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Recently, viral infectious diseases, including COVID-19 and Influenza, are the subjects of major concerns worldwide. One strategy for addressing these concerns focuses on nasal vaccines, which have great potential for achieving successful immunization via safe, easy, and affordable approaches. However, conventional nasal vaccines have major limitations resulting from fast removal when pass through nasal mucosa and mucociliary clearance hindering their effectiveness. Herein a nanoparticulate vaccine (NanoVac) exhibiting photochemical immunomodulation and constituting a new self-assembled immunization system of a photoactivatable polymeric adjuvant with influenza virus hemagglutinin for efficient nasal delivery and antigen-specific immunity against pathogenic influenza viruses is described. NanoVac increases the residence period of antigens and further enhances by spatiotemporal photochemical modulation in the nasal cavity. As a consequence, photochemical immunomodulation of NanoVacs successfully induces humoral and cellular immune responses followed by stimulation of mature dendritic cells, plasma cells, memory B cells, and CD4 + and CD8 + T cells, resulting in secretion of antigen-specific immunoglobulins, cytokines, and CD8 + T cells. Notably, challenge with influenza virus after nasal immunization with NanoVacs demonstrates robust prevention of viral infection. Thus, this newly designed vaccine system can serve as a promising strategy for developing vaccines that are active against current hazardous pathogen outbreaks and pandemics., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The role of Th17 cells in the pathogenesis of autoantibody-mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR-specific CD4(+) T cells is producing IL-17. IL-17(ko) mice developed fewer or no EAMG symptoms, although the frequencies of tAChR-specific CD4(+) T cells secreting IL-2, IFN-γ, or IL-21, and the percentage of FoxP3(+) Treg cells were similar to WT mice. Even though the total anti-tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL-17(ko) as compared to WT mice. Most importantly, pathogenic anti-murine AChR antibodies were significantly lower in IL-17(ko) mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR β/δ(ko) mice with WT or IL-17(ko) CD4(+) T cells. In conclusion, we show that the level of IgG2b and the loss of B-cell tolerance, which results in pathogenic anti-murine AChR-specific antibodies, are dependent on IL-17 production by CD4(+) T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody-mediated autoimmunity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein-protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development of mAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 μg of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available for monoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the high-throughput monoclonal antibody process providing a vast array of high quality antigens at very low cost., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IL-33 is associated with atopic and autoimmune diseases and, as reported here, it interacts synergistically with Ag to markedly enhance production of inflammatory cytokines in rodent mast cells even in the absence of degranulation. Investigation of the underlying mechanisms revealed that synergy in signaling occurred at the level of TGF-β-activated kinase 1, which was then transmitted downstream through JNK, p38 MAP kinase, and AP-1. Stimulation of the Ca(2+) /calcineurin/NFAT pathway by Ag, which IL-33 did not, was critical for the synergy between Ag and IL-33. For example, selective stimulation of the NFAT pathway by thapsigargin also markedly enhanced responses to IL-33 in a calcineurin-dependent manner. As indicated by luciferase-reporter assays, IL-33 failed to stimulate the transcriptional activities of NFAT and AP-1 but augmented the activation of these transcription factors by Ag or thapsigargin. Robust stimulation of NF-κB transcriptional activity by IL-33 was also essential for the synergy. These and pharmacologic data suggested that the enhanced production of cytokines resulted in part from amplification of the activation of AP-1 and NFAT as well as co-operative interactions among transcription factors. IL-33 may retune mast cell responses to Ag toward enhanced cytokine production and thus determine the symptoms and severity of Ag-dependent allergic and autoimmune diseases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many pathogens are initially encountered in the gut, where the decision is made to mount an immune response or induce tolerance. The mesentric lymph node (mLN) has been shown to be involved in immune response and much more in oral tolerance induction. Furthermore, using an in vivo transplantation model, we showed recently that lymph node (LN) stromal cells can affect T-cell function and influence the IgA response by supporting a site-specific environment. To elucidate the importance of LN stromal cells for tolerance induction, mLN or peripheral LN were transplanted into mice (mLNtx or pLNtx) and oral tolerance was induced via ovalbumin. A reduced delayed-type hypersensitivity (DTH) response was detected in pLNtx compared to mLNtx mice. Reduced IL-10 expression, reduced percentages of Tregs, and increased proportions of B cells were identified within the pLNtx. The increase of B cells resulted in a specific immunoglobulin production undetectable in mLNtx. Moreover, transferred IgG(+) cells of tolerized peripheral LN induced a strong reduction of the delayed-type hypersensitivity response, whereas CD4(+) cells were less efficient. Thus, stromal cells have a high impact on creating a unique environment. Furthermore, the environment of pLNtx induces a tolerogenic phenotype by B-cell accumulation and antibody production., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide-2, is a potent adjuvant for cross-priming against cellular antigens. Systemic administration of BPP-induced maturation of CD8alpha+ DC and CD8alpha- DC in the spleen and resulted in enhanced cross-presentation of intravenously co-administered antigen in mice. In addition, administration of BPP and cell-associated OVA generated an effective CTL response against OVA in vivo in a CD4+ T helper cell-dependent manner, but independent of IFN-alpha. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut-down of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross-priming.

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.

CD155, originally known as the cellular receptor for poliovirus, is the founding member of a subfamily of immunoglobulin-like adhesion receptors. Apart from its function in establishing adherens junctions between contacting epithelial cells, the engagement of CD155 with two recently identified ligands, CD226 and CD96, mediates immunologically relevant processes such as NK cell-driven killing of tumor cells in humans. Here we report on the generation and immunological analysis of mice constitutively deficient of CD155. Moreover, the expression profile of CD155 on hematopoietic cells has been determined using newly established antibodies. CD155-deficient mice develop normally without displaying an overt phenotype. However, the animals are distinguished by distinct deficits in the development of a regular humoral immune response. Whereas systemic challenges revealed no differences, orally administered antigen evoked less efficient IgG and IgA antibody responses despite of normal IgM titers when compared to wild-type mice. Therefore, CD155 may assist in an efficient humoral immune response generated within the intestinal immune system.

