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T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC-activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC-imprinted T cells protect against influenza more effectively than do gut and skin DC-imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC-activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC-activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4.

Airborne exposure to microbial cell wall lipids such as lipopolysaccharide triggers innate immune responses that regulate susceptibility to allergic airway inflammation. α-Glycosylceramides represent another widespread class of microbial lipids that directly stimulate innate-like, IL-4- and IL-13-producing, CD1d-restricted NKT cells. In this study, we demonstrate that NKT cells constitutively accumulate and reside in the microvasculature of the mouse lung. After a single airborne exposure to lipid antigen, they promptly extravasate to orchestrate the formation of peribronchiolar and interstitial lymphohistiocytic granulomas containing numerous eosinophils. Concomitant airborne exposure to ovalbumin (OVA) induces the priming of OVA-specific Th2 cells and IgE antibodies by the same dendritic cell coexpressing CD1d and MHC class II. Although NKT cell activation remains confined to the lipid-exposed lung and draining lymph nodes, Th2 cells recirculate and seed the lung of a parabiotic partner, conferring susceptibility to OVA challenge months after the initial exposure, in a manner independent of NKT cells and CD1d. Thus, transient recruitment and activation of lung-resident intravascular NKT cells can trigger long-term susceptibility to allergic airway inflammation.

Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)-specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10-secreting CD4(+) T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4(+) T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-gamma to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN-gamma and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9-deficient mice fail to develop experimental oral antigen-induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9-mediated mast cell responses have an important role in food allergy.

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c(+) monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid.

Antigen-mediated cross-linking of IgE bound to mast cells via the high affinity receptor for IgE triggers a signaling cascade that results in the release of intracellular calcium stores, followed by an influx of extracellular calcium. The collective increase in intracellular calcium is critical to the release of the granular contents of the mast cell, which include the mediators of acute anaphylaxis. We show that the sensitivity of the mast cell to antigen-mediated degranulation through this pathway can be dramatically influenced by the A2b adenosine receptor. Loss of this Gs-coupled receptor on mouse bone marrow-derived mast cells results in decreased basal levels of cyclic AMP and an excessive influx of extracellular calcium through store-operated calcium channels following antigen activation. Mice lacking the A2b receptor display increased sensitivity to IgE-mediated anaphylaxis. Collectively, these findings show that the A2b adenosine receptor functions as a critical regulator of signaling pathways within the mast cell, which act in concert to limit the magnitude of mast cell responsiveness when antigen is encountered.

We explored the relationship between the time of naive CD4+ T cell exposure to antigen in the primary immune response and the quality of the memory cells produced. Naive CD4+ T cells that migrated into the skin-draining lymph nodes after subcutaneous antigen injection accounted for about half of the antigen-specific population present at the peak of clonal expansion. These late-arriving T cells divided less and more retained the central-memory marker CD62L than the T cells that resided in the draining lymph nodes at the time of antigen injection. The fewer cell divisions were related to competition with resident T cells that expanded earlier in the response and a reduction in the number of dendritic cells displaying peptide-major histocompatibility complex (MHC) II complexes at later times after antigen injection. The progeny of late-arriving T cells possessed the phenotype of central-memory cells, and proliferated more extensively during the secondary response than the progeny of the resident T cells. The results suggest that late arrival into lymph nodes and exposure to antigen-presenting cells displaying lower numbers of peptide-MHC II complexes in the presence of competing T cells ensures that some antigen-specific CD4+ T cells divide less in the primary response and become central-memory cells.

Oral tolerance induction is a key feature of intestinal immunity, generating systemic nonresponsiveness to ingested antigens. In this study, we report that orally applied soluble antigens are exclusively recognized in the intestinal immune system, particularly in the mesenteric lymph nodes. Consequently, the initiation of oral tolerance is impeded by mesenteric lymphadenectomy. Small bowel transplantation reveals that mesenteric lymph nodes require afferent lymph to accomplish the recognition of orally applied antigens. Finally, oral tolerance cannot be induced in CCR7-deficient mice that display impaired migration of dendritic cells from the intestine to the mesenteric lymph nodes, suggesting that immunologically relevant antigen is transported in a cell-bound fashion. These results demonstrate that antigen transport via afferent lymphatics into the draining mesenteric lymph nodes is obligatory for oral tolerance induction, inspiring new therapeutic strategies to exploit oral tolerance induction for the prevention and treatment of autoimmune diseases.

The surface of the intestinal mucosa is constantly assaulted by food antigens and enormous numbers of commensal microbes and their products, which are sampled by dendritic cells (DCs). Recent work shows that the mesenteric lymph nodes (MLNs) are the key site for tolerance induction to food proteins and that they also act as a firewall to prevent live commensal intestinal bacteria from penetrating the systemic immune system.

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

Tolerance is the usual outcome of inhalation of harmless antigen, yet T helper (Th) type 2 cell sensitization to inhaled allergens induced by dendritic cells (DCs) is common in atopic asthma. Here, we show that both myeloid (m) and plasmacytoid (p) DCs take up inhaled antigen in the lung and present it in an immunogenic or tolerogenic form to draining node T cells. Strikingly, depletion of pDCs during inhalation of normally inert antigen led to immunoglobulin E sensitization, airway eosinophilia, goblet cell hyperplasia, and Th2 cell cytokine production, cardinal features of asthma. Furthermore, adoptive transfer of pDCs before sensitization prevented disease in a mouse asthma model. On a functional level, pDCs did not induce T cell division but suppressed the generation of effector T cells induced by mDCs. These studies show that pDCs provide intrinsic protection against inflammatory responses to harmless antigen. Therapies exploiting pDC function might be clinically effective in preventing the development of asthma.

