Normal Humoral Immune Responses Research Articles

T cell help shapes B cell tolerance.

T cell help is a crucial component of the normal humoral immune response, yet whether it promotes or restrains autoreactive B cell responses remains unclear. Here, we observe that autoreactive germinal centers require T cell help for their formation and persistence. Using retrogenic chimeras transduced with candidate TCRs, we demonstrate that a follicular T cell repertoire restricted to a single autoreactive TCR, but not a foreign antigen-specific TCR, is sufficient to initiate autoreactive germinal centers. Follicular T cell specificity influences the breadth of epitope spreading by regulating wild-type B cell entry into autoreactive germinal centers. These results demonstrate that TCR-dependent T cell help can promote loss of B cell tolerance and that epitope spreading is determined by TCR specificity.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies

TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating Bcells but overall normal humoral immune responses. Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

Venom composition of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae) and functional characterization of a major venom peptide

Endoparasitoid wasps use complex biochemical arsenals to suppress the normal humoral and cellular immune responses of their hosts in order to transform them into a suitable environment for development of their eggs and larvae. Venom injected during oviposition is a key component of this arsenal, but the functions of individual venom toxins are still poorly understood. Furthermore, there has been little investigation of the potential biotechnological use of these venom toxins, for example for control of agricultural pests. The endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) is a biocontrol agent reared in biofactories and released extensively in Brazil to control the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). The objectives of this work were to reveal venom components produced by C. flavipes and explore the function of a major venom peptide, Cf4. Using a combined proteomic/transcriptomic approach, we identified 38 putative venom toxins including both linear and disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins, and proteins of the odorant binding families. Because of its high abundance in the venom, we selected Cf4, a 33-residue peptide with three disulfide bonds, for synthesis and further characterization. We found that synthetic Cf4 reduced the capacity of D. saccharalis hemocytes to encapsulate foreign bodies without any effect on phenoloxidase activity, consistent with a role in disruption of the cellular host immune response. Feeding leaves coated with Cf4 to neonate D. saccharalis resulted in increased mortality and significantly reduced feeding compared to caterpillars fed untreated leaves, indicating that Cf4 is a potential candidate for insect pest control through ingestion. This study adds to our knowledge of endoparasitoid wasp venoms composition, host regulation mechanisms and their biotechnological potential for pest management.

Reshaping the murine immunoglobulin heavy chain repertoire with bovine DH genes

Having a limited number of VH segments, cattle rely on uniquely long DH gene segments to generate CDRH3length variation (3-70aa) far greater than that in humans or mice. Bovine antibodies with ultralong CDRH3s (>50aa) possess unusual structures and abilities to bind to special antigens. In this study, we replaced most murine endogenous DH segments with bovine DH genes, generating a mouse line termed B-DH. The use of bovine DH genes significantly increased the length variation of CDRH3 and consequently the Ig heavy chain repertoire in B-DH mice. However, no ultralong CDRH3 was observed in B-DH mice, suggesting that other factors, in addition to long DH genes, are also involved in the formation of ultralong CDRH3. The B-DH mice mounted a normal humoral immune response to various antigens, although the B-cell developmental paradigm was obviously altered compared with wild-type mice. Additionally, B-DH mice are not predisposed to the generation of autoantibodies despite the interspecies DH gene replacement. The B-DH mice reported in this study provide a unique model to answer basic questions regarding the synergistic evolution of DH and VH genes, VDJ recombination and BCR selection in B-cell development.

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B‐cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B‐cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro‐proliferative, pro‐GC program. In addition, DOT1L indirectly supports the repression of an anti‐proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B‐cell naivety and GC B‐cell differentiation.