In the spleen, exogenous antigen is preferentially presented by CD8alpha+CD11b- DC to CD8 T cells and by CD8alpha-CD11b+ DC to CD4 T cells. However, it is not yet clear whether the same rule applies to other secondary lymphoid organs. To address this issue, we first classified secondary lymphoid tissues into three categories based on the expression pattern of CD8alpha and CD11b in C57BL/6 and BALB/c mice: (a) spleen, (b) mesenteric lymph node (MLN) and (c) other peripheral lymph nodes (PLN). We then analyzed the OVA-specific T cell-stimulating capacity of each DC subset after intravenous injection with soluble OVA. Our results show that, regardless of tissue origin, CD8alpha-CD11b+ DC generally present OVA to CD4 T cells, a finding that held true as well for CD8alpha+CD11b+ DC in PLN. In striking contrast, CD8alpha+CD11b- DC in spleen, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN mainly cross-present OVA to CD8 T cells in their respective tissues. Of note, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN present OVA to both CD4 T and CD8 T cells. Therefore, the antigen-presenting capacity of each distinct DC subset is determined by its anatomic environment in combination with its surface phenotype.

Epicutaneous sensitization with a protein antigen was demonstrated to induce a predominant type 2 CD4 T cell response with high IgE production in mice. On the other hand, its CD8 T cell responses have not been addressed probably partly because of the generally accepted concept that cross-priming of soluble protein is an inefficient process. Here, we used an established patch-applied murine model to demonstrate that cross-priming with an epicutaneously introduced soluble protein antigen, though inefficient, generated mainly Tc1 cells, but not Tc2 cells. In the presence of an irritant or hapten, the efficiency of this cross-priming process could be enhanced and more Tc1 cells were generated. CpG oligonucleotides also promote the generation of Tc1 cells. In contrast, lipopolysaccharide and poly (inosinic-cytidylic) acid [poly (I:C)] have no effect. Together, these results provide supportive evidence of the epicutaneous sensitization of human cutaneous lymphocyte-associated antigen-positive CD8 T cells found in the peripheral blood or tissues of patients. The surprising observation of the type 1 character of the generated CD8 T cells will also help us to better understand the complicated pathogenesis of atopic and cutaneous inflammatory diseases.

Based on our previous observation that anergic T lymphocytes lose their migratory ability in vitro, we have proposed that anergic T cells are retained in the site where they have been generated to exert their regulatory function. In this study we have analyzed T lymphocyte trafficking and motility following the induction of tolerance in vivo. In a model of non-deletional negative vaccination to xenoantigens in which dendritic cells (DC) localize to specific lymphoid sites depending on the route of administration, tolerant T cells remained localized in the lymph nodes colonized by tolerogenic DC, while primed T cells could traffic efficiently. Using an oral tolerance model that enables the 'tracking' of ovalbumin-specific TCR-transgenic T cells, we confirmed that T cells lose the ability to migrate through syngeneic endothelial cell monolayers following tolerance induction in vivo. Finally, we show that tolerant T cells (both in vitro and ex vivo) can inhibit migration of responsive T cells in an antigen-independent manner. Thus, hyporesponsive T cells localize at the site of tolerance induction in vivo, where they exert their anti-inflammatory properties. In physiological terms, this effect is likely to render immunoregulation a more efficient and controllable event.

We employed an optimized ex vivo enzyme-linked immunospot assay for enumerating and defining the peptide specificity of all the hen egg lysozyme (HEL)-specific Th cells producing IL-2, IFN-gamma, or IL-4, in different lymphoid organs of HEL-immunized BALB/c and CBA mice. Previous studies, employing T cell proliferation assays, demonstrated that lymph node cells from BALB/c mice immunized with HEL emulsified in complete Freund's adjuvant (CFA) are specific for HEL(105-120). In contrast, we found that the spleens of BALB/c mice immunized with HEL/CFA, or with heat-aggregated HEL on aluminum hydroxide adjuvant, contain IL-4-producing T cells specific for other HEL peptides, previously characterized as "cryptic", with consistent responses to HEL(11-25). The Th repertoire expressed in different lymphoid organs of the same immunized mouse can be different, as can the repertoire of Th cells producing different cytokines and present in one lymphoid organ. In addition, we found that the repertoire of Th cells generated depends upon the adjuvant employed. Lastly, the summation of responses elicited by a panel of non-overlapping HEL peptides is equal to that elicited by HEL. This high-resolution study thus illustrates that the Th repertoire generated upon HEL immunization depends upon diverse parameters, and that the natural processing of HEL gives rise to more diverse peptides then previously evident from studies employing T cell proliferation assays.

Infant food allergies are increasing, and many breast-fed infants now sensitize to maternally-ingested antigens. As low-dose oral tolerance requires generation of suppressor lymphocytes producing TGF-beta1 (Th3 cells), we studied these cells in duodenal biopsies after diagnostic endoscopy. Spontaneous production of Th1, Th2 and Th3 cytokines by duodenal lymphocytes was studied using flow cytometry in 20 children with no eventual clinico-pathological diagnosis (controls), 30 children with multiple food allergy, nine with celiac disease and six with inflammatory enteropathies. Immunohistochemistry and in situ hybridization were used to localize TGF-beta1 protein and mRNA in matched biopsies. We found no significant Th1/Th2 skewing amongst mucosal lymphocytes in allergic children compared to controls, although celiac and inflammatory enteropathy patients showed increased Th1 responses. By contrast, the allergic children showed reduction of TGF-beta1(+) lymphocytes in both epithelial and lamina propria compartments. Reduction of TGF-beta1 expression was also seen in mononuclear cells and epithelium in food allergy by immunohistochemistry and in situ hybridization. The dominant mucosal abnormality in food allergic children was, thus, not Th2 deviation but impaired generation of Th3 cells. As generation of these cells requires innate immune response to enteric bacteria, we suggest that changing infectious exposures may inhibit primary establishment of basic oral tolerance mechanisms.

