2B4 Is Dispensable for T-Dependent B Cell Immune Responses,but Its Deficiency Leads to Enhanced T-Independent Responses Due to an Increase in Peritoneal Cavity B1b Cells

The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244 ^-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG₁ antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244 ^-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG₃ were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell responses due to a reduction in peritoneal cavity B cells, but has minimal impact on T-dependent B cell responses.     

Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax?23

Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax^®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA⁺ but not IgM⁺ or IgG⁺ memory B cells and a predominant generation of PnPS-specific IgA⁺ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax^®23. Our results demonstrate that a characteristic TI response induced by Pneumovax^®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.     

Critical role of IL-25-ILC2-IL-5 axis in the production of anti-Francisella LPS IgM by B1 B cells

B1 cells, a subset of B lymphocytes whose developmental origin, phenotype, and function differ from that of conventional B2 cells, are the main source of “natural” IgM but can also respond to infection by rapidly producing pathogen-specific IgM directed against T-independent antigens. Francisella tularensis (Ft) is a Gram-negative bacterium that causes tularemia. Infection with Ft Live Vaccine Strain activates B1 cells for production of IgM directed against the bacterial LPS in a process incompletely understood. Here we show that immunization with purified Ft LPS elicits production of LPS-specific IgM and IgG₃ by B1 cells independently of TLR2 or MyD88. Immunization, but not infection, generated peritoneum-resident memory B1 cells that differentiated into LPS-specific antibody secreting cells (ASC) upon secondary challenge. IL-5 was rapidly induced by immunization with Ft LPS and was required for production of LPS-specific IgM. Antibody-mediated depletion of ILC2 indicated that these cells were the source of IL-5 and were required for IgM production. IL-25, an alarmin that strongly activates ILC2, was rapidly secreted in response to immunization or infection and its administration to mice significantly increased IgM production and B1 cell differentiation to ASC. Conversely, mice lacking IL-17RB, the IL-25 receptor, showed impaired IL-5 induction, IgM production, and B1 ASC differentiation in response to immunization. Administration of IL-5 to Il17rb^-/- mice rescued these B1 cells-mediated responses. Il17rb^-/- mice were more susceptible to infection with Ft LVS and failed to develop immunity upon secondary challenge suggesting that LPS-specific IgM is one of the protective adaptive immune mechanisms against tularemia. Our results indicated that immunization with Ft LPS triggers production of IL-25 that, through stimulation of IL-5 release by ILC2, promotes B1 cells activation and differentiation into IgM secreting cells. By revealing the existence of an IL-25-ILC2-IL-5 axis our results suggest novel strategies to improve vaccination against T-independent bacterial antigens.     

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127⁻KLRG1⁺) or memory precursors cells (MPECs, CD127⁺KLRG1⁻) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8⁺ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6^−/− OT-I T-cells showed that the augmented memory formation is CD8⁺ T-cell intrinsic. Although the relative difference between the Nr2f6^+/+ and Nr2f6^−/− OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8⁺ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8⁺ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8⁺ T cells in vivo.Subject terms: Interferons, Bacterial infection     

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.     

Gamma Interferon Regulates Contraction of the Influenza Virus-Specific CD8 T Cell Response and Limits the Size of the Memory Population

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ^−/− and IFN-γR^−/− compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ^−/− mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ^−/− and IFN-γR^−/− mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.     

Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency

Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to N^ε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE^−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE^−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE^−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.     

Differential and Site Specific Impact of B Cells in the Protective Immune Response to Mycobacterium tuberculosis in the Mouse

Cell-mediated immune responses are known to be critical for control of mycobacterial infections whereas the role of B cells and humoral immunity is unclear. B cells can modulate immune responses by secretion of immunoglobulin, production of cytokines and antigen-presentation. To define the impact of B cells in the absence of secreted immunoglobulin, we analyzed the progression of Mycobacterium tuberculosis (Mtb) infection in mice that have B cells but which lack secretory immunoglobulin (AID^−/−µS^−/−mice). AID^−/−µS^−/− mice accumulated a population of activated B cells in the lungs when infected and were more susceptible to aerosol Mtb when compared to wild type (C57BL/6) mice or indeed mice that totally lack B cells. The enhanced susceptibility of AID^−/−µS^−/− mice was not associated with defective T cell activation or expression of a type 1 immune response. While delivery of normal serum to AID^−/−µS^−/− mice did not reverse susceptibility, susceptibility in the spleen was dependent upon the presence of B cells and susceptibility in the lungs of AID^−/−µS^−/−mice was associated with elevated expression of the cytokines IL-6, GM-CSF, IL-10 and molecules made by alternatively activated macrophages. Blocking of IL-10 signaling resulted in reversal of susceptibility in the spleens and lungs of AID^−/−µS^−/− mice. These data support the hypothesis that B cells can modulate immunity to Mtb in an organ specific manner via the modulation of cytokine production and macrophage activation.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

