Single and coexpression of CXCR4 and CXCR5 identifies CD4 T helper cells in distinct lymph node niches during influenza virus infection

Influenza virus infection results in strong, mainly T-dependent, extrafollicular and germinal center B cell responses, which provide lifelong humoral immunity against the homotypic virus strain. Follicular T helper cells (T(FH)) are key regulators of humoral immunity. Questions remain regarding the presence, identity, and function of T(FH) subsets regulating early extrafollicular and later germinal center B cell responses. This study demonstrates that ICOS but not CXCR5 marks T cells with B helper activity induced by influenza virus infection and identifies germinal center T cells (T(GC)) as lymph node-resident CD4(+) ICOS(+) CXCR4(+) CXCR5(+) PSGL-1(lo) PD-1(hi) cells. The CXCR4 expression intensity further distinguished their germinal center light and dark zone locations. This population emerged strongly in regional lymph nodes and with kinetics similar to those of germinal center B cells and were the only T(FH) subsets missing in influenza virus-infected, germinal center-deficient SAP(-/-) mice, mice which were shown previously to lack protective memory responses after a secondary influenza virus challenge, thus indicting the nonredundant functions of CXCR4- and CXCR5-coexpressing CD4 helper cells in antiviral B cell immunity. CXCR4-single-positive T cells, present in B cell-mediated autoimmunity and regarded as "extrafollicular" helper T cells, were rare throughout the response, despite prominent extrafollicular B cell responses, revealing fundamental differences in autoimmune- and infection-induced T-dependent B cell responses. While all ICOS(+) subsets induced similar antibody levels in vitro, CXCR5-single-positive T cells were superior in inducing B cell proliferation. The regulation of T cell localization, marked by the single and coexpression of CXCR4 and CXCR5, might be an important determinant of T(FH) function.     

Ontogeny of the antigen-reactive lymph follicle-forming capacity of the popliteal lymph node in neonatal mice

The ontogenetic development of the reactive lymph follicle-forming capacity of the popliteal lymph node was investigated immunohistochemically in young mice which had received a single injection of hemocyanin (KLH) in a rear footpad at a predetermined age (between 1 and 21 days). The mice were sacrificed at various intervals after injection. In non-stimulated young mice, primary lymph follicles first appeared in the popliteal node at 11 days of age. When KLH was given to 7-day-old or older mice, each draining popliteal node showed a marked increase in B lymphocytes in the extrafollicular zone 3 days after injection and produced a number of "new" lymph follicles outside the pre-existing follicles over the next few days. In mice injected at 2-4 days of age, these nodes showed an increase in B lymphocytes in the outer cortex and had produced several lymph follicles by 8 days of age. The number of lymph follicles produced by each node tended to increase in line with age at injection. These results indicate that neonatal popliteal nodes become able to produce lymph follicles in response to exogenous antigens some time before ontogenetically developing follicles appear. The formation of new lymph follicles observed in draining popliteal nodes after KLH injection at an early postnatal age is discussed in relation to the ontogenetic development of stromal cells (precursors of follicular dendritic cells) that are capable of interacting with B lymphocytes and the extent of B lymphocyte influx into the node induced by KLH stimulation.     

Arthropod- and host-specific Borrelia burgdorferi bbk32 expression and the inhibition of spirochete transmission

Antisera to BBK32 (a Borrelia burgdorferi fibronectin-binding protein) and BBK50, two Ags synthesized during infection, protect mice from experimental syringe-borne Lyme borreliosis. Therefore, B. burgdorferi bbk32 and bbk50 expression within Ixodes scapularis ticks and the murine host, and the effect of BBK32 and BBK50 antisera on spirochetes throughout the vector-host life cycle were investigated. bbk32 and bbk50 mRNA and protein were first detected within engorged ticks, demonstrating regulated expression within the vector. Then bbk32 expression increased in mice at the cutaneous site of inoculation. During disseminated murine infection, bbk32 and bbk50 were expressed in several murine tissues, and mRNA levels were greatest in the heart and spleen at 30 days. BBK32 antisera protected mice from tick-borne B. burgdorferi infection and spirochete numbers were reduced by 90% within nymphs that engorged on immunized mice. Moreover, 75% of these ticks did not retain spirochetes upon molting, and subsequent B. burgdorferi transmission by adult ticks was impaired. Larval acquisition of B. burgdorferi by I. scapularis was also inhibited by BBK32 antisera. These data demonstrate that bbk32 and bbk50 are expressed during tick engorgement and that BBK32 antisera can interfere with spirochete transmission at various stages of the vector-host life cycle. These studies provide insight into mechanisms of immunity to Lyme borreliosis and other vector-borne diseases.     