We used 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester labeled TCR-transgenic CD4(+) T cells to investigate the contribution of B7 costimulation to T cell activation and clonal expansion. B7 costimulation was blocked with the fusion protein cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Ig, which prevents the interaction of B7 with its receptor CD28 on T cells. CTLA4-Ig had different effects depending on the density of antigen (Ag)/MHC ligands available by T cells. In the presence of CTLA4-Ig, tenfold higher concentrations of Ag were required for T cells to undergo cell division in vitro. At high Ag concentrations, T cell division occurred at comparable rates whether in the presence or absence of CTLA4-Ig; however, T cell survival and clonal expansion were strongly inhibited. Addition of IL-2 restored T cell survival but not responsiveness to low doses of Ag. In vivo, B7 costimulation was similarly required for the survival of Ag-specific T cells but not for cell division in response to high amounts of Ag. Thus, B7 costimulation regulates CD4(+) T cell responses by promoting cell division in the presence of limiting amounts of Ag, and by protecting T cells from the onset of apoptosis.

This study addresses the relative importance of CD134 (OX40) and CD137 (4-1BB) in the costimulation of CD4+ and CD8+ T cells under comparable conditions of antigenic stimulation. We demonstrate that CD134 is capable of directly costimulating CD8+ T cells. However, costimulation of CD8+ T cells by CD134 is less potent than that triggered by CD137. The higher costimulatory activity of CD137, when compared with CD134, correlates well with its faster expression kinetics and higher levels on CD8+ T cells. Furthermore, induction of CD137 expression on CD8+ T cells is highly sensitive to low levels of TCR stimulation, which is in contrast with CD134. Conversely, CD134 is more effective than CD137 in costimulating CD4+ T cells. This, however, could not be attributed to differential expression. We also demonstrate that the transient nature of CD134 and CD137 expression on activated CD4+ T cells is the resultof proteolytic shedding. Consistent with the greater ability of CD137 to costimulate CD8+ T cells, stimulation of CD137 in vivo is considerably more effective than CD134 in augmenting anti-tumor immune responses. Therefore, agents that stimulate signaling via CD137 are likely to be more useful in clinical conditions where highly effective CD8+ CTL responses are required.

Allergic diseases are associated with excessive type 2 immunity, yet the endogenous controls - if any - responsible for limiting induction of such responses remain unclear. Using IL-12-deficient mice, we recently showed that endogenous IL-12 is not essential for preventing development of allergic responses. Here, we examine the roles of endogenous IFN-gamma and IL-18 production in limiting the occurrence and severity of type 2 immunity. Lack of endogenous IFN-gamma synthesis results in significantly higher type 2 cytokine (IL-4, IL-5, IL-10 and IL-13) and chemokine (TARC) production following exogenous antigen exposure. Thus, IFN-gamma, but not IL-12, is a key negative regulator of the type 2 immune response. IL-18, in addition to its established role as an inducer of IFN-gamma, also negatively regulates CD4(+) T cell-derived IL-4 synthesis via an IFN-gamma-independent mechanism, thereby further limiting the development of type 2 immune responses. Together, these results identify endogenous IFN-gamma and IL-18 as potent, independent, negative regulators of the development of type 2 immunity to ubiquitous environmental antigens.

Surface expression of IgM is a characteristic feature of the development of most B cells. Only pre-B cells bearing functional IgM heavy chains mu chains) are selected for clonal expansion and differentiation. Cells lacking mu chains are normally eliminated. muMT mice carrying a deletion of the first exon coding for the transmembrane domain of the immunoglobulin mu chain gene were described as mice deficient for mature B cells, plasma cells and immunoglobulins in serum. In this study, we describe in muMT/BALB/c mice the presence of a novel B cell population, producing IgG, IgA and IgE in the absence of IgM membrane expression. Moreover, this small population of B cells is able to recognize antigens and to differentiate into plasma cells. These "non-conventional" mu(- / -) B cells produce functional immunoglobulins after immunization, undergo germinal center reactions, and maintain B cell memory. Our findings support the concept, that a small percentage of mu -non-expressing pre-B cells can escape elimination, switch to downstream immunoglobulin heavy chains and respond to antigens. It remains an open question how the reactivity of these B cells is regulated and in which extent such B cells play a role in physiological and pathological processes such as autoantibody production and autoimmunity.

Naive CD4(+) T cell populations rapidly produce small amounts of IL-4 in response to T cell receptor-mediated stimulation and may undergo Th2 differentiation without exogenous IL-4. Whether this is due to autocrine IL-4-stimulation or the production of IL-4 by an infrequent naive cell has not been determined. Here we show that single CD4(+) T cells from RAG2-/- T cells receptor transgenic mice primed with their cognate antigen give rise to IL-4-producing cells at a similar frequency whether primed with or without added IL-4, but not if anti-IL-4 is added to the culture. Thus, each founder cell or one or more of its early daughters can produce sufficient IL-4 to drive Th2 differentiation. This indicates that autocrine IL-4 production by naive CD4 T cells can drive the appearance of Th2 cells.

We have previously demonstrated the loss of oral tolerance (OT) in lymphotoxinalpha-/- (LTalpha-/-) and TNFalpha / lymphotoxinalpha deficient (TNFalpha / LTalpha-/-) mice which have defective Peyer's patches (PP) and lymph node (LN) development. We have now studied OT in BALB / c mice with differential defects of the gut-associated lymphoid tissue (GALT) caused by inhibition of LTbetaR signaling during fetal development. Treatment of pregnant mice with LTbetaR-IgG (LTbetaRIgG) and TNFR I55-IgG (TNFR55IgG) abrogates the formation of PP (LTbetaRIgG) or of PP and mesenteric LN (MLN) (LTbetaRIgG / TNFRIgG) without genetically deleting the respective cytokine pathways. OT was readily induced in mice without PP but retaining MLN (PP null / LN +). In contrast, OTcould not be induced in mice lacking both MLN and PP (PP null / MLN null) as shown by the inability of these mice to suppress IFN-gamma secretion or DTH reactions. We next assessed OT in 129 x B6 LTalpha-/- mice with and without MLN. Timed treatment of pregnant LTalpha-/- mice with an agonist anti-LTbetaR mAb induces formation of MLN but not of PP in LTalpha-/- mice. LN + LTalpha-/- mice developed OT while LN LTalpha-/- mice were resistant to OT induction. Taken collectively, the data show that in the presence of MLN PP are not required for OT induction and that the presence of MLN is sufficient for OT induction in the LTalpha-/- model.

