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Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice., This work was funded by a grant provided to JP by the Lung Foundation Netherlands (5.2.14.058JO), the NIHR Cambridge Biomedical Research Centre and NIHR Imperial Biomedical Research Centre. ERC and CS’ laboratories receive grant support from the Medical Research Council, Wellcome Trust, NIHR, GlaxoSmithKline, MedImmune Ltd., and Bristol-Myers Squibb. CLC is supported by Bloodwise (12033), CRUK (C36195/A1183) and European Research Council (ERC) (337066). CP is supported by Bloodwise (12033). The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is part-supported by funding from the Wellcome Trust (grant 104931/Z/14/Z) and BBSRC (grant BB/L015129/1). KDF is supported by funding from the Wellcome Trust (201356/Z/16/Z). LMC is supported by core funding from Cancer Research UK (A23983 and A17196).

Background: The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21.Results: The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined.Conclusions: The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.

Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc * OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.

Dendritic cells (DCs) are the most potent antigen presenting cells and possess an incomparable ability to activate and instruct T cells, which makes them one of the cornerstones in the regulation of the cross-talk between innate and adaptive immunity. Therefore, a deep understanding of DC biology lays the foundations to describe and to harness the mechanisms that regulate the development of the adaptive response, with clear implications in a vast array of fields such as the study of autoimmune diseases and the development of new vaccines. However, the great difficulty to obtain large quantities of viable non-activated DCs for experimentation have considerably hindered the progress of DC research. Several strategies have been proposed to overcome these limitations by promoting an increase of DC abundance in vivo, by inducing DC development from DC progenitors in vitro and by generating stable DC lines. In the past years, we have described a method to derive immortalized stable DC lines, named MutuDCs, from the spleens of Mushi1 mice, a transgenic mouse strain that express the simian virus 40 Large T-oncogene in the DCs. The comparison of these DC lines with the vast variety of DC subsets described in vivo has shown that all the MutuDC lines that we have generated so far have phenotypic and functional features of type 1 conventional DCs (cDC1s). With the purpose of deriving DC lines with characteristics of type 2 conventional DCs (cDC2s), we bred a new Batf3 -/- Mushi1 murine line in which the development of the cDC1 subset is severely defective. The new MutuDC line that we generated from Batf3 -/- Mushi1 mice was phenotypically and functionally characterized in this work. Our results demonstrated that all the tested characteristics of this new cell line, including the expression of subset-determining transcription factors, the profile of cytokine production and the ability to present antigens, are comparable with the features of splenic CD4 - cDC2s. Therefore, we concluded that our new cell line, that we named CD4 - MutuDC2 line, represents a valuable model for the CD4 - cDC2 subset.

T and B cell compartmentalization is a hallmark of secondary lymphoid organs and is maintained by chemokine-expressing stromal cells. How this stromal cell network initially develops and differentiates into two distinct subsets is poorly known, especially for the splenic white pulp (WP). Here, we show that perivascular fibroblast precursors are triggered by LTα1β2 signals to expand, express CCL19/21, and then differentiate into two functionally distinct fibroblast subsets responsible for B and T cell clustering and WP compartmentalization. Failure to express or sense CCL19 leads to impaired T zone development, while lack of B cells or LTα1β2 leads to an earlier and stronger impairment in WP development. We therefore propose that WP development proceeds in multiple steps, with LTα1β2 + B cells acting as major inducer cells driving the expansion and gradual differentiation of perivascular fibroblasts into T and B zone organizer cells.

IFN-gamma is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-gamma reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-gamma-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-g by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common gamma-chain (gamma c) genes (Rag2(-/-) gamma c(-/-)), indicating their innate nature and their gc cytokine independence. Rag2(-/-) gamma c(-/-) mice are as resistant to Mycobacterium avium as Rag2(-/-) mice, whereas Rag2(-/-) mice lacking IFN-g are more susceptible than either Rag2(-/-) or Rag2(-/-) gamma c(-/-). These lineage-negative CD45(+)/Thy1.2(+) cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2(-/-) gamma c(-/-) animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-gamma-producing cells. Accordingly, Thy1.2specific mAb administration to infected Rag2(-/-) gamma c(-/-) animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2(+) non-NK innate-like cells present in the liver expresses IFN-g and can confer protection against M. avium infection in immunocompromised mice., Supported by a structured program on bioengineered therapies for infectious diseases and tissue regeneration (Grant NORTE-01-0145-FEDER-000012) supported by Norte Portugal Regional Operational Programme (NORTE 2020, under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund); by Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation, Portugal 2020; by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência (FCT), Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (Grant POCI-01-0145-FEDER-007274); by the Trudeau Institute, Inc.; and by FCT Grant SFRH/BD/89871/2012 (to M.R.), info:eu-repo/semantics/publishedVersion