Lncrnas As New Partners of Novel Chimeric Transcripts in Multiple Myeloma

Deregulation of long non-coding RNAs (lncRNAs) is a common feature of cancer, including Multiple Myeloma (MM). In our previous studies, we detected 11,495 and 40,511 previously non-annotated lncRNAs during normal humoral immune response and MM patient samples, respectively. These results support an important role for the lncRNAs transcriptome in this hematological malignancy. lncRNAs are genes that differ from coding genes in that they do not give rise to a protein. Nevertheless, lncRNA could undergo the same genetic alterations as coding genes. In this study, we hypothesize that lncRNAs can be involved in the principal genetic alterations occurring in MM such as chromosomal translocations, affecting the Immunoglobulin loci (IG) and in the majority of which the fusion partner still remains unknown. In order to unveil the role of lncRNAs in fusion transcripts occurring in MM, we analyzed the strand specific RNA-seq (ssRNA-seq) data obtained from 35 samples from different subpopulations of B cells (Naïve, Centroblast, Centrocyte, Memory and Plasma cells (PCs)), purified PCs from 32 MM patients and 3 MM cell lines. Chimeric transcripts were detected with the STAR-Fusion software, identifying 1,347 novel fusion transcripts ranging from 1 to 142 chimeric transcripts per sample. Strikingly, healthy PC samples (from tonsils and bone marrow) yielded the highest number of fusion transcripts, while other B cell subpopulations showed overall low numbers and MM samples turned out highly variable. 96% of all fusion transcripts detected in healthy PCs occurred with IG genes and harbored few reads per transcript, suggesting that the hyperactive transcription of the IG loci in PCs may be the cause for their formation and are probably not involved in the pathogenesis of the disease. We also found that HLA fusion transcripts were abundant in Naïve B cells, disappearing progressively during the humoral immune response (Figure 1). Interestingly, fusion transcripts identified in different B cell subpopulations were not detected in MM samples. Next, we focus on myeloma samples and identified 362 chimeric transcripts (312 unique) expressed specifically in MM (ranging from 2 to 24 chimeric transcripts per sample), most of them (84%) identified for the first time. 69% of these transcripts partnered with the IG genes, while the other fusions involved two non-IG genes. Interestingly, as we hypothesized, 26,5% of the chimeric transcripts in MM occurred with lncRNA as a partner, increasing the relevance of lncRNAs in this disease. Furthermore, using the read distribution per chimeric transcript, we identified a prevalent reciprocal transcript or a prevalent transcript expressed with >1 FFPM in 47% of MM samples, suggesting that they could derived from underlying genomic rearrangements. Besides these prevalent transcripts, we observed that 40% of the non-IG fusion transcripts occurred between adjacent genes, defining novel MM-transcriptional read-throughs, possibly caused by the oncogenic stress suffered by MM cells. Some of these chimeric transcripts were validated in different cell lines of MM by conventional PCR and sequencing. In summary, our findings show that ssRNA-seq data is an adequate strategy for the detection of chimeric transcripts in MM, being able to detect highly expressed chimeric transcripts that probably were derived from an underlying genomic rearrangements and also new categories of chimeric transcripts. In addition, our study reveals a complex landscape of fusion transcripts in the MM, many of them including a lncRNA, which could be potential therapeutic targets for the development of new treatment strategies for MM. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity.

Regulated apoptosis of germinal centre (GC) B cells is critical for normal humoral immune responses. ELL-associated factor 2 (EAF2) regulates transcription elongation and has been shown to be an androgen-responsive potential tumour suppressor in prostate by inducing apoptosis. Here we show that EAF2 is selectively upregulated in GC B cells among various immune cell types and promotes apoptosis of GC B cells both in vitro and in vivo. EAF2 deficiency results in enlarged GCs and elevated antibody production during a T-dependent immune response. After immunization with type II collagen, mice lacking EAF2 produce high levels of collagen-specific autoantibodies and rapidly develop severe arthritis. Moreover, the mutant mice spontaneously produce anti-dsDNA, rheumatoid factor and anti-nuclear antibodies as they age. These results demonstrate that EAF2-mediated apoptosis in GC B cells limits excessive humoral immune responses and is important for maintaining self-tolerance.

Review of Elephant Endotheliotropic Herpesviruses and Acute Hemorrhagic Disease

More than 100 young captive and wild Asian elephants are known to have died from a rapid-onset, acute hemorrhagic disease caused primarily by multiple distinct strains of two closely related chimeric variants of a novel herpesvirus species designated elephant endotheliotropic herpesvirus (EEHV1A and EEHV1B). These and two other species of Probosciviruses (EEHV4 and EEHV5) are evidently ancient and likely nearly ubiquitous asymptomatic infections of adult Asian elephants worldwide that are occasionally shed in trunk wash secretions. Although only a handful of similar cases have been observed in African elephants, they also have proved to harbor their own multiple and distinct species of Probosciviruses-EEHV2, EEHV3, EEHV6, and EEHV7-found in lung and skin nodules or saliva. For reasons that are not yet understood, approximately 20% of Asian elephant calves appear to be susceptible to the disease when primary infections are not controlled by normal innate cellular and humoral immune responses. Sensitive specific polymerase chain reaction (PCR) DNA blood tests have been developed, routine monitoring has been established, the complete large DNA genomes of each of the four Asian EEHV species have now been sequenced, and PCR gene subtyping has provided unambiguous evidence that this is a sporadic rather than epidemic disease that it is not being spread among zoos or other elephant housing facilities. Nevertheless, researchers have not yet been able to propagate EEHV in cell culture, determine whether or not human antiherpesvirus drugs are effective inhibitors, or develop serology assays that can distinguish between antibodies against the multiple different EEHV species.