Mast cell degranulation and de novo cytokine production is a consequence of antigen-aggregation of the immunoglobulin E (IgE)-occupied high affinity receptor for IgE (Fc epsilon RI). Herein, we report that lymphokines that promote allergic inflammation, like MCP-1, were potently induced at low antigen (Ag) concentrations or at low receptor occupancy with IgE whereas some that down-regulate this response, like interleukin (IL)-10, required high receptor occupancy. Weak stimulation of mast cells caused minimal degranulation whereas a half-maximal secretory response was observed for chemokines and, with the exception of TNF-alpha, a weaker cytokine secretory response was observed. The medium from weakly stimulated mast cells elicited a monocyte/macrophage chemotactic response similar to that observed at high receptor occupancy. Weak stimulation also favored the phosphorylation of Gab2 and p38MAPK, while LAT and ERK2 phosphorylation was induced by a stronger stimulus. Gab2-deficient mast cells were severely impaired in chemokine mRNA induction whereas LAT-deficient mast cells showed a more pronounced defect in cytokines. These findings demonstrate that perturbation of small numbers of IgE receptors on mast cells favors certain signals that contribute to a lymphokine response that can mediate allergic inflammation.

Allergic asthma is an inflammatory lung disease initiated and directed by T helper cells type 2 (Th2). The mechanism involved in generation of Th2 responses to inert inhaled antigens, however, is unknown. Epidemiological evidence suggests that exposure to lipopolysaccharide (LPS) or other microbial products can influence the development and severity of asthma. However, the mechanism by which LPS influences asthma pathogenesis remains undefined. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 (Th1) responses, it is unclear if TLRs are needed for Th2 priming. Here, we report that low level inhaled LPS signaling through TLR4 is necessary to induce Th2 responses to inhaled antigens in a mouse model of allergic sensitization. The mechanism by which LPS signaling results in Th2 sensitization involves the activation of antigen-containing dendritic cells. In contrast to low levels, inhalation of high levels of LPS with antigen results in Th1 responses. These studies suggest that the level of LPS exposure can determine the type of inflammatory response generated and provide a potential mechanistic explanation of epidemiological data on endotoxin exposure and asthma prevalence.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen-major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 A(u)-restricted, acetylated NH(2)-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for A(u). In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- kappaB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50(-/-) mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50(-/-) mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50(-/-) mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-kappaB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)-restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4(-)CD8(-)B220(+) T cells occurs more frequently from a CD8(+) precursor than from CD4(+) T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8(+) cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC-restricted TCR. The findings show that CD4(-)CD8(-)B220(+) T cells preferentially derive from a CD8(+) precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.

The mechanisms that maintain memory in T cells are not completely understood. We have investigated the role of antigen and interleukin (IL)-2 in the growth and maintenance of CD8+ T cells using a cytolytic T cell line specific for ovalbumin (OVA)257-264 presented by H-2Kb. This line does not secrete IL-4 or IL-2; hence, stimulation with the OVA-transfected EL4 line (E.G7-OVA) does not induce proliferation without addition of exogenous growth factors. Furthermore, this line can be maintained continuously by weekly addition of irradiated, splenic filler cells and IL-2, with or without E.G7-OVA. Although IL-2 induced proliferation of these cytotoxic T lymphocytes (CTLs), production of interferon gamma and tumor necrosis factor alpha required stimulation of the CTL with E. G7-OVA. The kinetics of lymphokine secretion after stimulation by E. G7-OVA were the same whether the CTL had been maintained with or without antigen (Ag). In addition, both CTL lines killed E.G7-OVA target cells within 4 h. Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy. CTLs cultured with or without Ag also served as memory T cells when parked for 100 d in unirradiated, syngeneic recipients without OVA. In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice. The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory. These data also suggest that stimulation of OVA-specific CTLs by lymphokines seems to be more important to maintaining memory than stimulation of TCRs by cross-reactive peptides complexed to class I molecules.

Dendritic cells (DC) are widely distributed in the lung where they are distinguished by their morphology and class II major histocompatibility complex (Ia) antigen expression. Although a role for DC as pulmonary antigen-presenting cell (APC) has been suggested, little is currently known concerning how these cells respond to inhaled antigens in vivo. Hen-egg lysozyme (HEL) was injected intratracheally into Lewis rats; DC were subsequently purified from the lung and regional lymph nodes (LN) at intervals of up to 14 d and examined for their ability to stimulate the proliferation of HEL-immune T cells in vitro in the absence of added HEL. Pulmonary DC displayed APC activities at 3 h and for up to 7 d after the injection of antigen. Dendritic cells in the draining hilar LN showed APC activities that appeared at 24 h, peaked at day 3, and then diminished progressively. After the primary sensitization, HEL-immune T cells were detected in hilar LN but not in the lung. A second airway challenge with HEL at day 14 yielded an antigen-specific pulmonary immune response, characterized histologically by the accumulation of mononuclear cells around lung venules. We conclude that APC activities shift from lung to lymph node during the response to inhaled antigen.

