposed and IL-12 treated mice. Recipients that received CD4 depleted spleen cells from UV-exposed and IL-12 treated donors, were still fully sensitizable. IL-12 also blocked transfer of UV-induced suppression when it was injected into UV-exposed donor animals at a time point when suppressor cells had already developed. CD4 depletion of such splenocytes did not result in a loss of the reconstitutive effect of IL-12. This suggests that IL-12 may break UV-induced tolerance not by inducing CD4+ effector T cells, but rather by inhibiting or inactivating suppressor T cells belonging to the CD8 subtype.

Macrophage synthesis of nitric oxide in the mouse mixed leucocyte reaction

The production of nitric oxide (•NO) in the splenocyte mixed leucocyte reaction (MLR) results in inhibition of allospecific lymphocyte effector function. In order to more clearly define the circumstances which promote •NO synthesis in the MLR, responder accessory cell depleted spleen cells (ACDSC) were co-cultured with allogeneic macrophage cell lines or peritoneal macrophages. •NO synthesis and C57BL/6 (H-2 b) ACDSC proliferation were concurrently monitored in cultures comparing RAW 264.7 (H-2 d, a high •NO producer), P388D1 (H-2 d, a low •NO producer) and BALB/c (H-2 d) peritoneal macrophages as allogeneic antigen presenting cells (APC). A concentration-dependent increase in lymphocyte proliferation was observed in the presence of 1×10 4 to 1×10 5 P388D1. In contrast, addition of N G-monomethyl- L-arginine (NMA), a competitive inhibitor of •NO synthase, was necessary in order to observe lymphocyte proliferation in the presence of increasing numbers of RAW 264.7 and BALB/c peritoneal macrophages. The addition of both anti-IL-2 and anti-IFNγ (interferon-gamma) monoclonal antibodies inhibited •NO synthesis in alloantigen-stimulated cultures. The IFNγ induced expression of class II antigen, as well as the constitutive expression of class I antigen, on RAW 264.7 was similar in the presence or absence of NMA, indicating that induction of •NO synthesis by IFNγ does not inhibit H-2 antigen expression. Thus, cytokines produced as a result of alloimmune interaction initiate macrophage •NO synthesis. However, allogeneic APC function, as assessed by H-2 antigen expression and subsequent stimulatory capacity in MLR, is not affected by initiation of the •NO pathway.

Resting B cells can act as antigen presenting cells in vivo and induce antibody responses.

Although it is well established that B lymphocytes are able to present antigen in vitro, the ability of small resting B cells to act as antigen presenting cells in vivo remains controversial. In this report we have studied the antigen presentation and the antibody response induced by mouse B cells after in vivo or in vitro targeting antigens to membrane Ig (mIg), using rat mAbs. Our results show that injection of these mAbs coupled to 2,4-dinitrophenyl (DNP) strongly enhances the IgG1 antibody response against DNP and rat Ig. T cell depleted spleen cells pulsed in vitro with rat Ig without specificity for B cells induced an antibody response when re-injected into mice, this response being much higher if the antigen was specific for mIg. Moreover, purified resting B cells were shown to induce a specific IgG1 response in vivo only when they were cultured with rat mAb against mIgM or mIgD but not with myeloma rat Ig of the same isotype. B cells do not need to be activated to present antigen since the induction of the specific antibody response does not correlate with the mitogenic activity of rat mAb nor with the IgG1 polyclonal synthesis in vivo. These data clearly show that resting B cells can present antigen in vivo and induce an antibody response, and underline the importance of mIgM and mIgD as targets for antigens.

CD3+4-8- alpha beta T cell population with biased T cell receptor V gene usage. Presence in bone marrow and possible involvement of IL-3 for their extrathymic development.

Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.

Ultraviolet light-induced increase in tumor cell susceptibility to TNF-dependent and TNF-independent natural cell-mediated cytotoxicity

Previously we demonstrated that two consecutive in vitro irradiations of MCA 102 cells with high doses of UVC light (610 and 457 J/m 2) resulted in a selection of a permanent line MCA102UV that manifested high sensitivity to natural cell-mediated cytotoxicity (NCMC). In the present study analysis of the effector cells involved in lysis of these tumor cells was performed by comparing the cytotoxicity of normal spleen cells which mediated both NK and NC cell activity with (a) normal spleen cells in which NC activity was neutralized by anti-TNF Abs (NK +, NC −), (b) NK-depleted or NK-deficient spleen cells (NK −, NC +), and (c) NK-deficient or -depleted spleen cells with NC activity neutralized by anti-TNF Abs (NK −, NC −). Results of these studies indicate that lysis of the original MCA 102 tumor cells was relatively low and was mediated by NC cells. UV irradiation significantly increased MCA 102 tumor cell sensitivity to lysis by both NK and NC cells. Analysis of the mechanisms involved in UV-induced NK sensitivity revealed that UV irradiation increased tumor cell susceptibility to lytic NK-derived granules. NC sensitivity of MCA102UV tumor cells was associated with their increase in sensitivity to TNF and selection of MCA102UV cells for resistance to rTNF resulted in a decrease in their susceptibility to NC cells. To determine how fast UV-induced sensitivity to NCMC and rTNF can be established, 51Cr-labeled MCA 102 cells were irradiated in vitro with 38–304 J/m 2 of UVC light and their sensitivity to lysis by spleen cells and rTNF was tested immediately in an 18-hr cytotoxicity assay. UV treatment with the same doses was repeated 12 days later. The data obtained showed that tumor cell sensitivity to NCMC and TNF appeared shortly after UV irradiation, was stable, and was further substantially augmented by the second round of UV treatment. Thus, in vitro UV irradiation of tumor cells could be an effective modulator of tumor cell sensitivity to TNF-dependent and TNF-independent cell-mediated cytotoxicity.

