Response Of Lymph Node Cells Research Articles

Cross‐reactive idiotypes on rabbit anti‐SEA antibodies stimulate anti‐idiotype spleen and lymph node cell responses of mice infected with Schistosoma mansoni

Antibodies to Schistosoma mansoni soluble egg antigens (SEA) generated in outbred rabbits from two different sources were used to study cross-reactive idiotype/anti-idiotype (Id/anti-Id) interactions in S. mansoni-infected mice in two different locations. Immunoaffinity purified rabbit polyclonal anti-SEA antibody preparations (RabId) were predominantly Ig by SDS-PAGE and demonstrated anti-SEA activity by ELISA and Western blot. RabId prepared from three separate rabbits and used at 40 micrograms/ml in vitro stimulated lymphocyte proliferative responses of spleen cells from mice with eight week infections (Mean +/- SEM [E-C]) of 3869 +/- 764, 18594 +/- 3046 and 8962 +/- 1734 cpm. Spleen cells from uninfected mice exposed to the same RabId preparations in vitro had respective [E-C] values of 206 +/- 144, 494 +/- 154 and 363 +/- 180. Lymph node cells from mice infected for 8 weeks demonstrated [E-C] of 123 +/- 400, 5073 +/- 828 and 2361 +/- 656 upon exposure to these 3 RabId preparations. Lymph node cells from uninfected mice had [E-C] of 220 +/- 76, 194 +/- 82 and 143 +/- 72 when exposed to these RabId. Maximum in vitro proliferative response to Ig from unimmunized rabbits was 761 +/- 400 by spleen cells from mice with eight week infections. These data indicate the presence of cross-reactive Id on rabbit anti-SEA antibodies from different rabbits that can stimulate in vitro lymphoproliferative responses of spleen or lymph node cells from S. mansoni-infected mice.

Leishmania (viannia) panamensis-induced cutaneous leishmaniasis in susceptible and resistant mouse strains.

We studied the susceptibility to Leishmania (Viannia) panamensis in strains of mice. The C57BL/6 strain was resistant and showed self-controlled lesion at the injected foot pad. The BALB/c and DBA/2J strains were susceptible and showed a foot swelling that started day 20 post-infection and progressed to a tumour-like lesion in later period of observation. The CBA/HJ strain was found to be of intermediary resistance. In contrast to other known cutaneous leishmaniasis in mice, the lesion in L. (V.) panamensis-infected mice was restricted to the inoculation site in the skin. In addition, we studied the development of cellular response and antibodies against Leishmania antigen in BALB/c and C57BL/6 strains. The proliferative response of lymph node cells against L. (V.) panamensis antigen was biphasic in both strains. An initial response was seen on day 20, followed by a refractory period between 40 and 80 days and a second response around fourth month post-infection. The response in the latter period was higher in C57BL/6 strain than in BALB/c strain. BALB/c strain presented much higher anti-Leishmania antibody level than C57BL/6 strain. The model and the correlation of immunological variables and the course of the infection are discussed.

Induction of tolerance to experimental anti-phospholipid syndrome (APS) by syngeneic bone marrow cell transplantation.

Previously, we have shown the ability to induce experimental autoimmune conditions (e.g. SLE, APLS, Wegener's granulomatosis) following active immunization with the pathogenic autoantibody emulsified in adjuvant. The mice first develop anti-autoantibodies (Ab2: anti-id) and eventually generate anti-anti-autoantibodies (Ab3: anti-anti-id) which carry the same antigen binding characteristic as the autoantibody (Ab1). The appearance of the specific autoantibodies in mice sera was associated with the emergence of the compatible laboratory and with the clinical findings characteristic to the respective autoimmune disease. The effects of syngeneic bone marrow transplantation (BMT) on experimental anti-phospholipid syndrome (APS) were investigated. BALB/c mice were immunized with anti-cardiolipin monoclonal antibody (MoAb) named CAM and developed elevated serum titres of anti-phospholipid Abs accompanied by prolonged activated partial thromboplastin time, thrombocytopenia and a high percentage of fetal resorptions. These mice were then lethally irradiated and transfused with bone marrow (BM) cells (T cell depleted) from syngeneic naive mice. The titres of antiphospholipid antibodies were reduced in the recipients. The decrease in titre of autoantibodies was found to be related to depletion of antibody forming cells in vivo, associated with reduced proliferative response of lymph node cells to anti-cardiolipin MoAbs. The recipients showed improvement in clinical parameters following syngeneic BMT. The same recipients developed specific unresponsiveness to a second challenge with the anticardiolipin MoAb (CAM), but developed experimental systemic lupus erythematosus upon immunization with a monoclonal anti-DNA antibody. We conclude that acute myeloablative immunosuppression combined with syngeneic bone marrow transplantation may induce a state of tolerance to the pathogenic autoantibodies in mice with experimental APLS. Our results suggest that a similar approach may be useful in treating life-threatening autoimmune syndromes (e.g. catastrophic APLS) in clinical practice.

Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells.

Previous experiments from this laboratory have shown that Lewis rats were protected from experimental allergic encephalomyelitis (EAE) induced by the injection of myelin basic protein (MBP) in Freund's complete adjuvant if they were treated with the encephalitogenic peptide of MBP covalently linked to mouse anti-rat immunoglobulin (Ig) D. It was suggested that this protection developed because the antibody-peptide conjugate targeted the peptide to B cells and that this mode of presentation induced a Th2-like T cell response that controlled the concomitant encephalitogenic Th1 reaction to the autoantigen. The current experiments were carried out to test this hypothesis and to examine the alternative explanation for the protective effect of the conjugate pretreatment, namely that it induced a state of nonresponsiveness in the autoantigenspecific T cells. It was shown that EAE induction was suppressed in Lewis rats when the antibody-peptide conjugate was injected intravenously 14 and 7 d before immunization with MBP in adjuvant, but that anti-MBP antibody titers were at least as high in these animals as in controls that were not pretreated with the conjugate before immunization. Lymph node cells from these pretreated animals, while proliferating in vitro to MBP as vigorously as those from controls, produced less interferon gamma and were very inferior in their ability to transfer disease after this in vitro activation. In contrast, these same lymph node cells from protected rats generated markedly increased levels of messenger RNA for interleukin (IL)-4 and IL-13. When these in vitro experiments were repeated using the encephalitogenic peptide rather than MBP as the stimulus, the proliferative response of lymph node cells from pretreated donors was less than that from controls but was still readily detectable in the majority of experiments. Furthermore, the cytokine expression induced by the peptide was similar to that elicited by whole MBP. While these results support the original hypothesis that the anti-IgD-peptide conjugate pretreatment protected rats from EAE by inducing a Th2-type cytokine response, a totally unexpected finding was that this pretreatment greatly reduced the level of leukocyte infiltration into the central nervous system. This result provides a direct explanation for the protective effect of the pretreatment, but it raises questions regarding migratory and homing patterns of leukocytes activated by different immunological stimuli.

Specific T-cell response correlates with resistance of genetic heterogeneous mouse populations to mouse hepatitis virus 3 infection.

In a recently published study [Vassão RC, Mello IGC, Pereira CA (1994) Arch Virol 137: 277-288] we have shown that the genetically selected high antibody responder mice (HIII) are susceptible and the low antibody responder counterparts (LIII) are resistant to death induced by experimental infection with mouse hepatitis virus 3 (MHV3). This report shows that the MHV3 titers in the peritoneal exudate (PE) of HIII mice, 3 days after infection, were more than 2 log greater than in the resistant LIII mice, the interferon gamma (IFN gamma) titers in the PE of both mouse populations being not significantly different. The treatment with monoclonal antibodies (mAb) against CD4+ or CD8+ T cells induced susceptibility among LIII mice. The depletion of CD4+ T-cell subset in LIII mice was evidenced by, and led to a significant reduction in, the IFN gamma synthesis in their PEs with a 100 fold increase in MHV3 titers. When lymph node cells (LNC) were harvested from MHV3-infected mice and stimulated "in vitro" with MHV3 inactivated by ultraviolet radiation (uv-MHV3), only LNC from LIII mice were capable of proliferating and synthesizing significant amounts of interleukin 2 (IL-2). The LNC proliferation and IL-2 synthesis were inhibited by treatment with mAbs against CD4 or CD8 molecules. The MHV3 infection induced in both lines of mice a profound depression of the mitogenic response of LNC to phytohemaglutinin (PHA). A correlation between the specific T-cell response and the resistance to MHV3 infection is discussed.