Pretransplant donor lymphocyte infusion (DLI) has been shown to enhance donor-specific allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored donor cell subsets after DLI in a murine MHC class I L^d-mismatched skin transplantation model. We found that donor B cells, but not DCs, are the major surviving donor APCs in recipients following DLI. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Furthermore, mice that had received donor B cells showed higher expression of Ly6A and CD62L on antigen-specific TCRαβ⁺CD3⁺CD4⁻CD8⁻NK1.1⁻ double negative (DN) regulatory T cells (Tregs). B cells presented alloantigen to DN Tregs and primed their proliferation in an antigen-specific fashion. Importantly, DN Tregs, activated by donor B cells, showed increased cytotoxicity toward anti-donor CD8⁺ T cells. These data demonstrate that donor B cells can enhance skin allograft survival, at least partially, by increasing recipient DN Treg-mediated killing of anti-donor CD8⁺ T cells. These findings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specific immune therapy to enhance allograft survival.     

Epstein-Barr virus latent membrane protein 2A preferentially signals through the Src family kinase Lyn

Latent membrane protein 2A (LMP2A) is a viral protein expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor signal transduction and provides survival and developmental signals to B cells in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, we crossed LMP2A transgenic mice (TgE) with Lyn^−/−, Fyn^−/−, or Blk^−/− mice. TgE Lyn^−/− mice had a larger immunoglobulin M (IgM)-positive B-cell population than TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. Both TgE Fyn^−/− and TgE Blk^−/− mice have an IgM-negative population of splenic B cells, similar to the TgE mice. LMP2A was also transiently transfected into the human EBV-negative B-cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates, whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. From these results, we conclude that LMP2A preferentially associates with and signals through Lyn compared to its association with other SFKs. This preferential association is due in part to the SH2 domain of Lyn associating with LMP2A.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28^−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28^−/loxCre^+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28^−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28^−/loxCre^+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28^−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T_FH cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28^−/− mice and tamoxifen treated CD28^−/loxCre^+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28^−/loxCre^+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.     

Carotid body chemosensory responses in mice deficient of TASK channels

Background K⁺ channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in TASK1^−/− or double TASK1/3^−/− mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in TASK1^−/− cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either TASK1^−/− or double TASK1/3^−/− cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-K⁺ channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background K⁺ current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for O₂/CO₂ sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of TASK1/3^−/− glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-K⁺ channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

Establishment of Murine Gammaherpesvirus Latency in B Cells Is Not a Stochastic Event

Murid γ-herpesvirus-4 (MuHV-4) promotes polyclonal B cell activation and establishes latency in memory B cells via unclear mechanisms. We aimed at exploring whether B cell receptor specificity plays a role in B cell susceptibility to viral latency and how this is related to B cell activation. We first observed that MuHV-4-specific B cells represent a minority of the latent population, and to better understand the influence of the virus on non-MuHV-4 specific B cells we used the SW_HEL mouse model, which produce hen egg lysozyme (HEL)-specific B cells. By tracking HEL⁺ and HEL⁻ B cells, we showed that in vivo latency was restricted to HEL⁻ B cells while the two populations were equally sensitive to the virus in vitro. Moreover, MuHV-4 induced two waves of B cell activation. While the first wave was characterized by a general B cell activation, as shown by HEL⁺ and HEL⁻ B cells expansion and upregulation of CD69 expression, the second wave was restricted to the HEL⁻ population, which acquired germinal center (GC) and plasma cell phenotypes. Antigenic stimulation of HEL⁺ B cells led to the development of HEL⁺ GC B cells where latent infection remained undetectable, indicating that MuHV-4 does not benefit from acute B cell responses to establish latency in non-virus specific B cells but relies on other mechanisms of the humoral response. These data support a model in which the establishment of latency in B cells by γ-herpesviruses is not stochastic in terms of BCR specificity and is tightly linked to the formation of GCs.     

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

Background

Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.

Methodology and Results

During the effector phase in BALB/c mice, only depletion of CD4⁺ and CD8⁺ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4⁺, CD8⁺ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4⁺ and CD8⁺ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4^−/−CD8^−/− and CD4^−/−, but not in CD8^−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4⁺, only CD8⁺, or CD4⁺ and CD8⁺ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.

Conclusion

Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.     

Targeted Deletion of FGL2 Leads to Increased Early Viral Replication and Enhanced Adaptive Immunity in a Murine Model of Acute Viral Hepatitis Caused by LCMV WE

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4⁺CD25⁺ Foxp3⁺ regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2^−/−) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2^−/− had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2^+/+). Frequencies of CD8⁺ and CD4⁺ T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2 ^−/− mice. Increased frequencies of CD8⁺ T cells specific for LCMV tetramers GP₃₃ and NP₃₉₆ were detected within the liver of fgl2^−/− mice. Plasma from fgl2^−/− mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2^−/− mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.