Residual antigen presentation after influenza virus infection affects CD8 T cell activation and migration

Activated virus-specific CD8 T cells remain in the lung airways for several months after influenza virus infection. We show that maintenance of this cell population is dependent upon the route of infection and prolonged presentation of viral antigen in the draining lymph nodes (DLN) of the respiratory tract. The local effects on T cell migration have been examined. We show retention of virus-specific CD8 T cells in the mediastinal lymph node (MLN) and continuing recruitment of blood-borne migrants into the lung airways during antigen presentation. These data show that antigen that is retained after pulmonary influenza virus infection controls the migratory pattern and activation state of virus-specific CD8 T cells near the site of virus amplification.     

Role of IgM antibodies versus B cells in influenza virus-specific immunity

We have compared the role of IgM antibodies with the role of B cells in control of primary influenza virus infection. Mice deficient in IgM (IgM(-/-)), but capable of producing other Igisotypes, exhibited increased pulmonary virus titers compared to wild-type mice. However, IgM(-/-) mice were less susceptible compared to B cell-deficient micro MT) mice. CD4(+) T cells from spleen and lung draining lymph nodes of infected micro MT mice showed reduced proliferation upon virus re-stimulation in vitro. Furthermore, numbers of IFN-gamma-producing CD4(+) effector T cells were reduced in the alveolar lavage (BAL) of micro MT mice but not IgM(-/-) mice. In contrast, total number of virus-specific CTL was almost comparable in BAL of micro MT and wild-type mice. Pulmonary recruitment of inflammatory macrophages and neutrophils occurred normally in both micro MT and IgM(-/-) mice. Interestingly, virus-specific IgG2a and IgG2b antibody responses were affected locally in the BAL and in the serum of IgM(-/-) mice, while IgG1 responses remained largely normal. Taken together, our data suggest a role for B cells to promote effector T cell responses and a role of both IgM and IgG antibodies in the defense against acute influenza virus infection.     

Humoral immune responses during asthma and influenza co-morbidity in mice

Humoral immunity serve dual functions of direct pathogen neutralization and enhancement of leukocyte function. Antibody classes are determined by antigen triggers, and the resulting antibodies can contribute to disease pathogenesis and host defense. Although asthma and influenza are immunologically distinct diseases, since we have found that allergic asthma exacerbation promotes antiviral host responses to influenza A virus, we hypothesized that humoral immunity may contribute to allergic host protection during influenza. C57BL/6J mice sensitized and challenged with Aspergillus fumigatus (or not) were infected with pandemic influenza A/CA/04/2009 virus. Negative control groups included naïve mice, and mice with only ‘asthma’ or influenza. Concentrations of antibodies were quantified by ELISA, and in situ localization of IgA- and IgE-positive cells in the lungs was determined by immunohistochemistry. The number and phenotype of B cells in spleens and mediastinal lymph nodes were determined by flow cytometry at predetermined timepoints after virus infection until viral clearance. Mucosal and systemic antibodies remained elevated in mice with asthma and influenza with prominent production of IgE and IgA compared to influenza-only controls. B cell expansion was prominent in the mediastinal lymph nodes of allergic mice during influenza where most cells produced IgG₁ and IgA. Although allergy-skewed B cell responses dominated in mice with allergic airways inflammation during influenza virus infection, virus-specific antibodies were also induced. Future studies are required to identify the mechanisms involved with B cell activation and function in allergic hosts facing respiratory viral infections.     

Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate

BACKGROUND: Many different types of phthalate ester are used as plasticizers and are thus found in the air. There have been several studies that suggest an association between allergies and phthalate esters. We previously found that di-butyl phthalate (DBP) has an adjuvant effect in a mouse contact hypersensitivity model, in which fluorescein isothiocyanate (FITC) is involved as an immunogenic hapten. OBJECTIVE: We examined whether other phthalate esters enhance the process of sensitization to FITC by facilitating the trafficking of FITC-presenting dendritic cells or macrophages from skin sites to draining lymph nodes. METHODS: Mice were epicutaneously sensitized with FITC dissolved in acetone containing a phthalate ester. Sensitization was evaluated as ear swelling after a challenge with FITC. Draining lymph node cells obtained 24 h after skin sensitization were examined for FITC fluorescence by means of flow cytometry. FITC-positive cells were characterized with anti-CD11c and anti-CD11b by three-colour flow cytometry. RESULTS: When mice were sensitized with FITC in acetone containing DBP or di-n-propyl phthalate (DPP), strong enhancement of the ear-swelling response was observed. Di-methyl phthalate (DMP) and di-ethyl phthalate (DEP) were less effective but produced some enhancement. Consistent enhancement was not observed with di-(2-ethylhexyl) phthalate or di-isononyl phthalate. Upon sensitization in the presence of DBP or DPP, the number of FITC-positive dendritic cells (total CD11c+ as well as CD11c+/CD11b+) was increased in draining lymph nodes. As to the other four phthalate esters, there was no significant increase in the FITC-positive cell number in the draining lymph nodes. CONCLUSION: During the process of sensitization to FITC, DBP, and DPP exert strong adjuvant effects that are associated with enhancement of trafficking of antigen-presenting dendritic cells from the skin to draining lymph nodes.     

Increased B Regulatory Phenotype in Non‐Metastatic Lymph Nodes of Node‐Positive Breast Cancer Patients

Tumour‐draining lymph nodes (TDLNs) are centre in orchestrating the immune responses against cancer. The cellularity and lymphocyte subpopulations change in the process of cancer progression and lymph node involvement. B lymphocyte subsets and their function in breast cancer‐draining lymph nodes have not been well elucidated. Here, we studied the influence of tumour metastasis on the frequencies of different B cell subsets including naïve and memory B cells as well as those which are known to be enriched in the regulatory pool in TDLNs of 30 patients with breast cancer. Lymphocytes were obtained from a fresh piece of each lymph node and stained for CD19 and other B cell‐associated markers and subjected to flow cytometry. Our investigation revealed that metastatic TDLN showed a significant decrease in active, memory and class‐switched B cells while the frequencies of B cells with regulatory phenotypes were not changed. However, CD27^hiCD25⁺ and CD1d^hiCD5⁺ B regulatory subsets significantly increased in non‐metastatic lymph nodes (nMLNs) of node‐positive patients compared with node‐negative patients. Our data provided evidence that in breast cancer, metastasis of tumour to axillary lymph nodes altered B cell populations in favour of resting, inactive and unswitched phenotypes. We assume that the lymphatic involvement may cause an increase in a subset of regulatory B cells in non‐metastatic lymph nodes.     

Accumulation of CD8+ Cells after Immunization with Soluble Antigen

Rats were immunized with ovalbumin, either subcutaneously or by aerosol inhalation. The lymphocyte distribution in lymph nodes, peripheral blood, and spleen was investigated by flow cytometry after labelling with T pan (OX19 and W3/13), T helper lymphocytes (W3/25), T cytotoxic/suppressor lymphocytes (OX8), kappa light chain (MAR 18-5), or MHC class II (OX6) monoclonal antibodies. The influence of the neurotoxic agent capsaicin on the lymphocyte distribution was also analysed. Subcutaneous immunization resulted in an increased number of OX8+ cells in mesenteric lymph nodes, spleen, and peripheral blood but not in the draining lymph nodes, axillary, brachial, and mediastinal lymph nodes. The number of positive cells for the other cell markers used were not affected by immunization. The neuromodulatory effect of capsaicin had no effect on the lymphocyte distribution. The results showed that the type of immunization used, low amounts of antigen without adjuvant given during a prolonged period, selectively induced OX8+ cells. The patterns were unaffected by neuromodulation using capsaicin.     