Age-associated B cells: key mediators of both protective and autoreactive humoral responses.

A subset of B cells with unique phenotypic and functional features-termed Age-associated B cells (ABCs)-has recently been identified in both mice and humans. These cells are characterized by a T-BET driven transcriptional program, robust responsiveness to TLR7 and TLR9 ligands, and a propensity for IgG2a/c production. Beyond their age-related accumulation, these cells play roles in both normal and pathogenic humoral immune responses regardless of host age. Thus, B cells with the ABC phenotype and transcriptional signature appear during viral, bacterial, and parasitic infections, but also arise during humoral autoimmune disease in both mouse models and humans. These observations suggest that both autoantigens and certain classes of pathogens provide the signals required for ABC differentiation. Herein, we review the discovery and features of ABCs, and propose that they are a memory subset generated by nucleic acid-containing antigens in the context of a promoting inflammatory cytokine milieu.

Histone deacetylase inhibitor suppresses virus-induced proinflammatory responses and type 1 diabetes

Microbial infections are hypothesized to play a key role in the mechanism leading to type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced islet destruction to better understand how virus infection triggers T1D. Inoculation of the LEW1.WR1 rat with KRV results in systemic inflammation followed by insulitis and islet destruction 2-4 weeks post-infection. In this study, we evaluated the effect of treatment with the anti-inflammatory histone deacetylase inhibitor (HDACi) ITF-2357 on KRV-induced immunity and disease progression. Administering LEW1.WR1 rats with KRV plus ITF-2357 on 14 consecutive days beginning on the day of infection protected animals from islet infiltration and T1D. ITF-2357 reversed KRV-induced T and B cell accumulation in the spleen or pancreatic lymph nodes on day 5 following infection. Moreover, ITF-2357 reduced the expression level of KRV-induced p40 subunit of IL-12/IL-23 in spleen cells in vitro and in the peripheral blood in vivo. ITF-2357 suppressed the KRV-induced expression of transcripts for IRF-7 in the rat INS-1 beta cell line. ITF-2357 increased the virus-induced IL-6 gene expression in the spleen, but did not alter the ability of LEW1.WR1 rats to develop normal KRV-specific humoral and cellular immune responses and clear the virus from the pancreatic lymph nodes, spleen, and serum. Finally, ITF-2357 reversed virus-induced modulation of bacterial communities in the intestine early following infection. The data suggest that targeting innate immune pathways with inhibitors of HDAC might represent an efficient therapeutic strategy for preventing T1D. Microbial infections have been implicated in triggering type 1 diabetes in humans and animal models. The LEW1.WR1 rat develops inflammation and T1D following infection with Kilham rat virus. The histone deacetylase inhibitor ITF-2357 suppresses virus-induced inflammation and prevents diabetes. ITF-2357 prevents T1D without altering virus-specific adaptive immunity or virus clearance. ITF-2357 therapy may be an efficient approach to prevent T1D in genetically susceptible individuals.

AB103, a CD28 antagonist peptide: a new therapeutic agent in a model of severe sepsis

AB103 is a novel CD28 antagonist peptide currently in clinical development that modulates CD28 signaling in T cells, without affecting the normal humoral immune response. In experimental models of Gram-positive, Gram-negative and polymicrobial sepsis, AB103 demonstrated significant activity, increasing overall survival.

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1-2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNalpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNalpha were titrated into (15)N-labeled SCR1-2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn(11), Arg(13), Ala(22), Arg(28), Ser(32), Arg(36), Lys(41), Lys(57), Tyr(64), Lys(67), Tyr(68), Arg(83), Gly(84), and Arg(89). With regard to IFNalpha, the binding is similar to the CR2-C3d interaction with specific residues being Arg(13), Tyr(16), Arg(28), Ser(42), Lys(48), Lys(50), Tyr(68), Arg(83), Gly(84), and Arg(89). We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K(d) values of 0.13 and 160 microm, whereas the CR2-gp350 and CR2-IFNalpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 microm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

Contribution of Chinese Pekin duck complement component C3d-P29 repeats to enhancement of Th2-biased immune responses against NDV F gene induced by DNA immunization