Interleukin 4-targeted (IL-4-/-) mice are defective in T helper (Th)2 cytokine production as determined after nematode infection. As Th2 cells appear to be selectively induced by oral immunization we investigated the ability of IL-4-/- mice to respond to perorally administered antigen. We found that IL-4-/- mice failed to respond to soluble protein antigens given perorally together with cholera toxin (CT) as a mucosal adjuvant. In contrast to wild-type mice no or poor anti-keyhole limpet hemocyanin (KLH) or anti-ovalbumin (OVA) B cell responses were observed in gut lamina propria, spleen, or serum of IL-4-/- mice after oral immunization. In addition, mucosal immunization failed to stimulate antigen-specific T cell responses in these mice. The lack of responsiveness was specific for mucosal administration of antigen and was not seen after intravenous injections with antigen and CT-adjuvant. The systemic adjuvant effect of CT was not impaired in IL-4-/- mice as evidenced by the strong enhancement of anti-KLH responses after intravenous immunization with KLH plus CT as opposed to KLH alone. However, CT as an immunogen, in contrast to KLH or OVA, stimulated significant mucosal and systemic immune responses in IL-4-/- mice after oral immunization. Both serum and intestinal IgA anti-CT antibodies were demonstrable in IL-4-/- mice as well as in wild-type mice. Total IgA levels in gut lavage and in serum of immunized IL-4-/- mice were of similar magnitude as in wild-type mice, suggesting that the ability of naive B cells to undergo isotype switch-differentiation from IgM to IgA in IL-4-/- mice did not appear to be impaired. Immunohistochemical analysis of Peyer's patches demonstrated a complete inability to form germinal centers in IL-4-/- mice in contrast to wild-type mice. Our data suggest that IL-4-/- mice are unable to respond to oral/mucosal immunization due to a failure to stimulate antigen-specific cells required to induce germinal center reactions in the Peyer's patches. Our findings demonstrate that IL-4 and probably functional Th2 cells are required for induction of gut mucosal antibody responses.

To determine the role of vascular cell adhesion molecule 1 (VCAM-1)/very late activation antigen 4 (VLA-4) and intercellular adhesion molecule 1 (ICAM-1)/lymphocyte function-associated antigen 1 (LFA-1) interactions in causing antigen-induced eosinophil and T cell recruitment into the tissue, we studied the effect of the in vivo blocking of VCAM-1, ICAM-1, VLA-4, and LFA-1 by pretreatment with monoclonal antibodies (mAb) to these four adhesion molecules on the eosinophil and T cell infiltration of the trachea induced by antigen inhalation in mice. The in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, prevented antigen-induced eosinophil infiltration into the mouse trachea. On the contrary, the in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, increased blood eosinophil counts after antigen challenge, but did not affect blood eosinophil counts without antigen challenge in sensitized mice. Furthermore, the expression of VCAM-1 but not ICAM-1 was strongly induced on the endothelium of the trachea after antigen challenge. In addition, pretreatment with anti-IL-4 mAb decreased the antigen-induced VCAM-1 expression only by 27% and had no significant effect on antigen-induced eosinophil infiltration into the trachea. The in vivo blocking of VCAM-1 and VLA-4 inhibited antigen-induced CD4+ and CD8+ T cell infiltration into the trachea more potently than that of ICAM-1 and LFA-1. In contrast, regardless of antigen challenge, the in vivo blocking of LFA-1, but not of ICAM-1, increased blood lymphocyte counts more than that of VCAM-1 and VLA-4. These results indicate that VCAM-1/VLA-4 interaction plays a predominant role in controlling antigen-induced eosinophil and T cell recruitment into the tissue and that the induction of VCAM-1 expression on the endothelium at the site of allergic inflammation regulates this eosinophil and T cell recruitment.

In the rat, mesenteric lymphadenectomy allows collection of dendritic cells (DC) derived from the small intestine after cannulation of the thoracic duct. We prepared rats this way and administered antigens by oral feeding or intraintestinal injection. DC enriched from the thoracic duct lymph collected over the first 24 h from these animals are able to stimulate sensitized T cells in vitro and to prime popliteal lymph node CD4+ T cells after footpad injection, while B and T cells from the same thoracic duct lymph are inert in priming. 500 or less DC pulsed in vitro with antigen can prime T cells in vivo, whereas 100 times more B cells or macrophages pulsed in vitro are quite inert. 1 mg of ovalbumin administered orally is sufficient to load DC for in vivo priming of T cells. Antigen could not be detected directly in DC but was present in macrophages in the lamina propria. Direct presentation of antigen by DC to T cells was demonstrated by injecting F1 recipients with parental DC and showing restriction of T cell sensitization to the major histocompatibility complex of the injected DC. Antigen-bearing DC do not induce a detectable primary antibody response but a small secondary antibody response can be detected after a boosting injection. These results show that acquisition of antigens by DC in the intestine is very similar to what occurs in vitro or in other tissues, suggesting that there may be no special difference in antigen handling at mucosal surfaces. One implication of these results is that hypotheses designed to explain oral tolerance must take into account the presence of immunostimulatory, antigen-bearing DC in animals that have received oral antigens.