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.     

MyD88 plays a unique role in host defense but not arthritis development in Lyme disease

To assess the contribution of TLR signaling in the host response to Borrelia burgdorferi, mice deficient in the common TLR adaptor protein, myeloid differentiation factor 88 (MyD88), were infected with B. burgdorferi. MyD88-deficient mice harbored extremely high levels of B. burgdorferi in tissues when compared with wild-type littermates and greater amounts of spirochetes in tissues than TLR2-deficient mice. These findings suggest that, in addition to TLR2, other MyD88-dependent pathways play a significant role in the host defense to B. burgdorferi. MyD88(-/-) mice maintained the ability to produce Abs directed against B. burgdorferi. Partial clearance of spirochetes was evident in long term infection studies and immune sera from MyD88-deficient mice were able to protect naive mice from infection with B. burgdorferi. Thus, the acquired immune response appeared to be functional in MyD88(-/-) mice, and the inability to control spirochete numbers was due to a failure of cells involved in innate defenses. Although macrophages from MyD88(-/-) mice responded poorly to Borrelia sonicate in vitro, MyD88(-/-) mice still developed an inflammatory arthritis after infection with B. burgdorferi characterized by an influx of neutrophils and mononuclear cells. The findings presented here point to a dichotomy between the recruitment of inflammatory cells to tissue and an inability of these cells to kill localized spirochetes.     

Delays and diversions mark the development of B cell responses to Borrelia burgdorferi infection

B cell responses modulate disease during infection with Borrelia burgdorferi, the causative agent of Lyme disease, but are unable to clear the infection. Previous studies have demonstrated that B. burgdorferi infection induces predominantly T-independent B cell responses, potentially explaining some of these findings. However, others have shown effects of T cells on the isotype profile and the magnitude of the B. burgdorferi-specific Abs. This study aimed to further investigate the humoral response to B. burgdorferi and its degree of T cell dependence, with the ultimate goal of elucidating the mechanisms underlying the failure of effective immunity to this emerging infectious disease agent. Our study identifies distinct stages in the B cell response using a mouse model, all marked by the generation of unusually strong and persistent T-dependent and T-independent IgM Abs. The initial phase is dominated by a strong T-independent accumulation of B cells in lymph nodes and the induction of specific Abs in the absence of germinal centers. A second phase begins around week 2.5 to 3, in which relatively short-lived germinal centers develop in lymph nodes, despite a lymph node architecture that lacks clearly demarcated T and B cell zones. This response failed, however, to generate appreciable numbers of long-lived bone marrow plasma cells. Finally, there is a slow accumulation of long-lived Ab-secreting plasma cells in bone marrow, reflected by a strong but ultimately ineffective serum Ab response. Overall, the study indicates that B. burgdorferi might evade B cell immunity by interfering with its response kinetics and quality.     

The cell-mediated immune response to Borrelia afzelii, garinii and burgdorferi in C57BL/6 mice is dependent on antigen specificity

Inbred C57BL/6 mice were challenged with Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen-specific T-cell response in vitro. The sonicated preparations of in vitro grown spirochetes were capable of stimulating polyclonal proliferation and specific cell-mediated response, depending on duration of the cell culture. Murine splenocytes previously sensitized to B. burgdorferi sensu lato (s.l. ), but not those from control mice, could be induced for antigen-specific proliferation in vitro. Moreover, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies as revealed by the [(3)H]thymidine incorporation assay. The current study considers that the strict B. burgdorferi s.l. antigen-specific response may also be expected in infections in humans and contributes to the explanation of the frequently poor antibody- and cell-mediated immune response observed in patients diagnosed with Lyme disease.     

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

Co-inoculation ofBorrelia afzelii with tick salivary gland extract influences distribution of immunocompetent cells in the skin and lymph nodes of mice

The impact of Ixodes ricinus salivary gland extract (SGE) on inflammatory changes in the skin and draining lymph nodes of mice, elicited by the infection with the important human pathogen, B. afzelii, was determined using flow cytometry. SGE injected together with spirochetes reduced the numbers of leukocytes and gammadelta-T lymphocytes in infected epidermis at early time-points post infection. In draining lymph nodes, the anti-inflammatory effect of SGE was manifested by the decrease of total cell count compared with that in mice treated with inactivated SGE. Changes in subpopulations of immunocompetent cells apparently reflected the effect of SGE on the proliferation of spirochetes in the host. The significance of tick saliva anti-inflammatory effect for saliva activated transmission of B. afzelii is shown.     