Background and aim: C3d, a split product of C3, interacts with its receptor (CR2 or CD21) on B cells and follicular dendritic cells (FDCs) and is crucial for induction and maintenance of a normal humoral immune response. This fragment of complement protein C3 (C3d) has also been shown to enhance B cell responses when complexed with antigen and C3d fusion increased Th2-biased immune response by inducing IL-4 production.Materials and Methods: The gene fragment coding for Chinese Pekin duck (Anas platyrhynchos) C3d gene (duC3d) was cloned and expressed as a component of fusion proteins destined for use in in vitro experiments. Two, four and six copies of CR2-binding domain duC3d-P29 were fused, respectively, to truncated Newcastle disease virus (NDV) F gene encoding soluble glycoprotein F in pcDNA3.1.All recombinant proteins were analyzed by SDS-PAGE and Western immunoblot. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine.Results: The result of immunogenicity detections of The IL-4 level for F-C3d-P29.6 DNA immunization approached that for the inactivated vaccine. Compared to C3d-P29.6, C3d-P29.4 enhanced F DNA immunogenicity to a lesser extent. Furthermore, C3d-P29.n fusion increased Th2-biased immune response by inducing IL-4 production.Conclusion: We demonstrated that C3d-P29 could enhance immunogenicity by directing Th1-biased to a balanced and more effective Th1/Th2 response. The expression of the duck C3d fusion proteins in this study which was the first reported, and the detections of the cytokine level for F-C3d-P29.n in DNA immunization using the BALB/c mice as the model animal, will provide the basis for immunization trials in chicken or other poultry, studies of receptor binding and cell activation of animal lymphocytes, and investigations of new types of vaccine, including genetic recombinant and DNA vaccines for the future against relevant pathogens.

Fusion of Ross Chicken Complement Component C3d-P29 Repeats with a Chicken Viral Newcastle Disease Virus F Gene Enhances Immune Responses Following DNA Immunization

C3d, a split product of C3, interacts with its receptor (CR2 or CD21) on B cells and follicular dendritic cells (FDCs) and is crucial for induction and maintenance of a normal humoral immune response. The gene fragment coding for Ross chicken C3d gene (chC3d) was cloned and expressed as a component of fusion proteins destined for its application in the vaccine study of chicken, and for in vitro experiments. Three potential vaccine construct units were engineered to contain two, four, and six copies of chC3d-P29 coding gene, which was linked to the F gene of Newcastle disease virus (NDV), an economically important pathogen of chicken that is classified as a list A contagious disease of poultry by the Office International des Epizooties (OIE). The cloned chC3d protein and different repeats of C3d-P29 proteins that contained the F gene of NDV (C3d-P29.n-F) was generated separately in Escherichia coli and CEF cells with the help of expression vectors. All recombinant proteins were analyzed by SDS-PAGE and Western blot. The result of immunogenicity detection of different numbers of repeats of C3d-P29.n-F revealed that C3d-P29 could enhance immunogenicity. Six or more repeats of C3d-P29 may be necessary for efficient enhancement of antigen-specific immune responses.

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo anti-polysaccharide, but not anti-protein, response to Streptococcus pneumoniae (43.24)

Abstract IgG anti-polysaccharide (PS) and anti-protein responses to Streptococcus pneumoniae (Pn) are dependent on CD4+ T cells, B7/CD28, and CD40/CD40-ligand interactions. However, the primary IgG anti-PS response terminates more quickly, requires a shorter period of T cell help, fails to generate memory, and is more dependent on Ig signaling. To determine a role for B cell apoptosis in these differences, we utilized mice expressing an Ig heavy chain enhancer-driven transgene encoding the anti-apoptotic proteins Bcl-xL or Bcl-2. Bcl-xL-Tg and Bcl-2-Tg mice immunized with Pn exhibited a prolongation of the primary IgM and IgG anti-PS, but not anti-protein, response resulting in up to 10-fold enhancements in specific Ig serum titers. Of note, Tg mice still failed to generate PS-specific memory. In contrast, Fas- or Fas-ligand-deficient mice elicited normal humoral immune responses to Pn. In vitro, Bcl-xL and Bcl-2 Tg B cells exhibited higher and more prolonged proliferative responses following multivalent Ig crosslinking, but not after stimulation with LPS or anti-CD40 mAb, relative to WT B cells. In contrast to intact Pn, in vivo anti-PS responses to purified PS or a soluble PS-protein conjugate were similar in Tg and WT mice. These data suggest that apoptosis may selectively limit clonal expansion and subsequent Ig secretory responses of PS-specific B cells during a primary immune response to an intact bacterium. N.I.H. R01 AI49192

B cell antigen receptor signaling and internalization are mutually exclusive events.

Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

Immune evasion by staphylococci

Staphylococcus aureus can cause superficial skin infections and, occasionally, deep-seated infections that entail spread through the blood stream. The organism expresses several factors that compromise the effectiveness of neutrophils and macrophages, the first line of defence against infection. S. aureus secretes proteins that inhibit complement activation and neutrophil chemotaxis or that lyse neutrophils, neutralizes antimicrobial defensin peptides, and its cell surface is modified to reduce their effectiveness. The organism can survive in phagosomes, express polysaccharides and proteins that inhibit opsonization by antibody and complement, and its cell wall is resistant to lysozyme. Furthermore, S. aureus expresses several types of superantigen that corrupt the normal humoral immune response, resulting in anergy and immunosuppression. In contrast, Staphylococcus epidermidis must rely primarily on cell-surface polymers and the ability to form a biolfilm to survive in the host.

The Antiapoptotic Protein Bcl-xL Is Dispensable for the Development of Effector and Memory T Lymphocytes

The antiapoptotic protein Bcl-x(L) is induced in activated T lymphocytes upon costimulation through CD28, 4-1BB, and OX40. Bcl-x(L) is also highly enriched in memory T lymphocytes. Based on this body of evidence, it was thought that Bcl-x(L) plays an essential role in the generation of effector and memory T lymphocytes. We report that mice with a conditional deletion of Bcl-x in T lymphocytes develop a normal CD8(+) T cell response to Listeria monocytogenes infection. Furthermore, Bcl-x conditional knockout mice exhibit normal T-dependent humoral immune responses. These results indicate that Bcl-x is dispensable for the generation of effector and memory T lymphocytes and suggest that costimulation of T lymphocytes promotes their survival through a Bcl-x(L) independent mechanism.

B Cells from Mice Prematurely Expressing Human Complement Receptor Type 2 Are Unresponsive to T-Dependent Antigens

Complement receptor type 2 (CR2/CD21), in association with CD19, plays an important role in enhancing mature B cell responses to opsonized Ags. We have shown that mice expressing a human CR2/CD21 (hCR2/CD21) transgene during the CD43(+)/CD25(-) late pro-B cell stage of B cell development demonstrate marked changes in subsequent B cell ontogeny. In the present study, we show that the humoral immune response to the T cell-dependent Ag, sheep RBC, is muted severely in a manner inversely proportional to B cell expression level of hCR2. Individual Ag-specific IgG isotypes vary in the degree to which they are affected but all are reduced while IgM titers are normal. A substantial reduction in germinal centers, both in size and frequency, in the spleens of immunized hCR2 transgenic mice demonstrates a failure to maintain germinal center reaction. However, both IgM expression levels and LPS-proliferative responses appear fully intact in B cells from hCR2-positive mice, suggesting that this alteration in B cell phenotype is different qualitatively from that of specific Ag-defined anergy models. These data suggest that the unresponsiveness to T-dependent Ags displayed by hCR2-positive B cells is linked to an increase in the level of stimulus required to propel the B cell into a fully activated state and thus a normal humoral immune response to Ags. We conclude that this phenotype and these mice may offer an additional means to dissect mechanisms underlying B cell tolerance and Ag responsiveness both in bone marrow and periphery.

Vaccine-induced protection against anthrax in cheetah ( Acinonyx jubatus) and black rhinoceros ( Diceros bicornis)

Institution of a policy of vaccination in endangered species with a vaccine not previously administered to it cannot be undertaken lightly. This applies even more in the case of cheetah ( Acinonyx jubatus) with their unusually monomorphic gene pool and the potential restrictions this places on their immune responses. However, the recently observed mortalities from anthrax in these animals in the Etosha National Park, Namibia, made it imperative to evaluate vaccination. Black rhinoceros ( Diceros bicornis), another endangered species in the park, have been vaccinated for over three decades but the effectiveness of this has never been evaluated. Passive protection tests in A/J mice using sera from 12 cheetahs together with enzyme immunoassay indicated that cheetah are able to mount seemingly normal primary and secondary humoral immune responses to the Sterne 34F2 live spore livestock vaccine. Overall protection rates in mice injected with the sera rose and fell in concert with rises and declines in antibody titres, although fine analysis showed that the correlation between titre and protection was complex. Once a high level of protection (96% of mice 1 month after a second booster in the cheetahs) had been achieved, the duration of substantial protection appeared good (60% of the mice 5 months after the second booster). Protection conferred on mice by sera from three of four vaccinated rhino was almost complete, but, obscurely, none of the mice receiving serum from the fourth rhino were protected. Sera from three park lions with naturally acquired high antibody titres, included as controls, also conferred high levels of protection. For the purposes of wildlife management, the conclusions were that vaccination of cheetah with the standard animal anthrax vaccine causes no observable ill effect in the animals and does appear to confer protective immunity. At least one well-separated booster does appear to be desirable. Vaccination of rhino also appears to be justified from the limited data obtained.