B‐cell fate decisions following influenza virus infection

Rapidly induced, specific Ab generated in extrafollicular foci are important components of early immune protection to influenza virus. The signal(s) that prompt B cells to participate in extrafollicular rather than germinal center responses are incompletely understood. To study the regulation of early B‐cell differentiation events following influenza infection, we exploited earlier findings of a strong contribution of C12 idiotype‐expressing B cells to the primary HA‐specific response against influenza A/PR/8/34. Using an idiotype‐specific mAb to C12 and labeled HA, in conjunction with multicolor flow cytometry, we followed the fate of C12Id‐expressing influenza HA‐specific B cells in WT BALB/c mice, requiring neither genetic manipulation nor adoptive cell transfer. Our studies demonstrate that HA‐specific C12Id⁺ B cells are phenotypically indistinguishable from follicular B cells. While they induced both extrafollicular and germinal center responses, extrafollicular responses were strongly predominant. Provision of increased HA‐specific T‐cell help increased the magnitude of the extrafollicular response, but did not shift the C12Id⁺ response toward germinal center formation. Collectively the data are consistent with the hypothesis that B‐cell fate determination following activation is a stochastic process in which infection‐induced innate signals might drive the preferential expansion of the early extrafollicular response.     

Active suppression in orally tolerized rats coincides with in situ transforming growth factor-beta (TGF-beta) expression in the draining lymph nodes

Adult rats were fed pellets containing ovalbumin (OvA) during 4 weeks, and were 2 weeks thereafter immunized subcutaneously with a mixture of OvA and human serum albumin (HSA) in Freund's complete adjuvant (day 0). As a result of the immunization, the draining lymph nodes of the nontolerized (control) rats were heavily enlarged from day 10 to day 18; however, this size increase was absent in the OvA-fed rats. This manifestation of active suppression in the tolerized rats was preceded by the appearance of scattered CD4+ TGF-beta-expressing T cells in the T cell area of their lymph nodes (days 5-8); correspondingly, the levels of TGF-beta mRNA in the nodes were elevated in the tolerant rats compared with the control rats. The anti-OvA antibody levels in sera from the rats revealed that there was an initial B cell priming in the OvA-fed group, with levels higher than in the control group during the first week. Thereafter, suppression governed the response, and from day 10 onwards the anti-OvA levels were considerably lower than in the controls. When other groups of animals were pretreated with neutralizing anti-TGF-beta antibodies 1 day before the immunization, the anti-OvA response of the OvA-fed rats was restored to the levels of the control group, demonstrating the importance of TGF-beta in the maintenance of suppression. In conclusion, we demonstrate that TGF-beta-producing cells appear in the draining lymph nodes shortly after immunization in rats made orally tolerant using a relatively high-dose feeding regime; these cells are probably responsible for the down-regulation of the immune response observed in the OvA-fed rats.     

Properties of murine (CD8+)CD27- T cells

In humans, loss of CD27 expression is associated with the stable acquisition of effector functions by CD8+ T cells. We found that murine (CD8+)CD27- T cells were confined to the primed CD62L(dull/-)CD44(bright)CCR7- T cell population. (CD8+)CD27- T cells were absent from lymph nodes but could be found in blood, spleen and in non-lymphoid organs such as lung and liver. Late after primary influenza virus infection, low percentages of antigen-specific CD27- cells emerged in the lung and spleen. After recovery from secondary influenza virus infection, high percentages of influenza-specific CD27- T cells were found in the lung and the loss of CD27 on lung CD8+ T cells coincided with high granzyme B expression. After murine cytomegalovirus infection, loss of CD27 expression on virus-specific CD8+ T cell populations was sustained and especially marked in liver and lung. We suggest that in mice, CD27 is lost from CD8+ T cells only after repetitive antigenic stimulation. Moreover, the high expression of both granzyme B and perforin in the CD27- T cells suggests that the lack of CD27 on murine CD8+ T cells can be used to identify memory T cells with expression of cytotoxic effector molecules.     