Immunization with cells expressing endogenous antigens can stimulate long-lived CD8(+) T cell memory. In many cases, the response is also stimulated by host antigen-presenting cells (APC) that have processed antigen from internalized apoptotic cells or cell fragments. This study investigated whether immunization with peptide-pulsed dendritic cells (DC) could prime long-lasting, peptide-specific CD8(+) T cell immunity in the absence of cross-priming by host APC. C57BL / 6 female mice immunized with syngeneic male splenic DC pulsed with the H-2K(b)-restricted ovalbumin peptide OVA(257 - 264) made memory CD8(+) CD44(high) T cell responses to OVA(257 - 264) and the male antigen HY more than 1 year after immunization. Establishment and maintenance of peptide-specific CD8(+) T cell memory did not require antibody or B cells. Immunization of H-2(bxd) mice with OVA(257 - 264)-pulsed minor-incompatible H-2(b) or H-2(d) DC demonstrated that CD8(+) T cells were primed exclusively by the injected cells, and not by peptide transferred to host APC, even though there was very effective cross-priming for CD8(+) T cell responses to the minor antigens expressed by the DC. Thus peptide-pulsed DC can prime long-lasting CD8(+) memory responses without any requirement for cross-priming by other APC.

Exogenous soluble proteins enter the endosomal pathway by endocytosis and are presented in association with MHC class II rather than class I. In contrast, the delivery of exogenous protein antigens (Ag) into the cytosol generates MHC class I-restricted cytotoxic T lymphocytes (CTL) responses. Although several immunization approaches, such as the utilization of liposomes, have induced the in vivo priming of MHC class I-restricted CTL responses to protein Ag, it remains unclear whether this priming results from the direct delivery of protein Ag to the cytosol. Here we report that fusogenic liposomes (FL), which are prepared by fusing simple liposomes with Sendai virus particles, can deliver the encapsulated soluble protein directly into the cytosol of cells cultured concurrently and introduce it into the conventional MHC class I Ag presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FL but not in simple liposomes results in the potent priming of OVA-specific CTL. Thus, FL function as an efficient tool for the delivery of CTL vaccines.

Delivery of foreign antigens to mucosal surfaces, such as the pulmonary airways, has been shown to preferentially induce Th2-mediated responses in humans and in mice. What is not clear from these studies is whether this preferential skewing in responses is the result of the limited types of antigen being administered and/or a bias towards using particular genetic strains of mice, or whether the lung environment in itself provides a favored site for the priming of Th2-type cells. We have addressed this issue using an antigen/mouse strain combination that, under typical conditions of immunization, is strongly biased towards priming for Th1 CD4+ T cells. We show that Leishmania major parasites delivered to C57BL/6 mice via an intranasal route fail to induce the expected Th1-dominated responses and instead preferentially prime for Th2 responses. These included an influx in lymphocytes and eosinophils into alveoli, as well as the induction of Th2-type foci of inflammation around pulmonary blood vessels and airways. Moreover, high levels of Th2-associated cytokines (IL-4 and IL-5) were generated when lung-draining lymph node and tissue cells were restimulated with L. major lysate. These data suggest that the lung environment per se favors Th differentiation towards the Th2 phenotype.

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.

We have studied the kinetics of the antigen induced response of naive and memory CD8 T cells expressing a transgenic T cell receptor (TCR) specific for the glycoprotein peptide amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV). Memory T cells were generated in vivo by adoptive transfer of LCMV TCR transgenic T cells into normal recipient mice, followed by LCMV infection. The results demonstrated that the cell cycle progression and kinetics of TCR down-modulation, CD25 and CD69 up-regulation were identical in naive and memory T cells after antigen recognition. Moreover, the two T cell populations did not differ in respect of activation thresholds and in their proliferative capacities neither in vitro nor in vivo. However, memory CD8 T cells could be more rapidly induced to become cytolytic and to secrete high levels of interleukin-2 and interferon-gamma than naive T cells. LCMV GP33-specific CD8 memory T cells were only slightly more efficient in reducing LCMV titers in the spleen but were far more effective than naive LCMV GP33-specific T cells in controlling subcutaneous tumor growth of B16.F10 melanoma cells which expressed the LCMV GP33 epitope as tumor-associated antigen. Thus, in our experiments the main difference between CD8 memory T cells and naive cells is the ability of the former to rapidly acquire effector cell functions.

The eye is endowed with a number of mechanisms that protect it from immune-mediated injury. One such mechanism, termed anterior chamber-associated immune deviation (ACAID), evokes the antigen-specific, systemic down-regulation of Th1 responses to antigen inoculated into the anterior chamber of the eye. ACAID has been correlated with the selective production of IL-10 by the antigen-presenting cells (APC) and the development of a cross-regulatory Th2-like response. A small subset of antigens do not induce ACAID, but instead provoke IL-12 and normal Th1 immunity. Remarkably, all soluble antigens tested are capable of inducing ACAID; only cell-associated antigens do not induce ACAID. We hypothesized that the nature of antigen plays a decisive role in the resultant immune response. This hypothesis was tested with two well-characterized antigens, ovalbumin (OVA) and SV40 large T antigen (SV40 Lg T Ag). The soluble forms of OVA and SV40 Lg T Ag induced ACAID in both in vivo and in vitro models of the eye. In contrast, the particulate forms of these antigens, i.e. OVA passively absorbed onto inert latex beads (OVA-latex) and SV40 Lg T Ag expressed in two different cell lines, 99E1 and SV-T2, did not induce ACAID in either in vivo or in vitro models of the eye. In addition, the cytokine profiles of ocular APC pulsed with OVA or OVA-latex showed that soluble OVA induced the production of IL-10, whereas OVA-latex induced the production of IL-12. These data suggest that the nature of the antigen in the eye, whether soluble or particulate, is a crucial determinant in the resultant immune response. Moreover, they suggest a mechanism in which soluble antigens preferentially induce the release of ACAID-inducing IL-10 whereas particulate antigens preferentially induce the release of Th1-inducing IL-12 by responding APC.

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-gamma is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.