The Preventive Effect of Oral Administration of Type I Interferon on Collagen-Induced Arthritis in Rats

We investigated the effect of oral administration of type I interferon (IFN) on an autoimmune disease collagen-induced arthritis (CIA) in rats that was induced by immunization with type II collagen (CII). The results showed that the oral administration of IFN before immunization with CII significantly suppressed the development of CIA. Delayed type hypersensitivity, in vitro proliferative responses of lymph node cells to CII, and IL-2 production were also inhibited by the fed cytokine. The serum from IFN-fed animals downregulated the development of CIA and proliferative responses to CII. In contrast, IFN given orally after the onset of CIA failed to affect the proliferation of T cells to CII as well as the progression of the disease. There was a decrease in the production of anti-CII antibody in rats fed IFN before, but not after, immunization with CII. Thus, orally administered IFN may have preventive, but not therapeutic, effects on autoimmune inflammatory joint diseases.

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant.

Macrophages, dendritic cells or B lymphocytes have been shown to play a major role in the presentation of soluble antigens to CD4+ T cells. In contrast, the capacity of these cells to present particulate antigens such as bacterial or parasitic antigens to T cells remains controversial. To investigate this question, well defined particulate antigens were prepared by covalent linkage of proteins or peptides to 1 micron in diameter synthetic microspheres. The T cell immunogenicity of such particulate antigens was analyzed in vitro and in vivo. In vitro, a soluble protein such as hen egg lysozyme (HEL) coupled to beads stimulated a strong proliferative T cell response of lymph node cells from HEL-primed mice or of specific T cell hybridomas. HEL coupled to beads was presented to the specific T cell hybridomas by splenocytes or by peritoneal macrophages, but not by lymphoma B cells. Immunization of mice with several different protein antigens or with a synthetic peptide covalently linked to beads induced strong CD4+ T cell responses in the absence of adjuvant. The strong in vivo immunogenicity of proteins coupled to beads did not result from a non-specific adjuvant effect of beads since covalent linkage of the antigen to beads was strictly required to induce T cell responses in the absence of adjuvant. In vivo treatment by carrageenan showed that macrophages are required for the in vivo stimulation of T cell responses by these particulate antigens. Thus, these results demonstrated the role of phagocytic cells, especially macrophages, for in vivo presentation of particulate antigens. These particulate antigens represent an interesting approach for the development of new vaccines, and for the in vivo analysis of the role of various antigen presenting cells in T cell activation and differentiation.

Kinetics of Somatic Mutation in Lymph Node Germinal Centres

Somatic mutation activity in immunoglobulin V kappa genes during the response to the hapten 2-phenyl-5-oxazolone was measured in lymph node B-cell populations at various timepoints after footpad immunization. When the V kappa Ox1 genes rearranged to the J kappa 5 segment were amplified from genomic DNA using the polymerase chain reaction and sequenced, somatic mutations could be detected as early as day 4 after immunization. Somatic mutations were also detected after sequencing RNA from oxazolone-specific hybridomas derived from lymph node cells at day 4 after immunization. These early mutations were found mostly in cells with a germinal centre phenotype. No indication of selection at the population level by apoptosis was detected until day 7 after immunization. These results suggest somatic mutations can be induced very early during the immune response in lymph node cells, prior to the peak of clonal expansion and selection with regard to antigen binding.

B- and T-cell responses in congenic mice to repeat sequences of the malaria antigen Pf332: effects of the number of repeats