Lymphocyte subsets in the lymph nodes of rats with autoimmune orchitis.

We determined temporal variations of cell subpopulations in the immunization draining lymph nodes during the development of an experimental autoimmune orchitis (EAO) induced in Wistar rats. A phenotypic characterization of T cells and their subsets (CD4+ and CD8+), B, and Ia+ cells was performed by immunofluorescent technique. At the end of the immunization period (30 days), rats injected with testicular homogenate plus adjuvants presented a considerable increase in absolute cell number but normal lymphocyte subset percentages. Testicular damage became evident at 50 days after the first immunization and increased its severity at 80 days: animals that developed EAO presented a lower number of CD8+ cells as compared with undamaged rats. This latter group showed a low CD4/CD8 ratio due to the high proportion of CD8+ cells, which could probably have a suppressor function. At 80 days massive testicular infiltration and decreased absolute cell number in lymph nodes suggest the possible migration of specific lymphocytes to the target organ.     

Development of antibody-secreting cells and antigen-specific T cells in cervical lymph nodes after intranasal immunization.

Intranasal (i.n.) immunization with bacterial protein antigens coupled to cholera toxin B subunit (CTB) effectively induces mucosal, especially salivary immunoglobulin A (IgA), and nonmucosal antibody responses in mice. To examine the regional distribution of antigen-specific B and T cells after i.n. immunization, antibody-secreting cells and antigen-responsive T cells in cervical lymph nodes (CLN) were compared with those found after intraoral or subcutaneous (in the neck) administration of the same antigen and with T cells found in mesenteric lymph nodes (MLN) and spleen after intragastric immunization. The i.n. immunization induced predominantly IgA antibody-secreting cells in salivary glands and IgA and IgG antibody-secreting cells in the superficial and central CLN; these responses were quantitatively enhanced if the antigen was coupled to CTB. Intraoral immunization also induced IgA and IgG antibody-secreting cells in the superficial and central CLN, but only if intact cholera toxin was included as an adjuvant. In contrast, subcutaneous (neck) immunization induced IgG antibody-secreting cells mainly in the draining facial lymph nodes. CLN cell populations resembled those of MLN, except that CLN lymphocytes had higher proportions of T cells and lower proportions of B cells and a slightly higher CD4+/CD8+ ratio among T cells than the MLN lymphocytes did. T cells that proliferated in response to antigen in vitro were found especially in central CLN 2 days after i.n. immunization and persisted for up to 6 months, whereas after intragastric immunization, responsive T cells were not found in the MLN for up to 14 days. After culture with antigen in vitro, T cells from the superficial CLN of i.n. immunized mice secreted both gamma interferon and interleukin-4. Therefore, after i.n. immunization, superficial and central CLN represent sites of regional lymphocyte development, and the central CLN in particular appear to be sites where memory T cells persist.     

Functionally distinct T cells in three compartments of the respiratory tract after influenza virus infection

This study aimed to resolve, firstly, whether T cell responses induced in one tissue site are similar to those induced by the same antigen in another site and, secondly, whether influenza virus infection induces one predominant type of T cell response locally in the respiratory tract. To address these questions, T cell responses in three compartments of the respiratory tract were compared after infection of mice with a sublethal dose of influenza virus: the draining mediastinal lymph nodes (MLN), the lung parenchyma and the airways. Each compartment harbored a T cell response substantially different from that found at the other sites. A preferential accumulation of ex vivo-cytolytic CD8⁺ T cells was found in the airways (CD4/CD8 ratio 1:2) and to a lesser extent in the lung parenchyma (CD4/CD8 ratio 1:1). T cells from both compartments expressed high levels of various cytokine mRNA, but showed differences in their respective expression pattern, with those from lung tissue showing particularly high levels of IFN-γ mRNA. The response in the draining lymph nodes, on the other hand, was dominated by CD4⁺ T cells (CD4/CD8 ratio 2:1) with a higher proliferative capacity (after TCR/CD3 cross-linking) and which provided better B cell help in vitro than CD4⁺ T cells isolated from lung tissue. T cells from MLN expressed mRNA for a variety of cytokines with only low levels of IFN-γ mRNA and they showed no CTL activity ex vivo. These functional differences were not due to differences in the kinetics of the response, or to the higher frequencies of activated T cells in lung tissue and airways compared to MLN, since the differences remained when cell-sorter-purified activated (CD18^hi, CD44^hi) T cells from MLN and lung tissue were compared in a time-course study. Taken together, these findings indicate that pathogens such as influenza virus induce a heterogenous set of T cell responses in different tissue sites affected by the infection.     