Previous studies in rodents have indicated that only a minor fraction of the immunoglobulin heavy chain variable region (IGHV-Cμ) transcripts carry somatic mutations and are considered memory B cells. This is in marked contrast to humans where nearly all marginal zone B (MZ-B) cells are mutated. Here we show in rats that the proportion of mutated IgM+ MZ-B cells varies significantly between the various IGHV genes analyzed, ranging from 27% mutated IGHV5 transcripts to 65% mutated IGHV4 transcripts. The observed data on mutated sequences in clonally-related B cells with a MZ-B cell or follicular B (FO-B) cell phenotype indicates that mutated IgM+ MZ-B and FO-B cells have a common origin. To further investigate the origin of mutated IgM+ MZ-B cells we determined whether mutations occurred in rearranged IGHV-Cμ transcripts using IGHV4 and IGHV5 genes from neonatal rat MZ-B cells and FO-B cells. We were not able to detect mutations in any of the IGHV4 and IGHV5 genes expressed by MZ-B cells or FO-B cells obtained from neonatal rat spleens. Germinal centres (GCs) are absent from neonatal rat spleen in the first few weeks of their life, and no mutations were found in any of the neonatal sequences, not even in the IGHV4 gene family which accumulates the highest number of mutated sequences (66%) in the adult rat. Therefore, these data do not support the notion that MZ-B cells in rats mutate their IGHV genes as part of their developmental program, but are consistent with the notion that mutated rat MZ-B cells require GCs for their generation. Our findings support that the splenic MZ of rats harbors a significant number of memory type IgM+ MZ-B cells with mutated IGHV genes and propose that these memory MZ-B cells are probably generated as a result of an antigen driven immune response in GCs, which still remains to be proven.

A cDNA encoding the chicken homologue of the human myelomonocytic differentiation antigen, CD14, was cloned by RT-PCR from chicken bone marrow cell RNA, using oligonucleotide primers based on the predicted cDNA sequence. The cloned chicken CD14 (chCD14) cDNA encodes an open reading frame of 465 amino acids (aa), with 31-34% aa identity to mouse, bovine and human (hu) CD14. As in mouse and man, chCD14 is a leucine-rich protein. In mammals, CD14 is a GPI-anchored protein. Protein structure analysis suggested that chCD14, by contrast, was potentially a trans-membrane protein. The predicted aa sequence comprises an extracellular domain of 417 aa, followed by a 23-aa trans-membrane segment, and a 25-aa intracytoplasmic region, the latter containing no obvious signalling motifs. COS-7 cells were transfected with p3XFLAG-CMV-8::chCD14 or pCDM8::huCD14, incubated with or without PI-PLC and stained with anti-FLAG or anti-huCD14 antibody respectively. PI-PLC cleaved huCD14 but not chCD14, suggesting that chCD14 is not GPI-anchored. Real-time quantitative RT-PCR analysis revealed that chCD14 mRNA was expressed in most lymphoid and non-lymphoid tissues, except muscle. ChCD14 mRNA was also expressed in most cells examined but strongly expressed in chicken peripheral blood monocyte/macrophages and KUL01+ splenocytes.