The Plasmodium falciparum antigen Pf332 comprises degenerated 11-amino-acid repeats with regularly spaced pairs of glutamic acid. Epitopes formed by such repeats are recognized by polyclonal and monoclonal antibodies that interfere with the life cycle of the blood stages of the malaria parasite. In order to study the immunogenicity of one such Pf332 repeat sequence (SVTEEIAEEDK), fusion proteins containing ZZ (two IgG binding domains of staphylococcal protein A) and dimers, trimers or tetramers of the malarial sequence were injected into mice. To analyse possible major histocompatibility complex class II restrictions of the immune response, mice of different H-2 haplotypes were used. A significant antibody response was elicited by administration of all the three fusion proteins in mice expressing the I-A k allele (B10.BR, B10.A(2R) and B10.A(4R)) whereas B10 and C57BL/6 (H-2 b) mice were low responders. In comparison, B10.D2 (H-2 d) mice were low responders to fusion proteins with 2 or 3 repeats but responded well to the protein containing 4 repeats. Lymph node cells from B10.BR (H-2 k) mice, primed in vivo with ZZ-fusion proteins containing either 2 or 4 repeats, proliferated in vitro in response to repeat sequences fused to ZZ or to an unrelated fusion partner, as well as to a synthetic peptide containing less than two repeats. In contrast, a response of lymph node cells from B10.D2 (H-2 d) mice was only obtained when a fusion protein containing 4 repeats was used both for in vivo priming and in vitro restimulation. Antibodies from all responding mice reacted with the native parasite protein in immunofluorescence. However, when measured by binding to a panel of synthetic peptides, the fine specificities of the antibodies were found to depend on the number of Pf332 repeats used for immunization. Thus, the number of repeat sequences influences both B- and T-cell responses. Since immunodominant repeat regions are a common feature for several malarial antigens involved in immune protection, the present findings have implications for the design of proper immunogens to be incorporated in malaria vaccines.

Lack of "Determinant Spread" to the Minor Encephalitogenic Epitope in Myelin Basic Protein-Induced Acute Experimental Autoimmune Encephalomyelitis in the Rat

Acute and monophasic experimental autoimmune encephalomyelitis (EAE) was induced in rats by immunization with myelin basic protein (MBP). Proliferative responses of lymph node cells to major and minor encephalitogenic and nonencephalitogenic determinants of the MBP molecules were measured at various time intervals after the immunization. Results of these experiments revealed that additional responses to minor determinants which had been observed at the late stage of mouse EAE (Lehmann et al., Nature 356, 155, 1992) were very weak and short-lived in the rat. Furthermore, the response to the minor encephalitogenic determinant was not recognized throughout the course of EAE. Coimmunization with synthetic peptides, corresponding to the major and minor determinants, induced T-cell response only to the major determinant. These findings suggest that poor generation of T cells reactive with the minor encephalitogenic epitope is attributable to peptide competition between these two determinants. The present results, together with those reported in mice, strongly suggest that differences in the clinical course of EAE, i.e., acute monophasic or chronic relapsing, is closely related to the presence or absence of "determinant spread" to minor encephalitogenic epitope(s).

Induction of T cell responses by chimeric bacterial proteins expressing several copies of a viral T cell epitope.

A viral T cell epitope was genetically inserted within the periplasmic MalE protein of Escherichia coli in two different permissive insertion sites and resulting hybrid proteins were used to study the in vitro and in vivo immunogenicity of the foreign T cell epitope. Purified hybrid MalE proteins containing the T cell epitope 120-132 (PreS:T) from PreS2 region of hepatitis B virus HBsAg inserted alone or with its adjacent B cell epitope (132-145) were able to induce strong peptide-specific T cell responses in mice. In vitro stimulation of primed lymph node cells or specific T cell hybridomas by the hybrid proteins required processing of the inserted T cell epitope and was inhibited by antigen-presenting cells fixation. The inserted T cell epitope was presented in vitro, in association with appropriate major histocompatibility complex molecules, as efficiently as free synthetic peptide. The in vitro immunogenicity of MalE hybrid proteins was increased by inserting four tandemly repeated copies of PreS:T, either at site 133 or 303. These results were confirmed in vivo by comparing the proliferative responses of lymph node cells from DBA/1 mice primed with MalE hybrid proteins containing one or four copies of PreS:T. Thus, the use of MalE hybrid proteins expressing multiple copies of a given foreign T cell epitope allows the induction of peptide-specific T cell response with a lower dose of priming antigen.

Local immunity in lung-associated lymph nodes in a murine model of pulmonary histoplasmosis.