Notexin对小鼠体内CD8+ T淋巴细胞活性的干扰作用

目的　探讨肌肉注射Notexin是否影响C57BL/6小鼠骨骼肌引流淋巴结内的外源性CD8+ T细胞（OT-I细胞）的活性和增殖。  方法　分别采用Notexin注射或机械挤压法制备B6小鼠的胫骨前肌（TA）急性肌损伤模型,并获得性转移（adoptive transfer, i.v.）OVA特异的OT-I细胞（CFSE标记）,随后肌注可溶性OVA蛋白。收集损伤侧TA引流淋巴结 (腘、腹股沟淋巴结),流式分析OT-I细胞的增殖程度。OVA静脉内注射免疫B6鼠,收集激活的脾脏树突状细胞（cDCs）,与CFSE标记的OT-I细胞体外联合培养,并添加不同稀释度的Notexin, 流式检测OT-I细胞的活性和增殖。 结果　CFSE的荧光递减结果证实,机械挤压损伤鼠TA引流淋巴结内OT-I细胞迅速活化增殖,但Notexin诱发的肌损伤小鼠引流淋巴结内OT-I细胞在4 d时无增殖反应,7 d的增殖率与非注射组无显著差异。与活化的cDCs细胞共培养的OT-I细胞活性良好,增殖显著,但即使添加高度稀释（1：1000）的Notexin也会严重干扰OT-I细胞的活性和增殖。  结论　肌内注射Notexin注射将干扰CD8+ T细胞的活性,这表明蛇毒血清诱导的骨骼肌损伤模型不适用于肌损伤诱发的T细胞功能改变的相关研究。     

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M? adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.     

Antigen-bearing dendritic cells from the sublingual mucosa recirculate to distant systemic lymphoid organs to prime mucosal CD8 T cells

Effector T cells are described to be primed in the lymph nodes draining the site of immunization and to recirculate to effector sites. Sublingual immunization generates effector T cells able to disseminate to the genital tract. Herein, we report an alternative mechanism that involves the recirculation of antigen-bearing dendritic cells (DCs) in remote lymphoid organs to prime T cells. Sublingual immunization with a muco-adhesive model antigen unable to diffuse through lymphatic or blood vessels induced genital CD8 T cells. The sublingual draining lymph nodes were not mandatory to generate these lymphocytes, and antigen-bearing DCs from distant lymph nodes and spleen were able to prime specific CD8 T cells in a time- and dose-dependent manner. This study demonstrates, for the first time, that antigen-bearing DCs originating from the site of immunization recirculate to distant lymphoid organs and provides insights into the mechanism of distant CD8 T-cell generation by sublingual immunization.     