Invasion of human neuronal and glial cells by an infectious strain of Borrelia burgdorferi

Human infection by Borrelia burgdorferi, the etiological agent for Lyme disease, can result in serious acute and late-term disorders including neuroborreliosis, a degenerative condition of the peripheral and central nervous systems. To examine the mechanisms involved in the cellular pathogenesis of neuroborreliosis, we investigated the ability of B. burgdorferi to attach to and/or invade a panel of human neuroglial and cortical neuronal cells. In all neural cells tested, we observed B. burgdorferi in association with the cell by confocal microscopy. Further analysis by differential immunofluorescent staining of external and internal organisms, and a gentamicin protection assay demonstrated an intracellular localization of B. burgdorferi. A non-infectious strain of B. burgdorferi was attenuated in its ability to associate with these neural cells, suggesting that a specific borrelial factor related to cellular infectivity was responsible for the association. Cytopathic effects were not observed following infection of these cell lines with B. burgdorferi, and internalized spirochetes were found to be viable. Invasion of neural cells by B. burgdorferi provides a putative mechanism for the organism to avoid the host's immune response while potentially causing functional damage to neural cells during infection of the CNS.     

Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.     

Lymph node cell responsiveness in BALB/c mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.     

Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs

B cells can either differentiate in germinal centers or in extrafollicular compartments of secondary lymphoid organs. Here we show the migration properties of B cells after differentiation in murine peripheral lymph node infected with mouse mammary tumor virus. Naive B cells become activated, infected, and carry integrated retroviral DNA sequences. After production of a retroviral superantigen, the infected B cells receive cognate T cell help and differentiate along the two main differentiation pathways analogous to classical Ag responses. The extrafollicular differentiation peaks on day 6 of mouse mammary tumor virus infection, and the follicular one becomes detectable after day 10. B cells participating in this immune response carry a retroviral DNA marker that can be detected by using semiquantitative PCR. We determined the migration patterns of B cells having taken part in the T cell-B cell interaction from the draining lymph node to different tissues. Waves of immigration and retention of infected cells in secondary lymphoid organs, mammary gland, salivary gland, skin, lung, and liver were observed correlating with the two peaks of B cell differentiation in the draining lymph node. Other organs revealed immigration of infected cells at later time points. The migration properties were correlated with a strong up-regulation of alpha(4)beta(1) integrin expression. These results show the migration properties of B cells during an immune response and demonstrate that a large proportion of extrafolliculary differentiating plasmablasts can escape local cell death and carry the retroviral infection to peripheral organs.     

Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi.

Borrelia burgdorferi lipoproteins activate inflammatory cells through Toll-like receptor 2 (TLR2), suggesting that TLR2 could play a pivotal role in the host response to B. burgdorferi. TLR2 does play a critical role in host defense, as infected TLR2(-/-) mice harbored up to 100-fold more spirochetes in tissues than did TLR2(+/+) littermates. Spirochetes persisted at extremely elevated levels in TLR2-deficient mice for at least 8 wk following infection. Infected TLR2(-/-) mice developed normal Borrelia-specific Ab responses, as measured by quantity of Borrelia-specific Ig isotypes, the kinetics of class switching to IgG, and the complexity of the Ags recognized. These findings indicate that the failure to control spirochete levels in tissues is not due to an impaired acquired immune response. While macrophages from TLR2(-/-) mice were not responsive to lipoproteins, they did respond to nonlipoprotein components of sonicated spirochetes. These TLR2-independent responses could play a role during the inflammatory response to B. burgdorferi, as infected TLR2(-/-) mice developed greater ankle swelling than wild-type littermates. Thus, while TLR2-dependent signaling pathways play a major role in the innate host defense to B. burgdorferi, both inflammatory responses and the development of the acquired humoral response can occur in the absence of TLR2.     