Influences of Cephem Antibiotics on the Immune Response in Mice

The effects of six cephem antibiotics, including ceftezole, cefmetazole, cefoxitin, cefotiam, cefoperazone, and cefotaxime, on murine humoral immunity were examined. In female BDF1 mice each cephem antibiotic was administered at a dose of 800 mg/kg/day i.v. for 7 consecutive days. Among the antibiotics tested, only ceftezole and cefoperazone induced a significant increase in serum total IgM, but not in serum total IgG. Especially in case of ceftezole, the mice developed splenomegaly due to the proliferation of IgM-producing cells in the germinal centers. The proliferation of splenic IgM-producing cells was also observed in female thymus-deficient Balb/c-nu/nu mice receiving intravenous ceftezole. Thus, the drug was indicated to enhance the polyclonal IgM production in mice by acting as a B cell mitogen. This is consistent with the in vitro finding that ceftezole exhibited a mitogenic effect on whole spleen cells from BDF1 mice, but not on B cell depleted spleen cells.

Anti-inflammatory and immuno-modulatory effects of novel retinoidlike 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10-23 and the level of plasma fibrinogen (MED approximately 25 mumoles/kg). When given therapeutically (75 mumoles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25-75 mumoles/kg). Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12-15) responded poorly to the T cell mitogen, Con A (2.5 micrograms/ml) and the B cell mitogen, LPS (10 micrograms/ml). The responses were partially restored (approximately 30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 mumoles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.

Influence of interferon γ on B cell replication

Semisolid agar culture techniques, recombinant murine gamma interferon, (Mu IFN γ) and monoclonal anti- Mu IFN γ antibodies were used to evaluate the role of interferon in mitogen-dependent B cell proliferation. Subtle, but significant, effects of interferon addition were noted when unseparated spleen cells were cultured. Depending on culture conditions, the cloning efficiency of the B cells was slightly increased by optimal concentrations of interferon. When the spleen cells were cultured over unstimulated peritoneal macrophages separated by an agar spacer, and especially when no other potentiators were added to the cultures, substantial enhancement of B cell proliferation routinely resulted. The mechanism of this effect was then investigated with macrophage depleted spleen cells as well as highly enriched, positively selected, B cell suspensions. Results of these experiments indicated that B cell replication can be modulated by direct interaction with interferon and again, the conditions of culture determined the magnitude and direction of the effect. Addition of anti-interferon antibodies, indomethacin, or interleukin 1 (IL-1) to the cultures did not influence the cloning efficiency of normal splenic B cells. Therefore, we found no evidence that endogenously produced interferon, prostaglandins, and IL-1 influence B cell colony formation. Since interferon stimulated macrophages dramatically improved B cell proliferation without direct cell contact, some other monokine may be involved. Therefore, gamma interferon can participate in humoral immune responses by influencing the expansion of activated B cells directly as well as indirectly via stimulation of macrophages.

Mechanism of action of the new immunomodulator LS2616 on T cell responses

Spleen cells from mice treated with LS2616 display a highly increased response to the polyclonal T cell lectin ConA. The total number of splenic T cells, and the relative ratios between L3T4 + and Lyt-2 + T cells were not altered by LS2616 treatment. By dissecting the overall ConA response it was found that the number of ConA-inducible, IL-2 reactive T cells was unaffected, while ConA-induced IL-2 production was enhanced after LS2616 treatment. Spleen cells from LS2616 treated mice, depleted of G10 adherent macrophages (Mφ) and reconstituted with Mφ from untreated mice displayed normal levels of ConA responses. Mφ depleted spleen cells from untreated animals, cocultured with Mφ enriched populations from LS2616 treated animals resulted in an increased ConA response. Furthermore, spleen cells from treated mice were found to be excellent stimulators for alloantigen-induced T cell responses; when used as responders in MLC, however, these cells were comparable to responders from non-treated animals. Taken together the results demonstrate that LS2616 exerts an immunostimulatory effect on Mφ, which indirectly facilitates polyclonal and antigen-specific T cell responses. The possible implications of this observation on various immunoregulatory events are discussed.