Antigen presentation in the rat: role of a nonadherent, nonphagocytic, W3/13, OX-6 positive T cell in the presentation of antigen to primed T lymphocytes

The nature of accessory cells in rat lymph nodes which can present antigen to primed T cells was investigated. Removal of adherent, phagocytic cells from antigen-primed lymph node cells by passage over glass-bead and nylon wool columns followed by treatment with carbonyl iron did not abrogate the antigen-specific proliferative response to keyhole limpet hemocyanin (KLH) or to the synthetic polypeptide L-glutamic acid-L-alanine-L-tyrosine (GAT). This T cell-enriched population was free of contaminating macrophages as determined by latex bead ingestion and morphological criteria during a 4-day culture period. Treatment of the T cell preparation with rabbit anti-rat IgG and complement or rosetting with IgG-coated sheep erythrocytes to remove any remaining B cells or macrophages did not significantly affect the proliferative response to antigen. Analysis of the T cell preparation by panning techniques with monoclonal antibodies to T cell surface markers suggested that both the responding T cell and the antigen-presenting cell were positive for the rat T cell marker, W3/13. The KLH-primed LN T cell-enriched fraction contained two distinct cell populations that were separable on the basis of their reactivity to OX-6 antibody. Two populations, an OX-6+ and an OX-6-, interacted synergistically in a KLH-dependent in vitro proliferative response. The cells within the T cell-enriched fraction that were positive for the OX-6 marker functioned primarily as the APCs, while the OX-6- cell fraction contained cells that proliferated to antigen when OX-6+ cells from either the T cell fraction or the adherent fraction were present. The implications of these findings are discussed.     

Augmentation of the antibody response by antigen-specific glycosylation-enhancing factor

BDF1 mice were immunized with a protein antigen, such as ovalbumin (OA) or keyhole limpet hemocyanin (KLH), absorbed to aluminum hydroxide gel, and their spleen cells were stimulated by homologous antigen for the formation of glycosylation-enhancing factor (GEF). It was found that GEF obtained from OA-primed spleen cells had affinity for OA, whereas those derived from KLH-primed spleen cells had affinity for KLH. Nonspecific GEF, which was obtained by stimulation of normal spleen cells with pertussis toxin, failed to bind OA or KLH. Both antigen-specific GEF and nonspecific GEF are inactivated by phenylmethylsulfonyl fluoride, but not by N-alpha-p-tosyl-L-lysyl-chloromethyl ketone. Both factors can be partially purified by binding to p-aminobenzamidine agarose and elution with benzamidine. These findings suggest that not only non-specific GEF but also antigen-specific GEF are serine protease(s). The antigen-specific GEF consisted of two m.w. species, of 65 to 85 kilodaltons (Kd) and 40 to 55 Kd, whereas nonspecific GEF consisted of 50 to 70 Kd and 20 to 30 Kd molecules. The OA-specific GEF augmented the in vitro secondary indirect PFC response of DNP-OA-primed cells to the homologous antigen, but failed to affect the PFC response of DNP-KLH-primed cells to DNP-KLH. Similarly, KLH-specific GEF enhanced the response of DNP-KLH-primed cells but not the response of DNP-OA-primed cells. However, OA-specific GEF failed to replace the requirement for antigen-primed helper T cells. Antigen-specific GEF bound to alloantibodies reactive to the products of the I region of the major histocompatibility complex. The results collectively suggest that antigen-specific GEF is identical to antigen-specific augmenting factors described by other investigators.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Combined detection of antigen specificity (anti-TNP) and (sub)class of intracellular antibodies

Mice and rabbits were immunized with trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin (KLH). Cells producing specific antibodies against the hapten TNP were detected in vivo in spleen and lymph nodes using a TNP--alkaline phosphatase (AP) conjugate. Using horseradish peroxidase (HRP)-conjugated anti-mouse (sub)class (IgG2A, IgG2B, IgM) antibodies and anti-rabbit class (IgG, IgM) antibodies and a double immunocytochemical staining technique for simultaneous demonstration of the enzymes AP and HRP, we were able to determine both the antigen specificity (anti-TNP) and the (sub)class of intracellular antibodies produced by individual antibody-forming cells in vivo.     

Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

The ovine nasal mucosa: an alternative tissue site for mucosal immunization

The ovine nasal mucosal environment has histological and ultrastructural features that resemble well-known inductive sites of mucosa-associated lymphoid tissue. In the present study, the nasal mucosa was assessed as a potential mucosal tissue site for delivering vaccines to sheep. Sheep were immunized by either injection with the model antigen, Keyhole Limpet Haemocyanin (KLH), and aluminium hydroxide gel (alum) or by aerosol spray with KLH with and without cholera toxin (CT). Sheep immunized by injection with KLH/alum and aerosol spray with KLH/CT induced strong anti-KLH IgG and IgA serum antibody responses as well as specific T cell memory. Anti-KLH IgG1 responses were significantly higher following immunization by injection and no significant differences in anti-KLH IgG2 responses were detected between groups. Sheep immunized with KLH by aerosol spray without CT did not produce serum antibody and T cell memory responses. Antibody-secreting cells were present in the parotid lymph nodes (draining lymph nodes) of sheep immunized with KLH/alum and KLH/CT, but secreted only Ag-specific IgG1, and not IgG2 or IgA. These results suggest that aerosolization of soluble antigen formulations with CT may provide an alternative method of delivering nasal vaccines to sheep and other large animal species, and that further improvements in antigen penetration of nasal tissues may dramatically improve the strength of the immune response.     

Macrophages and lymphoid cells exposed to antigenic stimulation

After antigenic stimulation (intraperitoneal administration of 6% emulsion of sheep erythrocytes) interaction of macrophages and lymphoid cells has been studied in non-inbred mice spleen, lymph nodes, lungs and skin. Histological, morphometrical and radiographic techniques demonstrate that dermal macrophages possess the least reactivity. In 15 days of the experiment activation of the macrophagial link of the spleen and lymph node coinsides with the most intensive transformation of lymphoid elements into the antibody-producing plasma cells. In the lung the phenomena described are observed on the 9th day of the experiment.     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

Induction by cells of immune responses to intestinal epithelial cell-associated components (ECAC): transfer with cultured murine mesenteric and popliteal/axillary lymph node cells

Although systemic and mucosal immune responses to intestinal epithelial self-antigens occur in several human disorders, there is no model system with which to study the physiology and regulation of the underlying cellular events. Therefore, we undertook to induce an immune response to purified epithelial macromolecules in the Lewis rat; characterize in vitro the reactive cells; and then transfer with immunocytes this antiepithelial reactivity to naive syngeneic rats, identifying the fine specificity and site of humoral and cell-mediated immunity induced in the cell recipient. Donor animals sensitized systemically (via footpad) or locally in gut mucosa (via Peyer's patches) to syngeneic or xenogeneic epithelial antigens generated specific immunoglobulin and were found to have T lymphocytes in the draining nodal areas (including the mesenteric nodes) which were (a) antigen-specific, having a [3H]thymidine uptake in the presence of antigen 30-fold the control; (b) generally of the Thelper/inducer subclass (W3/25+) which, upon further culture, developed phenotype surface markers for activation (IL-2R+); (c) able to induce an antigen-specific humoral and cell-mediated responses upon intravenous injection into naive syngeneic hosts; and (d) demonstrable in gut-associated lymphoid tissue (mesenteric lymph nodes) and, to a lesser extent in spleen, of the cell recipient. Further, lymphocytes cloned from reactive mesenteric lymph node cells demonstrated specificity for a gel-purified subfraction of epithelial antigen, designated P1, containing highly conserved organ-specific macromolecules thought to be autoantigenic for gut.     