The ability of B cells or macrophages and dendritic cells (DC) to elicit class II-restricted T cell responses in vivo was compared using a mouse chimera model. Severe combined immunodeficient (SCID) mice (H-2d), reconstituted either with T or T+B lymphocytes from (H-2d x H-2b) donors, were immunized subcutaneously with protein antigen (Ag) to induce a class II-restricted T cell response. The frequency and major histocompatibility complex restriction of the resulting Ag-specific T cells were analyzed to establish whether B cells were necessary for the induction of class II-restricted T cell responses, and to determine the cell type on which priming had occurred. The results indicated that: (a) B cells are not necessary for the induction of a class II-restricted T cell response in vivo, as the frequencies of interleukin 2 (IL-2)- or IL-3-secreting T cells induced in the presence or absence of B cells were comparable. (b) Activation of naive T cells requires presentation of Ag on DC; Ag presented only on B cells is not sufficient to elicit a response. No H-2b-restricted, IL-3-secreting cells could in fact be detected in SCID mice reconstituted with naive (H-2d x H-2b) T cells and nonimmune or antigen-primed (H-2d x H-2b) B cells. (c) Previously primed T cells are able to be stimulated by Ag presented by both B cells and DC. H-2b-restricted, IL-3-secreting cells could in fact be readily demonstrated in SCID mice reconstituted with antigen-primed (H-2d x H-2b) T and B cells. Irrespective of whether the T cells were naive or previously activated, B cells were able to respond with an Ag-specific immunoglobulin G response, indicating that B cells were functional and able to present Ag in order to receive specific T cell help. Therefore, it appears that B cells are not necessary and do not participate in the initial priming of T cells; however, Ag presented by B cells can reactivate previously primed T cells. Taken together, these data indicate that during the course of an immune response Ag is first presented to naive T cells via DC, and only subsequently primed T cells can be stimulated by Ag presented by B cells.

Immunization with antigens stimulates not only B lymphocytes secreting specific antibody but, in addition, results in the generation of very large numbers of splenic Ig-secreting cells which lack specificity for that antigen. The present report examined the nature of the antigens capable of eliciting this effect and the mechanisms whereby B cells could be nonspecifically activated. It is shown that the ability of T-dependent, but not T-independent antigens, to induce such increases requires the participation of T helper cells specific for the antigen so that any one antigen results in the activation of only a proportion of total B cells. Analysis of this nonspecific plaque-forming-cell response reveals that B cell activation is not random but occurs in a class-restricted manner. The magnitude of the increase and the isotype produced are shown to be characteristic of the immunizing antigen. Based on the data presented, the apparent nonspecific T-B collaboration can best be explained by invoking a second Ig-specific helper mechanism in which helper cells capable of recognizing determinants on Ig molecules, e.g. isotype or idiotype, cause the stimulation of B cells of any specificity providing they express that determinant.

We examined the effects of the developmental milieu on the capacity of B cells to undergo immune response gene-controlled, T cell-dependent polyclonal proliferation. Although I-Aq poly(Glu60 Ala30 Tyr10)n (GAT)-nonresponder T cells developing in a responder environment become phenotypic GAT-responders, I-Aq B cells remain unresponsive to GAT, even after maturation in a GAT-responder animal. Conversely, (B10.A x B10.Q)F1 ([GAT responder x GAT nonresponder]F1) T cells developing in a B10.Q GAT nonresponder host fail to respond to GAT, but F1 B cells from the same F1 leads to parent chimeras make excellent proliferative responses in the presence of GAT and responder T cells. Thus, by this assay, B cell immune response gene function is genetically determined and is not affected by the developmental milieu.

Three outcomes pertinent to contact sensitivity (CS) follow immunization with various forms of trinitrophenylated (TNP) substrates: (a) specific immunological unresponsiveness for CS is induced when immunization favors activation of splenic suppressor cells. This state is achieved by intravenous injection of trinitrophenyl-conjugated to various types of cells, such as peritoneal exudate cells (PEC). (b) A short-lived or evanescent form of CS is induced when immunization reduces activation of the suppressor circuit. This can be achieved by subcutaneous immunization with trinitrophenyl conjugated to syngeneic PEC, by pretreatment with cyclophosphamide to diminish suppression before intravenous immunization, or by altering the mode of antigen presentation by using TNP-substrate that has undergone phagocytosis. (c) A long-lived form of CS is induced when trinitrophenyl is presented to the immune system on skin cells either by contact skin painting with reactive trinitrophenyl, or by subcutaneous, or even intravenous injection of trinitrophenyl-conjugated epidermal cells. In fact, trinitrophenyl-conjugated epidermal cells induced CS even when the suppressor circuit was activated by intravenous coadministration of TNP-PEC. This implies that antigen presentation on epidermal cells induces sensitized cells that are relatively resistant to suppression. The cell type(s) in the skin that are primarily responsible for this potent form of antigen presentation are most likely Langerhans cells, because they can be concentrated by virtue of their Fc receptors and they are Ia positive. Thus, both the anatomical site where antigen is first encountered by the immune apparatus, as well as the nature of the cells which present the antigen, determine whether a CS response will ensue, as well as whether it will be evanescent or long-lasting.

Daily gastric intubation of lipopolysaccharide (LPS)-responsive C3H/HeN, BALB/c, and Swiss mice with SRBC for 2 wk resulted in oral tolerance, whereas similarly treated LPS-nonresponsive C3H/HeJ mice gave splenic anti-SRBC PFC responses, including the IgA isotype, after systemic challenge with antigen. Oral tolerance in LPS-responsive C3H/HeN mice was due to T suppressor (Ts) cells because significant Ts cell activity was demonstrated in both Peyer's patches (PP) and spleens of these animals. On the other hand, T cells from PP and spleens of identically treated C3H/HeJ mice exhibited mainly T helper cell activity. Prior treatment of PP or spleen cell preparations from tolerant C3H/HeN mice with anti-Lyt-2.1 resulted in good in vitro anti-SRBC PFC responses, especially IgA isotype responses in PP cell cultures. These results indicate that oral administration of a thymic-dependent antigen (SRBC) to LPS-responsive mice induced a Ts cell population in PP, which, after migration to peripheral lymphoid tissue (e.g., spleen), suppressed responses to systemically administered antigen. LPS-nonresponsive mice lack this Ts cell pathway and continually respond to oral administration of antigen.