Stimulation of systemic antigen-specific IgE production plays an important role in the mediation of food allergy; however, the mechanism of IgE production against food antigens is not fully understood. The development of relevant animal models may help to elucidate the pathogenesis of food allergy. We here show that DBA/2 mice receiving a casein diet without any adjuvant produced high levels of IgE specific for casein, accompanied by predominant Th2-like responses in liver lymphocytes, mesenteric lymph node cells and spleen cells. This model of IgE production produced by feeding protein antigen as a constituent of the diet can be applied to investigate the mechanism of IgE production and to develop reagents for controlling food allergy.

Analysis of antigen-induced negative selection of thymocytes in T cell receptor (TCR)-transgenic mice is complicated by the presence of an antigen-responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide-specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4+CD8+ (DP) thymocyte subset. However, our work shows that even though mature CD8+ T cells are inefficiently selected in TAP1-deficient mice, there was a striking degree of peripheral expansion and activation of CD8+ peripheral T cells. Furthermore, when cells from TCR-transgenic mice were adoptively transferred, we found that deletion of nontransgenic DP thymocytes occurred in Thy-1-congenic and even more efficiently in TAP1-deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR-mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1-deficient TCR-transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody-mediated depletion of CD8+ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.

Direct cell/cell communication occurs through gap junctions (GJ). We mapped GJ expression in secondary lymphoid organs and found, for the first time, a high density of connexin43 (Cx43) GJ in follicular dendritic cells (FDC) in close association with lymphocytes (Krenacs T. and Rosendaal M., J. Histochem. Cytochem. 1995. 43: 1125-1137). In this work, we used a combination of ultrastructural, immunocytochemical, molecular methods, and functional dye transfer experiments to study which germinal center cells are involved in direct cell/ cell communication and how GJ expression is regulated during antigen responses. One week after injecting the footpad of mice with 50 micrograms lysozyme, Cx43 GJ were detected on elongated cells in the paracortex of their popliteal lymph nodes. Repeated challenge led to the formation of secondary follicles with enlarged FDC meshwork full of Cx43 GJ. This positive correlation may reflect an importance for GJ in the pattern formation of FDC and lymphoid follicles. In human tonsil, the density of GJ and FDC was highest in the light zone of germinal centers where the fate of B cells is thought to be decided. Cx43 colocalized with CD21 and CD35 antigens in the vicinity of desmosomal junctions on FDC embracing lymphocytes. Freeze-fracture hallmarks of GJ of 200-400 nm were also found on FDC in the vicinity of desmosomal plaques. Furthermore, Northern blot analysis showed the consistent presence of Cx43 mRNA in human tonsil and spleen. Most Cx43 message was localized in situ to cells with FDC morphology and some to a few germinal center lymphocytes. To investigate functional cell coupling, we set up FDC/B cell cultures from the low density cell fractions of human tonsils. Cx43 plaques associated with lymphocytes were detected both on elongated FDC processes in early cultures (up to 4 h) and in established FDC/B cell clusters (between 4 and 24 h). In early cultures, we injected FDC with Lucifer Yellow, a fluorescent dye which passes through GJ: the dye spread into adjacent FDC and occasionally from FDC into CD19+ B cells. Based on these results, we propose that direct cell/cell communication through Cx43 GJ is involved in FDC/FDC and in FDC/B cell interactions. The functionally coupled FDC meshwork may serve as a communication channel synchronizing germinal center events. FDC may also deliver crucial direct signals through GJ involved in the rescue of high-affinity B cell clones from apoptotic cell death.

Intravenous injection of antigen-coupled splenocytes has been widely used to induce specific tolerance to a variety of antigens. In this study, we investigated the effects of such a treatment on Th1 and Th2 antigen-specific immune responses. Using both well-characterized model antigens and crude homogenates from Leishmania major promastigotes, we found that intravenous injection of antigen-coupled splenocytes strongly down-regulated antigen-specific Th2 responses but had no or only moderate effects on Th1 responses. Because the susceptibility of inbred strains of mice to murine leishmaniasis has been found to be correlated with a strong Th2 response against parasite antigens, we investigated whether administration of splenocytes chemically coupled to parasite antigens could protect susceptible mice from murine leishmaniasis. We found that this was indeed the case and further demonstrated that protection was associated with a strong decrease in the number of parasite-specific Th2-like cells. Because administration of antigen-coupled splenocytes is believed to induce ligation of the T cell receptor complex without inducing a co-stimulatory signal, our results further suggest that priming of Th1 cells is less dependent on co-stimulatory signals than the priming of Th2 cells.

Oral administration of retinal soluble antigen (S-Ag) suppresses the induction of S-Ag-mediated experimental autoimmune uveitis (EAU) in Lewis rats. EAU induced with interphotoreceptor retinoid-binding protein (IRBP), another retinal autoantigen, can also be suppressed by oral administration of IRBP. It has been speculated that feeding with one retinal autoantigen could suppress induction of uveitis with the other retinal protein by means of bystander suppression. Both uveitogenic effector and suppressor cells should find their antigens within the retina, where the suppressor cells would be expected to act on the effector cells. However, reciprocal combinations of antigens used for induction and suppression of uveitis failed to prevent onset of disease, demonstrating that bystander suppression obviously does not occur in the eye. To investigate further the localization of suppressor mechanisms, we fed Lewis rats either with retinal S-Ag or with ovalbumin (OVA) and then immunized the animals either with a mixture of S-Ag and OVA or with each antigen separately, injected into contralateral hind legs. Feeding of S-Ag prior to immunization led to suppression of uveitis, whereas feeding of OVA had no tolerizing effect when S-Ag and OVA were injected into different legs. Nevertheless, immunizing rats with a mixture of S-Ag and OVA after OVA feeding suppressed uveitis to a high degree. These findings suggest that orally induced bystander suppression might not occur in the target organ, but rather peripherally at the site of induction of the autoimmune T cells.