Local immunity against acute pulmonary histoplasmosis was studied in the lung-associated lymph nodes of normal nonimmune mice infected intratracheally with live Histoplasma capsulatum yeasts. The phenotypes and distribution of cells in lung-associated lymph nodes and spleens were determined by flow cytometry. In addition, the immune responsiveness of these cells was evaluated by in vitro blastogenesis. Anti-H. capsulatum antibodies in serum and H. capsulatum antigen in tissue were measured by immunoassays. Cellular immune responses were greater in the lymph nodes than in the spleens. In lymph nodes 7 days after infection, a marked increase in the number of B lymphocytes caused the percentage to rise to 43%, compared with 26% in controls, and it remained elevated throughout the course of infection. A CD3+ cell that did not express CD4 or CD8 increased in number until it constituted 21% of lymph node cells, compared with 5% in controls, by day 14. The numbers of CD4+ and CD8+ T lymphocytes were modestly increased from days 7 to 35, but their percentages dropped because of the greater numbers of B lymphocytes and CD3+4-8- cells. Macrophages consistently constituted 2 to 3% of lymph node cells during the study. In spleens 7 days after infection, the percentage of macrophages in infected mice rose to 21%, compared with 9% in controls, but the total spleen cell number did not increase until day 14, when all cell subsets were nearly double in number. The in vitro blastogenic response of lymph node cells to H. capsulatum peaked at day 7, but spleen cell response was minimal during the course of infection. Histoplasma-specific serum immunoglobulin G antibodies reached peak levels by day 21 and remained high to the end of the study. In contrast, levels of antigen-specific immunoglobulin M antibodies were very low. These data suggest that antigen-specific immune responses occur in lung-associated lymph nodes and that this draining lymph node response may be an important component in host defense against Histoplasma lung infection.

Inhibition of fertility in female mice by immunization with a B-cell epitope, the synthetic sperm peptide, P10G

Human patient sera containing antisperm antibodies (from vasectomized men and infertile women) immunologically react with the synthetic peptide P10G (PGGGTLPPSG), and affinity purified antibodies to P10G (anti-P10G) react with human spermatozoa (O'Rand et al., 1990). In this study P10G was used to elicit antibodies and the effect of the antibodies on fertility in female mice determined. The P10G sequence is derived from the 14-kDa rabbit sperm autoantigen, RSA (O'Rand and Widgren, 1990). The results of this study demonstrate that female mice can become infertile when immunized with the synthetic peptide P10G conjugated to the carrier protein keyhole limpet hemocyanin (KLH). However, the results also show that it is important to distinguish those mice with high serum antibody levels from those with lower levels. Infertility was clearly apparent in the high titer subgroup with an 80% decrease in pregnancy rate during the last of three matings and a 71% decrease in litter size over all three matings when compared to the low titer subgroup or the control groups. Significantly, mice immunized with P10G without carrier protein show no detectable antigen specific proliferation of lymph node cells in response to 100 μM of the peptide. P10G is not a T-cell epitope, but rather a B-cell epitope and it does not elicit an autoimmune response in the female mouse. This demonstration in mice is an important first step in the development of a safe human immunocontraceptive vaccine.

Selective inactivation of lymphocytes after psoralen/ultraviolet light (PUVA) treatment without affecting systemic immune responses.

Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of several proliferative diseases. It has been suggested that the effectiveness of this treatment is due to induction of a specific immune response by PUVA-treated lymphocytes that down-regulates untreated cells of the same specificity. The present studies show that the clinically used psoralen analogue 8-methoxypsoralen (8-MOP), as well as 4,8-dimethyl-5'-(pyridiniummethyl)psoralen bromide (4NQ), a derivative that does not cross the cell membrane, when activated by UVA light, render lymphocytes unresponsive to mitogenic, antigenic, or phorbol myristate acetate + ionomycin-induced stimulation. This state of unresponsiveness was not reversed by exogenous recombinant interleukin-2. Treatment of lymphocytes with 4NQ and UVA light had no effect on the viability or the homing pattern of the cells to spleen or liver, whereas 8-MOP induced toxicity in about 50% of cells after 3 days in culture. Using an adoptive transfer mouse model, we found that antigen-specific lymphocytes treated with PUVA (8-MOP or 4NQ) had no effect on the ability of these mice to respond to a subsequent challenge with the same or an irrelevant antigen. This was tested by measuring the T cell proliferative responses of lymph node cells from mice primed with keyhole limpet hemocyanin (KLH) or fowl gamma globulin (FGG) before or after adoptive transfer of large numbers of KLH- or FGG-specific PUVA-treated lymph node cells. No effects of antigen-primed PUVA-treated cells on antigen-primed untreated cells were observed in vitro in mixed lymphocyte cultures responding to the relevant antigen. These results suggest that, in our model system, PUVA induces cell inactivation but does not affect systemic T cell responses.