Poly-N-acetyl glucosamine (PNAG) is a major component of Staphylococcus epidermidis biofilms extracellular matrix. However, it is not yet clear how this polysaccharide impacts the host immune response and infection-associated pathology. Faster neutrophil recruitment and bacterial clearance was observed in mice challenged intraperitoneally with S. epidermidis biofilm cells of the PNAG-producing 9142 strain than in mice similarly challenged with the isogenic PNAG-defective M10 mutant. Moreover, intraperitoneal priming with 9142 cells exacerbated liver inflammatory pathology induced by a subsequent intravenous S. epidermidis challenge when compared to priming with M10 cells. The 9142-primed mice had elevated splenic CD4+ T cells producing IFN- and IL-17A, indicating that PNAG promoted cell-mediated immunity. Curiously, despite having more marked liver tissue pathology, 9142-primed mice also had splenic T regulatory cells with increased suppressive activity compared with the M10-primed counterparts. By showing that PNAG production by S. epidermidis biofilm cells exacerbates host inflammatory pathology these results altogether suggest that this polysaccharide contributes for the clinical features associated to biofilm originated infections., Fundação para a Ciência e a Tecnologia (FCT) and COMPETE [grant numbers PTDC/BIA-MIC/113450/2009, FCOMP-01-0124-FEDER-014309, FCOMP‐01‐0124‐FEDER‐022718, FCT PEst‐C/SAU/LA0002/2011, NORTE-01-0145-FEDER-000012]. The following authors received individual FCT fellowships: PF (SFRH/BD/76900/2011), AC (SFRH/BPD/91623/2012), VC (SFRH/BD/78235/2011), AF (SFRH/BPD/99961/2014). LT was supported by Fundo Social Europeu and Programa Operacional Potencial Humano through FCT Investigator [grant number IF/01241/2014].

NLRC5 is a transcriptional regulator of MHC class I (MHCI), which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK–T-cell crosstalk, raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly, NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice, we show that NK cells surprisingly break tolerance even towards ‘self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore, during chronic LCMV infection, the total CD8+ T-cell population is severely decreased in these mice, a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets, having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions., NK cell tolerance to self-MHCI levels is calibrated during their development. Here the authors show that this tolerance is overcome by an inflammatory environment and that NLRC5 protects T cells from NK cell-mediated elimination by maintaining high MHCI expression.

Background Interferon (IFN)-β exerts anti-inflammatory effects, coupled to remarkable neurological improvements in multiple sclerosis, a neuroinflammatory condition of the central nervous system. Analogously, it has been hypothesized that IFN-β, by limiting inflammation, decreases neuronal death and promotes functional recovery after stroke. However, the core actions of endogenous IFN-β signaling in stroke are unclear. Methods To address this question, we used two clinically relevant models of focal cerebral ischemia, transient and permanent middle cerebral artery occlusion, and two genetically modified mouse lines, lacking either IFN-β or its receptor, the IFN-α/β receptor. Subsets of inflammatory and immune cells isolated from the brain, blood, and spleen were studied using flow cytometry. Sensorimotor deficits were assessed by a modified composite neuroscore, the rotating pole and grip strength tests, and cerebral infarct volumes were given by lack of neuronal nuclei immunoreactivity. Results Here, we report alterations in local and systemic inflammation in IFN-β knockout (IFN-βKO) mice over 8 days after induction of focal cerebral ischemia. Notably, IFN-βKO mice showed a higher number of infiltrating leukocytes in the brain 2 days after stroke. Concomitantly, in the blood of IFN-βKO mice, we found a higher percentage of total B cells but a similar percentage of mature and activated B cells, collectively indicating a higher proliferation rate. The additional differential regulation of circulating cytokines and splenic immune cell populations in wild-type and IFN-βKO mice further supports an important immunoregulatory function of IFN-β in stroke. Moreover, we observed a significant weight loss 2–3 days and a reduction in grip strength 2 days after stroke in the IFN-βKO group, while endogenous IFN-β signaling did not affect the infarct volume. Conclusions We conclude that endogenous IFN-β signaling attenuates local inflammation, regulates peripheral immune cells, and, thereby, may contribute positively to stroke outcome. Electronic supplementary material The online version of this article (doi:10.1186/s12974-015-0427-0) contains supplementary material, which is available to authorized users.

Murine acquired immunodeficiency syndrome (MAIDS) is characterized by lymphoproliferation, polyclonal B cell activation resulting in the production of autoantibodies, and a progressive immunodeficiency. These are all hallmarks of some autoimmune diseases. Yaa is a Y-chromosome-linked gene that accelerates autoimmune diseases in some autoimmune-prone strains of mice. To further elucidate a possible relationship with autoimmunity, the effect of the Yaa gene on MAIDS was investigated. Analysis of phenotypic and functional disease parameters revealed that Yaa does not accelerate MAIDS disease. This is probably due to the generalized activation of most or all lymphoid cells in MAIDS, which cannot be enhanced by the Yaa gene. This result is in accordance with the selective enhancing effect of the Yaa gene on the immune response against self and foreign antigens in a specific genetic background. It suggests that the autoimmune response associated with MAIDS is a secondary phenomenon. Interestingly, even in wild-type C57BL/6 mice, autoantibody production may contribute overproportionally to the hypergammaglobulinemia associated with MAIDS.

Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.

International audience; Background: The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy.Methods: We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1.Results: Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells.Conclusion: Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

We explore herein the effect of TNF-related activation-induced cytokine (TRANCE) co-stimulatory pathway blockade on islet survival after allograft transplantation. Expression of TRANCE on murine C57Bl/6 (B6) CD4+ T cells after allogeneic activation was analyzed by fluorescence-activated cell sorter (FACS). The effect of a TRANCE receptor fusion protein (TR-Fc) and anti-CD154 antibody (MR1) on B6 spleen cell proliferation after allogeneic activation was assessed by mixed lymphocyte reaction (MLR). Three groups of B6 mice were transplanted with allogeneic islets (DBA2): Control; short-term TR-Fc-treatment (days 0-4); and prolonged TR-Fc-treatment (days -1 to 13). Donor-specific transfusion (DST) was performed at the time of islet transplantation in one independent experiment. Transplantectomy samples were analyzed by immunohistochemistry. TRANCE expression was upregulated in stimulated CD4+ T cells in vitro. In MLR experiments, TR-Fc and MR1 both reduced spleen cell proliferation, but less than the combination of both molecules. Short-course TR-Fc treatment did not prolong islet graft survival when compared with controls (10.6 +/- 1.9 vs. 10.7 +/- 1.5 days) in contrast to prolonged treatment (20.7 +/- 3.2 days; P < 0.05). After DST, primary non function (PNF) was observed in half of control mice, but never in TR-Fc-treated mice. Immunofluorescence staining for Mac-1 showed a clear decrease in macrophage recruitment in the treated groups. TRANCE-targeting may be an effective strategy for the prolongation of allogeneic islet graft survival, thanks to its inhibitory effects on co-stimulatory signals and macrophage recruitment.

International audience; CX(3)CR1 expression is associated with the commitment of CSF-1R(+) myeloid precursors to the macrophage/dendritic cell (DC) lineage. However, the relationship of the CSF-1R(+) CX(3)CR1(+) macrophage/DC precursor (MDP) with other DC precursors and the role of CX(3)CR1 in macrophage and DC development remain unclear. We show that MDPs give rise to conventional DCs (cDCs), plasmacytoid DCs (PDCs), and monocytes, including Gr1(+) inflammatory monocytes that differentiate into TipDCs during infection. CX(3)CR1 deficiency selectively impairs the recruitment of blood Gr1(+) monocytes in the spleen after transfer and during acute Listeria monocytogenes infection but does not affect the development of monocytes, cDCs, and PDCs.

International audience; Viral and non-viral vectors have been developed for gene therapy, but their use is associated with unresolved problems of efficacy and safety. Efficient and safe methods of DNA delivery need to be found for medical application. Here we report a new monopolar system of non-viral electro-gene transfer into the thymus in vivo that consists of the local application of electrical pulses after the introduction of the DNA. We assessed the proof of concept of this approach by correcting ZAP-70 deficient severe combined immunodeficiency (SCID) in mice. The thymic electro-gene transfer of the pCMV-ZAP-70-IRES-EGFP vector in these mice resulted in rapid T cell differentiation in the thymus with mature lymphocytes detected by three weeks in secondary lymphoid organs. Moreover, this system resulted in the generation of long-term functional T lymphocytes. Peripheral reconstituted T cells displayed a diversified T cell receptor (TCR) repertoire, and were responsive to alloantigens in vivo. This process applied to the thymus could represent a simplified and effective alternative for gene therapy of T cell immunodeficiencies.