Phenotypic and Functional Analysis of Mucosal Lymph Node T Cell Subpopulations Proximal and Distal to Chronic Staphylococcal Antigen Challenge

We investigated the functional and subset surface marker characteristics of supramammary lymph node T cell populations at sites proximal and distal to the mammary region of goats repeatedly injected with heat-treated Staphylococcus aureus antigen (HK-SAC). Flow cytometric studies showed quantitative differences in CD4⁺ and CD8⁺ T cell subsets among large and small lymphocyte populations in ipsilateral and contralateral supramammary lymph nodes of these animals. Although ipsilateral (draining) lymph nodes were enriched with CD4⁺ and CD8⁺ T cells, CD4/CD8 ratios were comparatively lower than those of contralateral (non-draining) lymph nodes (2.30 vs 2.60, respectively). Cell size analysis by flow cytometry showed that nearly 70% of the lymphocytes in ipsilateral nodes were of large cell phenotype with CD4/CD8 ratios of 2.52. In contrast, there were only 56.1% large lymphocytes in contralateral lymph nodes but with similar CD4/CD8 ratios of 2.55. The number of large lymphocytes in corresponding nodes of uninoculated control animals was significantly lower (50%) with much lower CD4/CD8 ratios (2.08). Alloantigenic responses of both ipsilateral and contralateral lymph node T cells were greater than those of uninoculated controls. Antigen-specific proliferation studies showed that ipsilateral lymph node T cells greatly enhanced both primed and non-primed lymph node B cell responses to HK-SAC, whereas those from contralateral lymph nodes were less stimulatory. In contrast, contralateral lymph node T cells had greater enhancing effects on PWM-induced polyclonal B cell responses. These studies indicate that repeated local infection with bacterial antigen induce changes in the numbers, ratios and antigen-specific and non-specific responses among ipsilateral (draining) and distal contralateral (non-draining) lymph node T cell populations in mucosal-associated immune systems such as the mammary gland.     

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.     

Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge

Mucosal surfaces are important for the induction of immunity against influenza virus. In a murine intranasal immunization model, we demonstrated that the attenuated Shigella flexneri Deltaasd strain 15D, carrying a DNA construct encoding the influenza virus hemagglutinin (HA), induces protective immunity against a lethal respiratory challenge with influenza A/WSN/33. Influenza virus-specific IFN-gamma T cells were detected among splenocytes, and anti-HA IgG was detected in serum post-immunization, albeit at low levels. Following influenza virus challenge, an accelerated anti-HA IgA antibody response was detected in bronchoalveolar lavage (BAL) washings from mice vaccinated with attenuated shigella containing the HA construct. These results suggest that S. flexneri Deltaasd strain 15D is a promising vector for mucosal DNA vaccine immunization against influenza virus and other mucosal pathogens.     

Kinetics, Localization and Isotype Profile of Antibody Responses to Immune Stimulating Complexes (Iscoms) Containing Human Influenza Virus Envelope Glycoproteins

The immune stimulating complex (iscom) is a particulate adjuvant formulation combining multimeric presentation of antigen with a built-in adjuvant, Quillaja saponin. Iscoms induce strong serum antibody responses that are readily boosted. To further characterize this property of iscoms, the development and maturation of primary and secondary antibody responses to iscoms containing influenza virus antigen were investigated, in serum by ELISA and on single B-cell level by ELISPOT. After a single subcutaneous injection, B cells secreting antigen-specific IgG (IgG-SC) were primarily observed in the draining lymph nodes (LN), showing peak numbers at day 7 which then declined rapidly. Serum IgG levels, as well as IgG-SC in the spleen, persisted for several weeks and, with time, IgG-SC cells also appeared in the bone marrow (BM). These results suggest that the IgG response to iscoms initially is located to the LN but that IgG-SC are redistributed with time and may persist for a long time in other organs, including the spleen and BM. Moreover, a booster dramatically enhanced the frequency of IgG-SC in LN, spleen and BM suggesting that iscoms induce a potent B-cell memory. Comparisons of antibody responses to iscoms with those to influenza virus antigen in Freund's complete adjuvant, TiterMax^TM or aluminium hydroxide suggest that the choice of adjuvant influences both the magnitude, kinetics, localization and isotype profile of antibody responses.