In this study T helper cells that recognize idiotypes as carriers for a hapten-specific B cell response were analyzed under limiting dilution conditions. T helper cells, induced by phosphorylcholine-hemocyanin (PC-Hy) priming, recognize trinitrophenylated TEPC-15 and MOPC-167 (TNP-T15, TNP-167) equally well. Limiting dilution analysis indicates identical frequencies of helper cells for TNP-T15 and TNP-167. Double immunization protocols using TNP-T15 and TNP-167 fail to demonstrate additive effects. Inhibition of carrier recognition in vitro using free hapten, PC, and unconjugated T15 or M167 indicates identical specificities of helper cells for T15 and M167. Collectively, these results provide strong evidence that PC-Hy priming induces only one population of idiotype-recognizing helper cells that are unable to distinguish between the T15 and the M167 idiotopes. The helper cell induction circuit was further analyzed. PC-Hy priming induces T15/167-specific helper T cells in X-linked immune defect-expressing F1 mice. This indicates that a B cell response to PC is not required to induce idiotype-recognizing T cells. Adoptive cotransfer of B cells from PC-Hy-primed mice together with normal T cells fails to induce idiotype-recognizing T cells. These results indicate the existence of a T helper1-T helper2 induction loop. In this scheme, the T helper1 cell carries T15-like receptors and the T helper2 cells, anti-T15-like receptors. Monoclonal antiidiotypic antibodies specific for T15 also induce a T15/167-recognizing T helper cell population. This finding demonstrates that idiotope-specific priming induces non-idiotype-specific T cells. Evidently, the idiotypic T cell network is based on a different selection of idiotope determinants than the selection of the B cell idiotype network.

Administration of cholera toxin/toxoid by either intraduodenal or parenteral routes increases the frequency of antigen-sensitive B cells in Peyer's patches (PP) and in distant lymphoid tissues greater than 50-fold. The special feature of mucosal priming with toxin is its unique effectiveness at generating secondary B cells, whose progeny express IgA exclusively, and such cells appear in highest frequency in PP and in appreciable numbers in spleen. Thus, this deliberate intraduodenal immunization seems to mimic the natural priming process induced by enteric bacterial colonization, which we have postulated to account for the high frequencies of IgA-committed cells specific for bacterial determinants in the PP of conventionally reared mice. furthermore, as a result of intraduodenal immunization, antigen-specific memory B cells are disseminated to sites distant form that of antigen application, including the lymphoid follicles associated with the respiratory mucosa. Direct antigenic stimulation of cells in the PP therefore results in effective cross-priming among mucosal and systemic sites through division, differentiation, and disemination of antigen-sensitive secondary B cells.

A macrophage-dependent, antigen-specific murine T-cell proliferation assay was utilized to examine the role of soluble products of murine and human adherent cells in the activation of T lymphocytes. Highly purified human leukocytic pyrogen, and supernates from both murine and human mononuclear phagocytes-macrophages stimulated the immune T-cell proliferative response to the multideterminant antigens dinitrophenyl-ovalbumin and keyhole limpet hemocyanin. The implications of these studies and the relationship of leukocytic pyrogen to human lymphocyte-activating factor are discussed.

Cell-mediated immunity includes both the generation of cytotoxic cells and initiation of delayed-type hypersensitivity (DTH). The resting T-cell population, before stimulation by antigen, already contains cells of the Lyl subclass that are programmed to initiate DTH (and helper function) but not cytotoxic responses, as well as Ly23 cells which can generate killer activity (and suppressive function) but not DTH. The central implication of these findings is that the broad division between humoral and cell-mediated immune responses does not precisely correspond to the division of labor among T-cell subclasses. The relative contribution of DTH-competent Lyl cells and cytotoxic Ly23 cells to the classical homograft response remains to be determined.

Neonatal mice given intact living Schistosoma mansoni eggs or soluble schistosome egg antigens intragastrically developed systemic cellular hypersensitivity as shown by markedly accelerated, augmented granulomatous inflammation around S. mansoni eggs subsequently injected intravenously into the pulmonary microvasculature. To achieve partial sensitization in adult mice schistosome eggs had to be administered intragastrically with bicarbonate; full sensitization occurred when the eggs were injected intraduodenally. These data indicate that under appropriate conditions intestinal administration of antigen can result in systemic cellular immune sensitization.

The feeding of sheep erythrocytes (SRBC) to mice leads to the production of two distinct T cell-derived suppressor factors by spleen cells. Each has been characterized for specificity, genetic restrictions, and cellular interactions. Fraction I has a 60,000-75,000 mol wt, is specific for antigen, and is suppressive of primary in vitro anti-SRBC responses at all times. It is not restricted by major histocompabitility complex (MHC)- or Igh-linked genes, but it fails to suppress spleen cells derived from any strain of mouse with a B10 background. It acts on an Lyt-2+ T cell to increase suppressive activity. An antiserum has been prepared against this factor that reacts with other, unrelated T cell suppressor factors. Fraction II has an approximately 30,000-40,000 mol wt, is specific for antigen, and has a dual effect on in vitro anti-SRBC responses. On day 3 of culture, it leads to augmentation of the response, whereas at day 5 it suppresses the response. It is not restricted by MHC genes, but it is restricted by Igh-linked genets. It acts by activating an Ly-1 t cell to both help and induce feedback suppression. These factors, and the antisera prepared against them, should allow more precise dissection of the molecular pathways by which immunoregulatory cells communicate with one another.