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

The Cd22 gene encodes a B cell-specific adhesion molecule that modulates B cell Ag receptor-mediated signal transduction, and is allelic to a lupus-susceptibility locus in New Zealand White (NZW) mice. In this study, we show that, in addition to the wild-type transcripts, NZW (Cd22a) mice synthesize aberrant CD22 mRNAs that contain approximately 20-120 nucleotide insertions upstream of the coding region between exons 2 and 3, and/or approximately 100-190 nucleotide deletions of exon 4. Sequence analysis revealed that these aberrant mRNA species arose by alternative splicing due to the presence in the NZW strain of a 794-bp sequence insertion in the second intron, containing a cluster of short interspersed nucleotide elements. Both the presence of sequence insertion and aberrantly spliced mRNAs were specific to mice bearing the Cd22a and Cd22c alleles. Up-regulation of CD22 expression after LPS activation appeared impaired in Cd22a spleen cells (twice lower than in Cd22b B cells). Furthermore, we show that partial CD22 deficiency, i.e., heterozygous level of CD22 expression, markedly promotes the production of IgG anti-DNA autoantibodies in C57BL/6 (Cd22b) mice bearing the Y chromosome-linked autoimmune acceleration gene, Yaa. Taken together, these results suggest that a lower up-regulation of CD22 on activated B cells (resulting from Cd22 gene anomaly in Cd22a mice or from CD22 heterozygosity in mutants obtained by gene targeting) is implicated in autoantibody production, providing support for Cd22a as a possible candidate allele contributing to lupus susceptibility.

Mutations in the putative transcription factor autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiosis-ectodermal dystrophy (APECED; OMIM#240300), a monogenic recessively inherited disease characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiosis of mucous membranes and ectodermal dystrophies. In this study the expression of murine homolog for AIRE protein, Aire, was detected in a fraction of thymic medullary epithelial cells. Subcellularly, in thymus the protein appears as concentrated into nuclear dot-like structures, whereas in transfected cells the protein is also bound along a cytosolic fibrillar network. By RT-PCR Aire mRNA was detected in thymus, lymph node, spleen and testis although the second round PCR amplified Aire specific band from most mouse tissues analyzed. Furthermore, the Aire mRNA was detected in dendritic cell (DC) populations isolated from thymus and spleen, representing both myeloid- and lymphoid-related lineages of DC. We also demonstrate that the Aire protein is absent in the thymus of RelB-deficient mouse and in NOD thymus most of the Aire positive cells showed an abnormal morphology. These results suggest that the Aire protein is associated with the normal development and/or action of a subset of thymic medullary stromal cells involved in tolerance induction.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cγ2a and Cγ1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon γ in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten–protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten–protein can proceed in a node where there is substantial Th1 activity.

The injection of semi-allogeneic F1 spleen cells into newborn mice of a parental strain induces a state of immune tolerance characterized by anti-donor CTL unresponsiveness and the appearance of a transient SLE-like autoimmune syndrome associating autoantibody production, hypergammaglobulinemia, splenomegaly and glomerulonephritis. Our previous experiments have demonstrated that host Th2-like CD4+ T lymphocytes activate donor F1 B cells persisting in the host to produce autoantibodies, and that this cellular interaction relies on the presence of alloMHC class II molecules on donor B cells. In order to investigate the role and the involvement of MHC alloantigens in the cellular T(host)-B(donor) interaction, newborn C57BL/6 (B6) mice were injected with F1 spleen cells differing from the host at the level of defined portions of the MHC class I (K) or class II (I-A and I-E) molecules. B6 mice injected at birth with spleen cells from different F1 strains were tolerant to each alloantigen (alloAg) tested, as assessed by specific anti-donor CTL unresponsiveness. However, the SLE-like autoimmune syndrome only developed in B6 mice injected at birth with F1 spleen cells differing at the level of MHC class II I-A or I-E molecules. Autoantibodies appeared later in B6 mice neonatally tolerized to I-E alloAg than those detected in B6 mice neonatally tolerized to I-A alloAg. These results show that the SLE-like autoimmune disease that develops concomitantly to neonatally-induced tolerance to alloAg is the consequence of cognate T host-B donor cellular interactions triggered by even minute differences in the MHC class II I-A or MHC class II I-E molecules.

zugleich Diss. LMU München 1982. lt. DNB: Diss. A

Mouse spleen lymphocytes synthesize a protein which comigrates with skeletal muscle alpha-actinin on two-dimensional gel electrophoresis and is immunoprecipitated by an antibody directed against skeletal muscle alpha-actinin. Mouse lymphocyte alpha-actinin is present in membrane fractions, and is immunoprecipitated from lymphocyte detergent lysates by an antiserum made against these purified membranes. The anti-alpha-actinin activity of this antiserum is not adsorbed after incubation with fixed intact lymphocytes. Lymphocyte alpha-actinin does not bind concanavalin A and it is inaccessible to lactoperoxidase-catalyzed surface iodination. Double immunofluorescence shows that alpha-actinin moves concurrently along the cell membrane with redistributed surface immunoglobulins and Thy-1 antigen, and remains associated up to 30 min with surface aggregates of these receptors. Our results suggest that lymphocyte alpha-actinin, as defined by molecular weight and cross reactivity with the antibody against the muscle protein, (a) is associated with the cell membrane, (b) is not expressed at the cell surface, and (c) participates in the movement of surface receptors.