Staining patterns for the anti-horseradish peroxidase antibody reaction in proplasma cells developing in the medulla of rat popliteal lymph nodes during the secondary response

The development of plasma cells from lymphocytes was studied in the medulla of popliteal lymph nodes of rats during the secondary response to horseradish peroxidase (HRP). Changes in the microscopic appearance of proplasma cells were compared with changes in the intensity of the anti-HRP antibody reaction in these cells. Early proplasma cells, appearing 2 to 3 days after the injection of HRP into the footpads, were relatively small cells similar in size to lymphocytes. Their small nuclei were eccentrically located due to the one-sided enlargement of the pyroninophilic cytoplasm. The reaction for the anti HRP antibody in these cells was weak or negative. Other proplasma cells located in the same medullary cord regions showed a more intense antibody reaction. This change was correlated, in many cases, with an enlargement of the nucleus, giving the cells a blast-like appearance. Three to 6 days after the reinjection of the antigen, the medullary cords contained many mature plasma cells characterized by an intense antibody reaction. The mature plasma cells were always accompanied by proplasma cells, the latter varying in microscopic appearance (stage of development) asd staining intensities (antibody contents). The staining intensities and the microscopic appearance of proplasma cells, and the proportion of proplasma cells to plasma cells, varied in different medullary cord regions of the same lymph nodes. The staining patterns, together with the microscopic appearance of the cells, seemed to show whether antibody formation was inhibited or stimulated.     

The effects of glutaraldehyde treatment and horesradish peroxidase conjugation on the immunoreactivity of bovine myelin encephalitogenic protein.

The effects of the immunoreactivity of bovine myelin encephalitogenic protein (EP) of treatment with glutaraldehyde and conjugation with horeradish peroxidase (HRP) were investigated by complement fixation and immunohistochemistry. Both glutaraldehyde treatment and HRP conjugation of EP decreased but did not abolish the reactivity of EP with rabbit anti-EP. Conjugation of EP to HRP by the two-step method of Avrameas had less detrimental effects on the immunochemical reactivity of EP than did the one-step procecure. The use of EP-HRP conjugates to probe for antibodies to EP is an immunochemically sound system with the major limitation that the number of antibody-producing cells is likely to be underestimated due to the failure to detect cells producing low levels of antibody or antibodies directed toward determinants altered by the modification procedures.     

IgE antibody-forming cells in rats infected with Nippostrongylus brasiliensis and immunized with antigens

The distribution of IgE antibody-forming cells was examined in rats infected with Nippostrongylus brasiliensis (Nb) or immunized with Nb antigen or with OA. The frequency of antigen-specific IgE antibody-forming cells was detected by a passive cutaneous anaphylactic (PCA) reaction using cell extract from lymphoid organs. In Nb-infected rats, anti-Nb and anti-4th stage larvae (L4) IgE-forming cells distributed mainly in the mesenteric and the bronchial lymph nodes (LN) near the parasite-harboring sites. After intraperitoneal (ip) immunization with Nb antigen mixed with Al(OH)3 and Bordetella pertussis (Bp) as adjuvants, anti-Nb IgE antibody-forming cells were detected in the mesenteric and the bronchial LN. Anti-Nb or OA IgE antibody-forming cells after subcutaneous (sc) immunization were found in the inguinal and the axillary LN. An effect of Bp on the distribution of IgE antibody-forming cells seems to be ruled out. The distribution of IgG2a antibody-forming cells was similar to that of IgE antibody-forming cells, indicating that the distribution of the IgE antibody-forming cells is not preferential. IgE antibody-forming cells were stimulated in the regional LN near the site of antigen administration. IgE antibody-forming cells induced by potentiated IgE antibody production were also examined. Rats were immunized ip or sc with OA and infected with Nb. Anti-OA IgE antibody-forming cells were found in all of the lymphoid organs and especially in the regional LN near the Nb parasite-harboring and antigen administration sites.     

Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice

Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.     

T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.     