The intragastric administration of homologous strain epididymal sperm to adult virgin NZBW (Rt 1) female rats was shown to induce short-to long-term infertility. Infertility was associated with an early rise of genital secretory fluid IgA antisperm antibody preceding mating.

Initiation of an autoimmune tubulointerstitial disease was achieved in strain XIII guinea pigs by passive transfer of functionally pure IgG1 or IgG2 fractions of isologous anti-tubular basement membrane (TBM) serum. IgG2 appeared to be somewhat more effective than IgG1. The immunopathologic features in the IgG1 and IgG2 recipients were similar at the time of sacrifice, 14 days after transfer. The recipients that developed disease had higher than expected anti-TBM titers at 14 days. Furthermore, anti-TBM antibodies were of both IgG isotypes. In contrast, simultaneously administered IgG1 or IgG2 anti-BGG antibodies declined in titer in the recipients and were never found in the isotype fraction that had not been transferred. These findings indicate that the recipients of anti-TBM antibodies of either IgG1 or IgG2 isotype were stimulated to produce anti-TBM autoantibodies, which participated in the pathogenesis of the renal disease. The model demonstrates that autoantibodies may provide a mechanism (autoimmune amplification) for the intensification and perpetuation of antibody-mediated autoimmune diseases.

Virus plaque-forming cell assay with vesicular stomatitis virus (VSV), which had been originally introduced by Bloom and his colleagues as a tool for the enumeration of activated lymphocytes, was first applied to the immune response of mice to a widely used antigen, i.e. sheep red blood cells (SRBC). When spleen cells taken from mice previously primed with SRBC were cultured in the presence of the antigen, lymphocytes capable of replicating VSV (antigen-induced virus plaque-forming cells, Ag-V-PFC) were generated in the culture. They seemed to appear as early as 1 day of culture, and the peak was attained by the 2nd day. Most of Ag-V-PFC belonged to T-cell population, since 80-90% of Ag-V-PFC was killed by the treatment of cultured cells with anti-thymocyte serum plus complement. In vitro generation of Ag-V-PFC seemed to be highly cross-reactive (about 40%) with a related antigen (horse red blood cells). Ag-V-PFC detected in the present experiment may not represent helper T cells, effector T cells, or their precursors because of the following: (a) The generation of Ag-V-PFC was completely suppressed by the addition of anti-SRBC mouse serum in the culture, though the helper activity was apparently augmented by the same treatment. (b) Development of Ag-V-PFC was almost completely suppressed by the pretreatment of mice with cyclophosphamide 2 days before immunization, by which delayed-type hypersensitivity (DTH) was markedly augmented. (c) After the immunization of mice, Ag-V-PFC began to develop just when the level of DTH declined, at which point helper activity of the spleen cells also diminished. A possible role of Ag-V-PFC in the immune response was discussed.

Shortly after intravenous immunization of mice with heterologous erythrocytes (RBC) antigen-specific Thy 1+ cells which form rosettes with the immunizing RBC (thymic-derived lymphocytes-forming rosettes [T-RFC]) appear in the spleen. These T-RFC are much less stable than Thy 1- RFC (non-thymic-derived [B-RFC]) although most if not all of both classes of RFC adhere to nylon. T-RFC are induced with low doses of antigen (which fail to induce B-RFC) and are inhibited by higher antigen doses which are optimal for induction of B-RFC. Pretreatment of mice with cyclophosphamide prevents the high dose inhibition of T-RFC. Although there are many parallels between the production of T-RFC and delayed-type hypersensitivity (DTH) it is unlikely that the T-RFC are essential for DTH reactions since DTH can be transferred with cells which pass through nylon, and such cells are almost totally depleted of T-RFC. Thus immunization can lead to the production of large numbers of antigen-specific T-RFC whose functional role in the immune response is unknown. However, the characteristics of the T-RFC suggest that they may play an important role in amplification of suppressor cell activity.

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4 degrees C followed by washing and incubation at 37 degrees C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti-H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition.

The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5-hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice. We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell. Spleen cells of immune, reserpine-treated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice. We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines. If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate. The secondarily arriving vasoactive amine-dependent cells are responsible for the micro- and macroscopic lesions of DTH reactions. Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called.

We have investigated the possibility of oral administration of ovalbumin (OVA) to prevent a secondary antibody response and interrupt reaginic antibody production. Repeated feeding seems necessary for both. Quality of results was dependent on the number of ingestions. Differences in abrogation of antibody response between mice strains were observed. Best results were obtained with AKR mice, good suppression was seen in C3H strain, and inconsistent results were obtained with DBA/2. 10 OVA oral doses were necessary to prevent a secondary antibody response in parenterally immunized mice, but 4 doses interrupted reaginic production in sensitized AKR mice. These results demonstrate that antigen feeding can prevent a secondary antibody response and interrupt reaginic antibody production.