Increasing the recruitment of neutrophils to the site of infection dramatically attenuates Borrelia burgdorferi infectivity

Borrelia burgdorferi infection causes an initial skin lesion called erythema migrans (EM) in human Lyme disease and in models of monkey and rabbit borreliosis. EM results from the inflammatory response triggered by spirochete replication and likely develops to contain the initial infection but allows bacterial dissemination to occur. The essential lack of neutrophil involvement in EM histopathology prompted us to examine the consequence of increasing their recruitment in the inflammatory response to the Lyme disease agent. B. burgdorferi was modified genetically to constitutively express and secrete the chemokine KC, a neutrophil chemoattractant. After inoculation into the dermis of the murine host, control spirochetes induced an infiltration of macrophages, neutrophils, and basophils within 6 h; however, the recruited neutrophils and basophils were quickly substituted by eosinophils, and the inflammatory response became macrophage dominant by 16 h. Such a response failed to contain the initial infection and allowed the spirochetes to disseminate. In contrast, B. burgdorferi with KC secretion induced an intensive neutrophil infiltration at the inoculation site, and as a result, the host's ability to control the initial infection was greatly enhanced. Taken together, this study suggests that the failure of sufficient neutrophil recruitment and activation during the initial inflammatory response may allow B. burgdorferi to effectively colonize the mammalian host.     

The Initial Draining Lymph Node Primes the Bulk of the CD8 T Cell Response and Influences Memory T Cell Trafficking after a Systemic Viral Infection

Lymphocytic choriomeningitis virus (LCMV) causes a systemic infection in mice with virus replication occurring in both peripheral tissues and secondary lymphoid organs. Because of the rapid systemic dissemination of the virus, the secondary lymphoid organs responsible for the induction of the LCMV-specific CD8 T cell response are poorly defined. We show that the mediastinal lymph node (MedLN) serves as the primary draining lymph node following LCMV infection. In addition, we demonstrate that the MedLN is responsible for priming the majority of the virus-specific CD8 T cell response. Following resolution of the acute infection, the draining MedLN exhibits characteristics of a reactive lymph node including an increased presence of germinal center B cells and increased cellularity for up to 60 days post-infection. Furthermore, the reactive MedLN harbors an increased frequency of CD62L⁻ effector memory CD8 T cells as compared to the non-draining lymph nodes. The accumulation of LCMV-specific CD62L⁻ memory CD8 T cells in the MedLN is independent of residual antigen and is not a unique feature of the MedLN as footpad infection with LCMV leads to a similar increase of virus-specific CD62L⁻ effector memory CD8 T cells in the draining popliteal lymph node. Our results indicate that CD62L⁻ effector memory CD8 T cells are granted preferential access into the draining lymph nodes for an extended time following resolution of an infection.     

Atrophy of mesenteric lymph nodes in experimental Chagas' disease: differential role of Fas/Fas-L and TNFRI/TNF pathways

It is currently accepted that experimental acute infection by Trypanosoma cruzi promotes changes in secondary lymphoid organs, with general T and B lymphocyte polyclonal activation. Here we show that mesenteric lymph nodes (MLN) of acutely infected mice show severe atrophy due to extensive lymphocyte apoptosis. Accordingly, clusters of apoptotic cells are detected in the initial phase of infection in MLN but not in subcutaneous nodes. Moreover, such atrophy is independent of the infection route, parasite load or the mouse strain used. Studies in Fas-L deficient (BALB gld/gld+/+) and in TNF type 1 receptor (p55-/-) knockout mice indicate that both molecules are involved in MLN atrophy: Fas-L participates in cell death of CD4+ as well as B lymphocytes, whereas the TNF type 1 receptor is important for the apoptosis of CD4+ and CD8+ T lymphocytes. In contrast, perforin does not play a role, as lymph nodes from perforin-deficient mice do not behave differently from the corresponding wild types. Our data support the concept that, even in a systemic infection, differential (even opposing) responses can be found in different lymph node chains.     

Antigenicity and recombination of VlsE, the antigenic variation protein of Borrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirochete

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.     

Effect of Prior Exposure to Noninfected Ticks on Susceptibility of Mice to Lyme Disease Spirochetes

To determine whether prior exposure to Nearctic Ixodes vector ticks protects native reservoir mice from tick-borne infection by Lyme disease spirochetes, we compared their infectivities for white-footed mice and laboratory mice that had been repeatedly infested by noninfected deer ticks. Nymphal ticks readily engorged on tick-exposed laboratory mice, but their feeding success on white-footed mice progressively declined. Tick-borne spirochetes readily infected previously tick-infested mice. Thus, prior infestation by Nearctic ticks does not protect sympatric reservoir mice or Palearctic laboratory mice from infection by sympatric tick-borne spirochetes.     

MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.