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology

The objective of the present study was to investigate the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus (SS) to regions of follicular retention in the cortex. The migration of horseradish peroxidase (HRP), used as a histochemically identifiable antigen, was followed by light and electron microscopy in C3H mouse popliteal lymph nodes obtained 1, 5, 15, and 30 min, and 5 and 24 hr after hindfoot pad injection of HRP. The observations showed that as early as 1 min after HRP injection, localization of antigen occurred at distinct sites in the SS and subjacent areas of the cortex on the afferent side. At these sites, between 1 min and 24 hr, the antigen formed light microscopically identifiable trails, which reached progressively deeper into the cortex with time toward individual follicular regions. By 24 hr this apparent migration of antigen was complete, and HRP was localized in follicles. This migration pattern did not occur on the efferent sides of lymph nodes, and it was dependent on the systemic presence of specific antibodies since it was observable only in passively immunized but not in nonimmune mice. Temporary retention of antigen by typical macrophages was also observed in the SS on the efferent side. This was minimal in nonimmune mice and was significantly enhanced in passively immunized mice. Electron microscopy indicated that the apparent migration of immune complexes was mediated by a group of cells observed in the migration path that had immune complexes sequestered on their surface or in plasma membrane infoldings. These antigen transporting cells (ATC) were relatively large nonphagocytic cells, with lobated or irregular euchromatic nuclei and cell processes of various complexity. ATC observed in or near the SS appeared to be less differentiated, were monocyte-like, and resembled non-Birbeck granule-containing Langerhans cell precursors or veiled cells. Others, located deeper in the cortex, appeared more differentiated, interdigitated with antigen-retaining dendritic cells, and shared morphologic characteristics with follicular dendritic cells (FDC). The results support the concepts that immune complexes are trapped in the SS and are transported by a group of non-phagocytic cells, other than lymphocytes, to follicular regions. The mechanism of transport may involve the migration of ATC with a concomitant maturation into FDC, or by a mechanism of ATC to FDC transport utilizing dendritic cell processes and membrane fluidity, or by a combination of the two mechanisms.     

Definition of the pulmonary antibody response to ovalbumin following local challenge in systemically immunized rats

The in vivo pulmonary immune response of rats to local stimulation with antigen was assessed by measuring antigen-specific antibody and antibody-secreting cells utilizing enzyme-immunoassay technology. Sprague-Dawley rats were immunized subcutaneously with ovalbumin (OA) emulsified in Freund's incomplete adjuvant, challenged with OA intratracheally on Day 19 and sacrificed 1, 2, 3, or 4 days later. Specific antibody-secreting cells in the lung-associated lymph nodes were enumerated with the ELISA-SPOT assay and antibody concentration in the pulmonary lavage fluids and sera was assessed with the ELISA. The greatest response for each parameter was on Day 2. Cellular infiltration of the lung was minimal. Cellular infiltrates consisted mainly of polymorphonuclear leukocytes and were most numerous in the lavage fluid on Days 1 and 2 and in the lung parenchyma on Day 2 after challenge. Local production versus serum transudation of antibody was evaluated by comparing the levels of OA-specific antibody to albumin in the lavage fluid and serum. The data showed that antibody in the lungs was primarily produced locally.     

Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells.

The distribution of functionally active hapten-specific B memory cells was investigated. Using antigen-pretreated lethally irradiated recipients, a marked accumulation of adoptively transferred B memory cells was demonstrated in lymph nodes containing specific antigen, but not in lymph nodes containing non-cross-reacting hapten conjugates. This difference in responsiveness between lymph nodes containing specific versus those containing nonspecific antigen developed over a period 3–5 days after memory cell transfer. The localization of antigen specific cells was T-cell independent; both carrier-primed T helper cells and specific antigenic challenge, however, were required to trigger the localized B memory cells into antibody production. Specific B memory cell accumulation did not result from an expansion of the antigen-specific cell population due to local proliferation induced by antigen depots in the lymph nodes to challenge. Rather, the results indicated that recirculating B memory cells had progressively accumulated through retention by antigen in the lymph node. These findings suggest that, in the absence of T-cell help and specific antigenic challenge, B memory cells accumulate in lymphoid tissue (follicles) without responding and provide persistent local memory for the humoral immune response.