Interest in the Ir genes of rheus monkeys stems from their phylogenetic relationship to man and the extensive data already available on the major histocompatibility complex of the monkey. At least two independent dominant H-linked Ir genes have been identified in the rhesus. These genes control the ability of monkeys to respond to the random linear copolymer of glutamyl alanine (GA), or the dinitrophenyl conjugate of glutamyl lysine (DNP-GL). These synthetic polymers can elicit weak delayed-type skin reactions and strong humoral responses in some monkeys. In a series of unrelated monkeys phenotyped for the serologically defined RhL-A specificities of both segregant series, there were no correlations between any RhL-A specificity and responder status to the GA or DNP-GL polymers. However, segregation analysis of 21 rhesus families sired by 3 fathers indicated the capacity of the offspring to form antibodies was associated with genes coded for in the RhL-A complex. In three monkeys, verified recombination within the RhL-A complex between the genes coding for the serologically defined determinants (SD loci) and the gene(s) controlling the lymphocyte-activating determinants (Lad loci) responsible for mixed lymphocyte reactivity was established. In two of these monkeys the immune response genes controlling the DNP-GL response segregated with the Lad genes, while in the third case the Ir-GL gene segregated with the SD loci, tentatively localizing the Ir-GL gene between the SD and Lad loci. In addition, we have shown that genetically distinct genes control responsiveness to DNP-GL and GA. These genes were separated by recombination, thus one monkey inherited the Lad, Ir-GL, and SD loci from one paternal haplotype and by crossing over inherited the gene controlling GA responsiveness from the other paternal haplotype. The fine structure mapping of the RhL-A gene complex is compared with the H-2 and HL-A gene complexes. Several striking similarities were noted.

Spleen cells educated in vitro with sheep red blood cells (SRBC) suppressed the plaque-forming cell response of Mishell-Dutton assay cultures challenged with optimal doses of SRBC. Changing conditions in the assay cultures changed the effect educated cells had on the assay culture responses. For example, educated cells helped rather than suppressed assay cultures of suboptimal numbers of spleen cells. Similarly, augmentation resulted upon addition of educated cells to assay cultures challenged with suboptimal doses of SRBC. Such a reversal of regulatory effects was not observed when assay cultures were challenged with supraoptimal antigen doses. Educated cells helped assay cultures of B spleen cells, and the addition of normal T cells reinstated suppression. Furthermore, maintenance of assay cultures under stationary rather than the usual rocking conditions allowed educated cells to help rather than suppress the antibody response of assay cultures. These results show that when the response of the target population (assay cultures) is low, the regulator (educated) cells augment the response, and vice versa, supporting the hypothesis that the effect regulator cells produce depends on the activity of the cells they regulate.

When thoracic duct lymphocytes (TDL) from mice given sheep erythrocytes (SRC) 1 day previously (1-day-primed TDL) were placed in microcultures with antigen for 5-6 days, the cells failed to produce antibody to SRC, but responded well to horse erythrocytes (HRC); reciprocal results were obtained with TDL from HRC-injected mice. The specific unresponsiveness of the TDL was observed despite the presence of an allogeneic factor in the cultures; this factor exerted not only a macrophage-like effect (TDL from normal mice failed to respond in the absence of this factor) but also a T-cell-replacing function. It was concluded therefore that the unresponsiveness of the TDL was the result of selective recruitment of specific B lymphocyte precursors from the recirculating lymphocyte pool to the lymphoid tissues; whether there was also recruitment of specific T lymphocytes was not investigated. Since addition of 1-day-primed TDL to cultures of normal TDL did not inhibit the response of the latter, there was no evidence that active suppression accounted for the unresponsiveness. The spleen appeared to be the main site for recruitment since 1-day-primed spleen cells placed in microcultures contained above normal numbers of specifically-reactive precursors. This was in striking contrast to the effect of transferring 1-day-primed spleen cells in vivo where, as previously reported, the cells fail to respond to the injected antigen within 1 wk of transfer. An explanation for this paradox is discussed.

We evaluated the effect of antigen feeding on the subsequent primary and secondary anti-ovalbumin (OVA) responses of C57BL/6 and NZB mice. When C57BL/6 mice were given a single 20-mg dose of OVA intragastrically, profound tolerance was observed after challenge, 7 d later, with 125 micrograms of OVA in complete adjuvant or after two injections of 5 micrograms of OVA adsorbed to alum given 7 and 21 d after antigen feeding. OVA-fed NZB mice failed to become tolerant to a primary challenge with OVA in complete adjuvant, but showed a degree of tolerance similar to that of C57BL/6 mice when challenged two or three times with OVA in alum. These studies demonstrate that NZB mice fail to show tolerance at the level of the primary response after antigen feeding; however, they are normally tolerant when a secondary response to a lower dose of antigen is evaluated. This study suggests that, after antigen feeding, different mechanisms of tolerance may be involved in the regulation of primary and secondary responses.

Specific immunological unresponsiveness was induced using thymus-dependent antigens in congenitally athymic (nu/nu) mice, in which no T-cell function has been demonstrated. The tolerance was induced in vivo by the injection of 5-10 mg of either FGG or DNP-HGG. Spleen cells from treated mice were tested in vitro for the ability to mount thymus-independent immune responses against FGG in the presence of polymerized flagellin POL, and the DNP determinant conjugated to POL. A specific deficiency in either the in vitro anti-FGG or anti-DNP response was demonstrated, depending on the antigen used for treatment of the spleen cell donor. Athymic mice treated with FGG were also tested by in vivo challenge with FGG given with POL as an adjuvant and were found to be hyporesponsive. Unresponsiveness to in vitro challenge was established by 24 h after the in vivo injection of FGG. It was found that the injection of POL with the FGG prevented the development of unresponsiveness, but not if the POL was given 24 h or more after the FGG. The unresponsiveness could not be overcome by confrontation with allogeneic spleen cells from CBA mice, although the presence of allogeneic spleen cells had a large amplifying effect on the response of control spleen cells. These experiments demonstrate a mechanism for the tolerization of bone marrow-derived cells by thymus-dependent antigens in the absence of the thymus.