Oral tolerance is a biologically relevant pathway for inducing peripheral tolerance to foreign antigens. The mechanisms responsible for the tolerant state following feeding with antigen have been shown to involve both anergy and suppression. The demonstration of anergic T lymphocytes following oral tolerance has so far been limited in in vitro systems, and a primary objective of the present study was to provide evidence, in vivo, for the existence of a state of anergy in mice orally fed with ovalbumin (OVA). In addition, it has been shown that peripheral anergy following the intravenous administration of antigen is selectively induced in Th1 lymphocytes. Thus, a second objective of this study was to investigate whether tolerance induced by a feeding regimen known to cause anergy could be selectively limited to Th1 lymphocytes, and whether tolerance induction could be explained by antigen absorption from the gut into the circulation. Oral tolerance was induced by a single feeding with OVA, and was demonstrated by diminished antibody production in vivo, and by reduced cytokine secretion or proliferation in vitro. Anergy, as a mechanism for tolerance, was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in recombinant interleukin-2 (rIL-2). Reversal of the tolerant state in vivo was established by antibody production in irradiated mice adoptively transferred with cells cultured in the presence of rIL-2. The possibility that suppression was also an in vivo mechanism for tolerance was studied by adoptive transfer experiments. Our results show: 1) that a single dose of orally administered OVA leads to the selective tolerization of Th1 responses (diminished IgG2a, IL-2 and interferon-gamma production) with intact Th2 responses (IgG1, and IL-4), 2) that tolerance in vivo is explained by anergy in the absence of active suppression, 3) that exposure of tolerant cells to rIL-2 in vitro abrogates the anergic state both in vitro (proliferation and cytokine secretion) and in vivo (IgG2a production), and 4) that the induction of oral tolerance is inhibited by the presence of antibodies specific for the tolerizing antigen. These findings indicate that the induction of anergy via the oral route might depend on the dissemination of antigen absorbed from the gut. It is suggested that tolerance is guaranteed by the fact that this absorbed antigen is presented to Th1 lymphocytes in the absence of inflammatory and co-stimulatory molecules; these foreign antigens are thus not different from self antigens.

The B cell immune response to 2,4-dinitrophenyl (DNP) keyhole limpet hemocyanin was compared in antigen-free, germ-free and conventional BALB/c mice. The numbers of total and of DNP-specific IgM-, IgG- and IgA-secreting cells in the spleen were determined by enzyme-linked immunosorbent plaque assays after primary, secondary and hyperimmunization. Three days after primary immunization a peak of DNP-specific IgM-secreting cells was seen in conventional mice only. However, this specific response was accompanied by a rise in the total number of IgM-secreting cells. At day 6 after primary immunization the total number and the frequency of DNP-specific IgG-secreting cells were higher in antigen-free mice, compared to germ-free and conventional mice. After secondary immunization in conventional mice only, a considerable bystander IgG response was seen together with the DNP-specific IgG response. At the end of the secondary response 90% of all IgG-secreting cells were DNP specific in antigen-free mice, while the corresponding figure in germ-free and conventional mice was 63% and 14%, respectively. After hyperimmunization, the absolute number of DNP-specific IgG-secreting cells in the spleen was 5-fold and 11-fold higher in antigen-free mice than in germ-free and conventional mice, respectively. Approximately 48% of all IgG-secreting cells were DNP specific in antigen-free mice, while the corresponding figure in germ-free and conventional mice was 17% and 12%, respectively. The results show that the exogenous antigenic load of animals influences the immune response to newly introduced antigens. The higher absolute and relative numbers of antigen-specific IgG-secreting cells after hyperimmunization in antigen-free mice compared to germ-free and conventional mice may provide a better source for antigen-specific B cells that eventually can be used for hybridoma production.

On the basis of expression of the T cell differentiation antigen CD27, human peripheral blood CD4+ memory cells can be divided into two subsets, a large CD45RA-CD27+ (82%) and a small CD45RA-CD27- (18%) population. Analysis of the functional properties of these memory T cell subsets showed that proliferative responses to the recall antigen tetanus toxoid (TT), shortly after booster immunization, were mainly confined to the CD27- population. Also, in atopic individuals, proliferative responses to allergens for which these individuals are sensitized, were limited to the CD45RA-CD27- population. After stimulation with CD3 monoclonal antibody and phorbol ester, CD27+ cells produced vast amounts of interleukin (IL)-2 but minimal amounts of IL-4, whereas in marked contrast, CD27- T cells secreted low levels of IL-2 and high levels of IL-4. The capacity of the vast majority of these latter cells to produce IL-4 was found to be a stable feature since high IL-4 secreting T cell clones were generated from the CD27- subset. These findings suggest that upon renewed as well as chronic antigenic stimulation in vivo, memory T cells acquire the CD45RA-CD27- phenotype and that, as a consequence, in this subset functionally differentiated CD4+ T cells are compartmentalized. Our results predict that analysis of the small CD27- subset of memory cells, that makes up approximately 10% of the peripheral blood T cell population, will provide information on the specificity and function of responding CD4+ T cells at a given point in time in healthy and diseased individuals.

A single intratracheal instillation of liposomes containing dichloro methylene diphosphonate into rats eliminated greater than 80% of the alveolar macrophage (AM phi) population, and the population was not significantly renewed during the ensuing week. AM phi depletion markedly increased local antibody production in the lung wall in pre-primed animals exposed to antigen aerosols. However, AM phi depletion did not affect the normal development of protective tolerance (particularly in the IgE antibody class) to inhaled antigen in immunologically naive rats. These results are discussed in relation to regional control of immune responses in the upper vs. the lower respiratory tract.

The in vitro recognition of acetylsalicilyl ovalbumin, penicilloylated bovine IgG (BPO-BGG) and the multichain copolymer poly-L-(Tyr, Glu)-poly-D L-Alapoly-L-Lys [(T, G)-A-L] by primed (2 x 13) F1 cells could be markedly and specifically blocked by alloantisera directed against "receptors" (R) or recognition structures for these antigens. The antisera were raised against lymphoid cells from various F2 hybrid guinea pigs of (2 x 13) histocompatibility type in which responsiveness to low doses of acetylsalicylic acid anhydride, BPO-BGG and (T, G)-A-L segregated independently of each other. One of the genes required for the expression of high responsiveness appears to be linked to the 13 MHC genes. The anti-R antisera did not contain antibodies capable of interacting directly with the stimulating antigen since passage of the sera through antigen-immunoadsorbent columns did not remove antibodies capable of suppressing in vitro recognition. Furthermore, the antisera did not appear to contain cytotoxic antibodies directed against specific antigen-sensitive cells. The activity of the anti-R sera was highly specific: their inhibitory activity could only be absorbed by lymphoid cells bearing the specific recognition structures for the appropriate antigen. Preliminary experiments yield no indication that the anti-R antisera are specific for immunoglobulin idiotypes.