Mice treated from birth with polyclonal, crude or affinity purified rabbit or monoclonal rat anti-mouse IgM antibodies [b-7-6 and C-2-23: Eur. J. Immunol. 14: 753-757, 1984] were found to be heavily suppressed with respect to B-cell activities. Crude or affinity purified rabbit or monoclonal rat anti-mouse IgM gave comparable results as follows: serum IgM was below detectable levels; serum IgG was reduced to about 1-3% of normal levels; free anti-IgM was always detectable; IgM and/or kappa-light-chain positive cells as well as IgM-secreting cells were absent in various lymphoid organs; the B-cell mitogen lipopolysaccharide was unable to induce proliferative responses; primary antibody responses could not be induced against sheep red blood cells and phosphorylcholine; lymphoid organs were reduced in size and B-cell areas were not populated with lymphocytes; besides a 40% reduction in absolute lymphocyte numbers in the blood, we found increased platelet counts and a 10% eosinophilia in anti-IgM-treated mice.

In view of the eminently important role of interleukin-2 (IL-2) in T-cell responses, and in view of reports about immune stimulatory effects of PGE2, we reinvestigated the question whether PGE2 inhibits IL-2 production. It was found that PGE2 does not inhibit IL-2 production in murine spleen cell cultures after optimal stimulation (5 micrograms/ml concanavalin A) but does inhibit at suboptimal stimulation conditions. The failure of PGE2 to inhibit IL-2 production at optimal concanavalin A concentration was demonstrated by two independent IL-2 assays namely by the co-stimulator assay and by the proliferation of IL-2-dependent T-cell clone W-2. Our observations indicated that the inhibitory effect of PGE2 depends on the strength of the stimulating signal. IL-2 production in cultures with 5 micrograms/ml concanavalin A was also not suppressed by PGE1, by prostaglandin D2, thromboxane B2 (T X B2), and prostaglandin F2.

Murine spleen and lymph node L3T4+ T cells were found to spontaneously produce high levels of interleukin 3 (IL3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in cultures containing 10% fetal calf serum (FCS) in the absence of other stimulation. The IL3 and GM-CSF activities in culture supernatants peake between the fifth and seventh day of culture. The specificity of the bioassays was attested by the use of rabbit anti-IL3 and anti-GM-CSF antibodies, as well as by the detection of a maximal accumulation of IL3 and GM-CSF mRNA on the fourth day. In contrast, no significant activities of IL2, IL4 or interferon-gamma were detected in these culture supernatants. The markedly limited production of IL3 and GM-CSF in cultures performed in 1% autologous normal mouse serum and the inhibitory effect of anti-Ia or anti-L3T4 monoclonal antibody strongly suggest that the selective production of most, if not all IL3 and GM-CSF by L3T4+ T cells is a result of activation of L3T4+ T cells by fetal calf serum. All the strains of mice tested except athymic nude mice produced substantial amounts of IL3 and GM-CSF during the culture. This is in contrast to a previous report (Palacios, Eur. J. Immunol. 1984. 14: 599), indicating that only spleen cells of the MRL strain homozygous for the lpr gene spontaneously release IL3 in cultures. We found that spleen and lymph node cells from MRL/MpJ-lpr/lpr or C57BL/6J-lpr/lpr mice released, in fact, much less IL3 and GM-CSF in cultures. This was, however, due to the high proportion of the peculiar lpr Ly-2-/L3T4-T cells in spleen and lymph nodes, since after depletion of this lpr T cell subset, lymph node cells from C57BL/6J-lpr/lpr mice produced IL3 and GM-CSF at levels comparable to those in C57BL/6J-+/+ mice. These results further support the notion that the lpr Ly-2-/L3T4- T cell subset is immunologically nonfunctional and its accumulation dilutes functional L3T4+ T cells in mice bearing the lpr mutation.