Previous studies have demonstrated that the B cells in immune and nonimmune mice manifest different major histocompatibility complex (MHC) collaborative phenotypes with antigen-specific T cells. Immune, or secondary B cells require syngeneic-like MHC recognition by collaborating T cells, and in its absence fail to be stimulated. Primary B cells manifest a much less stringent requisite for MHC recognition by T cells, and under conditions in which secondary B cells fail to be stimulated, primary B cells are stimulated to secrete IgM antibody. Experiments were conducted to determine whether the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference for IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference of IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was found to be dependent on the presence of T cells during in vivo immunization. Thus, the restriction imposed on T cell-B-cell-collaborative interactions in secondary humoral immune responses appears to be the result of T dependent antigen-driven events.

Using monoclonal antibody affinity chromatography, we isolated a 48,000 mol wt, glucose-rich glycoprotein (3M-1) from collagenase-solubilized rabbit renal tubular basement membrane (SRTA). The purified 3M-1 protein is noncollagenous, and is capable of inducing anti-TBM (tubular basement membrane) antibodies and interstitial nephritis in susceptible hosts. Further, when SRTA, at a normally nephritogenic dose, was selectively depleted of 3M-1, it lost its ability to induce disease. As shown by immunofluorescent techniques, 3M-1 appears to be localized on rodent TBM to the exclusion of the glomerular basement membrane, but was lacking in the TBM of the LEW rat, a strain devoid of the relevant antigen of anti-TBM disease. Immunoelectron microscopy revealed that 3M-1 was associated with the most lateral aspect of the TBM, which borders, and lies in the interstitium. These results indicate that 3M-1 is the nephritogenic antigen producing experimental anti-TBM disease.

The blood flow to individual lymph nodes of sheep and rabbits has been determined with 85Sr-labeled microspheres. A popliteal node of the sheep received 0.014% of the cardiac output and a comparable node in the rabbit 0.011%. A sheep lymph node weighing 1 g received an average of 24 ml/h of blood. It was calculated that there was a highly selective removal of lymphocytes by the node and that an equivalent to one in every four lymphocytes that entered a normal lymph node migrated out of the blood, through the substance of the node, and into the efferent lymph. During the immune response to either allogeneic lymphocytes or tuberculin, the blood flow to sheep lymph nodes, even without considering the increase in node weight, increased an average of fourfold. During the primary immune response in the rabbit to keyhole limpet hemocyanin, the blood flow increased threefold. The increase in blood flow preceded the antigen-induced increase in lymphocyte traffic recorded in the efferent lymph. The early phase of increased blood flow was considered to be due to hyperemia, whereas the latter phase had a significant angiogenesis component. It was calculated that an equivalent to 60% of the entire mobilizable pool of lymphocytes could pass through an average lymph node in the blood during an immune response lasting 5 days.

The mechanisms underlying the X-linked thymus-independent (B) lymphocyte functional defect in the CBA/N (CN) mice and their F1 progeny were studied. Immune defective mice were unable to respond to the T-independent antigen 2,4-dinitrophenyl-lysyl-derivative of Ficoll (DNP-lys-Ficoll) but were able to form antibody against the highly cross-reactive hapten (trinitrophenyl) when it was coupled to an erythrocyte carrier. Immune defective CN X DBA/2N (DN) F1 male mice, which do not normally respond to T-independent antigens, were able to respond to both polyribosinic-polyribocytidylic acid and DNP-lys-Ficoll after the administration of CN X DN F1 female spleen cells even if these cells had been depleted of T lymphocytes. In addition, it was shown that the inability of the CN mice and their F1 progeny to respond to T-independent antigens was not due to an intrinsic abnormality of their microenvironment or the suppressive actions of a T lymphocyte. Our data present evidence that the X-linked defect in the CN mice is due to an intrinsic defect in B-lymphocyte development.

The mechanism of selective anti-hapten IgE antibody production was studied in SJL mice. Using an adoptive transfer method of spleen cells into syngeneic recipients irradiated with a sublethal dose of 600 rads, it was demonstrated that for the suppression of anti-dinitrophenyl (DNP) IgE antibody production the interaction of two subsets of T cells is necessary. DNP-primed B cells and carrier-primed T helper cells are taken from donors primed with small amounts of DNP-carrier conjugates. Without injection of other cells, high titer and persistent anti-DNP antibodies are produced in the recipients. The two subsets of T cells that are active in suppression of IgE are taken from two types of donors: one donor is immunized (hyperprimed) with larger amounts of carrier protein twice, the other is an unprimed donor. The carrier for hyperpriming the first type of donor may be unrelated to the carrier used for priming the helper T cells. To bring about anti-DNP IgE suppression it is necessary that the animals should be challenged with the same DNP-carrier conjugate used for priming the B and T helper cells. If the hyperprimed donors were immunized with a heterologous, unrelated carrier, then this heterologous unconjugated carrier must also be injected together with the homologous DNP-carrier conjugate. In these conditions, anti-DNP IgE antibody production is suppressed, but the production of anti-DNP IgG1 antibody is not diminished.