A single injection of the retinal soluble antigen (S-Ag, 30 micrograms) to the Lewis or the heterozygous (rnu/+) rats induces a severe bilateral uveitis, characterized initially by infiltration of the retina with inflammatory cells. The athymic nude rat (homozygous rnu/rnu), which lacks the thymus gland and T cell-mediated functions, does not develop ocular inflammatory disease, clinically of histologically, after repeated challenges with S-Ag. Circulating anti-S-Ag antibodies were found in S-Ag-immunized Lewis rats and in the heterozygous, but not the athymic nude rats. Good proliferative responses to concanavalin A, S-Ag and purified protein derivitive of tuberculin (PPD) were found in lymphocyte cultures prepared from the draining lymph nodes of immunized heterozygous rats, but not when lymphocytes from the athymic nude rats were used. Uveitis could be induced in the athymic nude rat when lymphocytes from S-Ag-immunized heterozygous rats were transferred to them. By stimulating the donor lymphocytes in vitro with S-Ag before transfer, the number of recipients that developed uveitis was increased. On the other hand, it was impossible to transfer disease with hyperimmune serum alone. The possible role of T lymphocytes in the induction of experimental autoimmune uveitis with S-Ag is discussed.

In BALB/c mice, antibodies to the alpha-(1-3) glucosidic linkage of some dextrans (Dex) carry the idiotype of the BALB/c myeloma protein J558. Both specific antibody and idiotype are inherited in a dominant fashion, linked to the immunoglobulin (Ig) (heavy chain) allotype Igla of BALB/c mice (Eur. J. Immunol. 1975. 5: 775). In F1 hybrid mice from the parent strains SJL and BALB/c, we were able to suppress the expression of anti-Dex antibodies by immunizing prospective SJL mothers to the J558 idiotype. The state of suppression in the progeny was ascertained by immunization with Dex, and tests for the following were carried out: (a) antibodies specific for Dex; (b) inhibition of such antibodies (if present) by antiidiotypic serum to protein J558; (c) presence of the J558 idiotype; and (d) concentration of lambda1 chains (which are associated with the 558 idiotype) in the serum. SJL mothers, once immunized, conferred suppression upon several successive litters, spanning a period of 4-5 months. Suppression in F1 progeny animals lasted for 16 weeks or more. Spleen cells from suppressed F1 mice which had neither been treated with Dex nor with J558 protein, were able to confer suppression to further F1 newborn mice.

The purpose of these experiments was to determine whether the B cell clonotype profile expressed in mice which have recovered from antigen-induced unresponsiveness is similar to that of nontolerized mice. Unresponsiveness to phosphorylcholine (PC) was initiated by injection of neonatal mice with PC-coupled human gamma globulin, resulting in the inability to respond to challenge with PC-lipopolysaccharide at 1.5 months of age. By 3 months of age, pretreated mice were 50% responsive while by 5 and 7.5 months of age, full responsiveness was observed. At each time point two differences distinguished the anti-PC antibody from nonpretreated mice. First, whereas nonpretreated mice displayed T15 dominance, pretreated mice did not. Second, the average avidity of T15-negative antibody produced in pretreated mice was greater than that in nonpretreated mice and similar to that of T15-positive antibody. Possible mechanisms for this "permanent" alteration of the antibody profile are discussed.

Peyer's patch (PP) cells transferred into sublethally irradiated recipients generated substantial IgM, IgG and IgA anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses in the recipient spleen. If donor mice were given SRBC orally for 4-5 weeks prior to transfer, the adoptively transferred PP IgG and IgA responses were considerably suppressed, although the IgM responses were often unaffected. Co-injection of PP cells from antigen-fed mice with PP cells from normal mice resulted in marked suppression of the normal PP IgG and IgA response. However, treatment of PP cells from antigen-fed mice with anti-Thy-1.2 plus complement prior to cotransfer completely abrogated suppression of the IgG PFC response and partially abrogated the suppressed IgA response. B cells from the PP of antigen-fed mice, when transferred into SRBC-primed irradiated recipients (to provide T cell help) generated 2-3 times more IgG and IgA PFC than comparable numbers of B cells from the PP of normal mice. Thus antigen feeding generates suppressor T cells in PP which can mask the expression of B cell priming to orally administered antigen.

Hapten (2,4-dinitrophenyl (DNP))-substituted type 3 pneumococcal polysaccharide (DNP-lys-S3) effectively suppresses both primary and secondary anti-DNP antibody responses to DNP-proteins. The present experiments show that tolerizing doses of DNP-lys-S3 have much less effect on B cell memory (re)generation in three distinct situations: (i) in serial transfers of DNP-protein-primed cells, (ii) in virgin mice tolerized before priming with DNP-hemocyanin, and (iii) in tolerized, hemocyanin-primed mice boosted with DNP-hemocyanin. Under some conditions tolerized mice developed 10-50% of normal memory in the absence of significant antibody formation. Isoelectric focusing analyses revealed that many B cell clones proliferate in tolerant mice, but produce very little antibody until transferred to further hosts. Clones that escape suppression in partially tolerant mice do not appear to be resistant to DNP-lys-S3, when retested. Since there is independent evidence that the generation of B memory cells is less T cell-dependent than the development of antibody-forming cells, these data are best explained by assuming that DNP-lys-S3 blocks lymphocyte cooperation. This would be expected to preferentially suppress antibody formation, and to spare memory generation.

The adjuvant activity of 5 different lysolecithin analogs (LLA) has been studied in delayed-type hypersensitivity (DTH) using fowl gamma globulin, bovine serum albumin and the terpolymer L-glutamic acid 60-L-alanine30-L-tyrosine10(GAT), as antigens. Increased DTH responses, by factors of 1.8--2.0 in CBA and BALB/c mice, showed that LLA are immunopotentiators if given intraperitoneally (i.p.) or subcutaneously (s.c.) together with the antigen. The concentration range, within which LLA are active, is limited to 10--20 micrograms/mouse s.c. and 100--300 micrograms/mouse i.p. Adjuvanticity was tested as a function of the LLA structure. The most pronounced immunopotentiation was obtained with racemic 1-octadecyl-2-methylglycero-3-phosphorylcholine (ET18-O-CH3). The LLA became less active with decreasing numbers of C atoms in the alkyl chain.

The effect of a splenic or lymph node anti-sheep red blood cell response on lymphocyte migration patterns in mice was studied. It was found that trapping of lymphocytes in these stimulated organs was indiscriminate and was followed by a period of restricted cell entry or localization; furthermore, reduced cell localization in the spleen, during the splenic response, was accompanied by a reduction in the number of cells localizing in unstimulated brachial and axillary lymph nodes. These results were taken to indicate that major changes occur in lymphocyte circulation during strong splenic immune responses.

Although xid/Y mice fail to make a detectable primary antibody response to a variety of antigens such as Type III pneumococcal capsular polysaccharide (SIII), 2,4, 6-trinitrophenyl (TNP)-Ficoll, pneumococcal C carbohydrate, group A streptococcal vaccine and several kinds of related antigenic determinants such as phosphorylcholine (PC) and N-acetyl-glucosaminyl (GlcNAc), even when the latter are coupled to hemocyanin (Hy), they do show: (a) an antigen-dependent development of splenic B cells which can act as successful, productive fusion partners for SP2 cells giving hybridomas making monoclonal anti-SIII, anti-PC, anti-GlcNAc, anti-TNP, etc., and (b) an antigen-dependent appearance of antigen-binding plasmablasts in their spleens. The frequencies of specific B cells arising in xid/Y males with either of these properties are of the same order of magnitude as those found in immunocompetent xid/X female littermates. Further, both PC-Hy and GlcNAc-Hy prime xid/Y and xid/X mice for quantitatively and qualitatively similar secondary responses. All three of these expressions of a specific, primary response occur in xid/Y mice in the absence of any rise in circulating antibodies. The properties of successful, productive normal fusion partners leading to secretory hybridoma lines are unknown. Thus we cannot decide whether the antigen-binding plasmablasts that arise in xid/Y mice can also play the role of productive fusion partners. Neither do we know whether the development of specific IgM and IgG3 plasmablasts in xid/Y mice after antigen stimulation is an abnormality reflecting the xid mutation. It cannot be excluded that the development of productive fusion partners, of nonsecretory plasmablasts and of memory cells are all interrelated and reflect a process that also normally occurs in xid/X, X/X and X/Y mice following similar immunization regimens. It is tempting to speculate that such cells initially responding may lag in the development of normal secretory mechanisms and that the "transformed" fusion partner may complement this deficiency.

Partial protection of (C57BL/6 X DBA/2)F1 mice against infection with Schistosoma mansoni has been achieved by immunization with small amounts (0.2 to 2.0 microgram) of crude cercarial sonicate adsorbed on aluminium hydroxide gel adjuvant (alum). A decrease of 34-90% in the adult worm burden, of the immunized mice, as compared to that of untreated mice or those injected with adjuvant alone, has been found in five experiments by liver perfusion six weeks after percutaneous challenge infection. High titers of anti-cercarial IgE antibodies have been found in the sera of the immunized mice by two independent techniques, radioimmunoassay and degranulation of rat basophilic leukemia cells (RBL-2H3), determined by 3H-serotonin release. By counting the live worms in the lungs of immunized and uniummized mice on days 4-7 after infection it was observed that the schistosomula were killed before they reached the lungs, probably at the skin. Mice immunized with the same amounts of cercarial antigen in Freund's complete adjuvant were not protected against infection with S. mansoni. These animals developed high titer of total anti-cercaria antibodies (determined by radioimmunoassay) but had low levels of antiparasite IgE. The possible role of IgE in protective immunity is discussed.

The involvement of rheumatoid factor (RF)-secreting cells in normal immune responses was examined by screening hybridomas derived from 129/Sv mice during primary and secondary immune responses against foreign proteins. No RF-secreting cells were detected during primary responses, but up to 10% of the total number of clones obtained during secondary responses produced IgM with anti-IgG activity. Like typical mouse RF, these anti-IgG autoantibodies had a strict isotypic specificity, mostly for IgG1, and a much stronger avidity for immune complexes than for native IgG. The selective activation of IgG1-specific RF during these secondary immune reactions was not due to fortuitous antigenic similarities between mouse IgG1 and the antigens used for immunization, nor did it result from the use of adjuvants for priming, or from contamination of antigen preparations with lipopolysaccharide. It is therefore concluded that, in the 129/Sv mouse, RF production during secondary immune responses is a physiological phenomenon.

Priming with the T-dependent antigen trinitrophenylated horse red blood cells led to an oscillatory response of plaque-forming cells (PFC) with peaks of response after 4, 8, 12 and 16 days. Limiting dilution (LD) analysis of regulatory T cells indicated that the response is partly due to activation of helper T cells (Th), but mainly is the result of release from suppression by activation of a third regulatory T cell (T3) population (Zöller, H. and Andrighetto, G., Immunology 1985. 55: 703). Priming resulted in consecutive activation of suppressor T cells (Ts; day 1-3), a population regulating Ts (T3; day 2-4) and of Th (day 4). Furthermore, regulatory elements displayed a cyclical behavior like PFC. Th and T3 oscillated within a period of 4 days in phase with PFC, while Ts oscillated phase-shifted to PFC, Th and T3. Hence, oscillation of PFC after priming with a T-dependent antigen is the result of interactions among regulatory T cells. The data are interpreted as follows. Th are the primum movens of the oscillatory behavior of PFC response, since Th oscillate in phase with PFC and display the same pattern of decreasing amplitudes as PFC. However, the basic regulatory elements are Ts and T3 which are interlinked and oscillate phase shifted. As revealed by the time-course analysis of LD curves, T3 function as suppressor cells for Ts. Hence the status of mutual interactions of Ts and T3 determines suppression of Th or release from suppression, i.e. when T3 are found at high frequencies, Th are released from suppression, since T3 suppresses Ts. Instead, the increase in the frequency of Ts and their predation on Th determine the decrease of the frequency of Th. Thus, these data support the hypothesis of a circular network, the central regulatory core being composed of two elements, Ts and T3, with mutual suppressive activity.