Sympathetic Regulation of T-Helper Cell Function

The role of sympathetic neurotransmission in regulation of T-cell function was examined in mice. After in vivo priming with a model protein antigen, hen egg white lysozyme (HEL), the specific proliferative recall response of lymph node cells (LNCs) was examined. The release of endogenous catecholamines by amphetamine (5 mg/kg, 45 min prior to sacrifice) markedly inhibited the proliferative response of LNCs. Combined alpha- and beta-adrenergic blockade (phentolamine, 1.0 mg/kg + propranolol, 2.5 mg/kg) prevented this effect of amphetamine on proliferation. In vitro, the alpha-adrenergic agonist phenylephrine, but not the beta-agonist isoproterenol, inhibited proliferation in a dose-dependent manner, up to 80%. The effect of phenylephrine was blocked by the alpha-adrenoceptor antagonist phentolamine. The effects of catecholamines appear to be mediated at the level of antigen processing/presentation: Amphetamine given prior to sacrifice inhibited interleukin 2 production when irradiated spleen cells were used to present HEL and HEL-related peptides to HEL-specific T-cell hybridomas. In summary, the sympathetic nervous system seems to inhibit antigen processing/presentation and, indirectly, T-helper responses.

Induction of Experimental Autoimmune Thyroiditis in Rats with the Synthetic Peptide (2495-2511) of Thyroglobulin

We have previously shown that the synthetic 17 mer peptide TgP1 (a.a. 2495-2511) from the rat thyroglobulin (Tg) sequence is thyroiditogenic in mice and consists of nonimmunodominant but highly immunogenic determinants at the T or B cell level. Although TgP1 appears to be phylogenetically conserved, it remains uncertain whether it is a self-peptide in mice since the mouse Tg amino acid sequence is unknown. In this report, we demonstrate that TgP1 also induces experimental autoimmune thyroiditis (EAT) in WKY, F344, and WF strains of rats that are known to be EAT-susceptible after Tg challenge. Priming of all strains with TgP1 led to strong proliferative lymph node cell responses to the peptide in vitro that were abrogated by MAbs against CD4, OX-6, and OX-17 determinants, suggesting TgP1 recognition by class II-restricted T cells. In vivo priming with autologous or heterologous Tg did not lead to TgP1-specific proliferation in vitro confirming the cryptic nature of this sequence in rats. In contrast to findings in the mouse, strong peptide-specific IgG responses were not observed in rats despite the presence of EAT. These data demonstrate that TgP1 is immunopathogenic in an autologous system and provide the first evidence of a defined autoantigenic Tg peptide in rats.

Identifying polymorphic regions of the p190 protein from different Plasmodium falciparum strains by using specific T cells.

The p190 protein (also called MSA1 or MSP1) of the asexual blood stage forms of Plasmodium falciparum, a human malaria vaccine candidate, shows polymorphism between different isolates. Mice were immunized with p190-3, a recombinant protein which contains mostly conserved sequences derived from the p190 protein of the K1 parasite isolate. Proliferative T-cell responses of lymph node cells from immunized mice were assessed by stimulation in vitro with p190-3 or preparations of parasitized red blood cells (PRBC) containing the native protein. The p190-3-specific T cells from C57BL/6 mice consistently responded to some P. falciparum isolates, representing either the K1 or MAD20 serotype of p190, but not to other P. falciparum strains or to rodent malaria parasite-infected red blood cells. p190-3-specific T-cell responses from other mouse strains (BALB/c, C3H/He) did not distinguish between P. falciparum isolates. The polymorphic epitopes which were preferentially recognized by T cells from C57BL/6 mice were identified.

Differences of draining lymph node cell proliferation among mice, rats and guinea pigs following exposure to metal allergens

Contact sensitivities of three well known metal allergens (nickel sulfate, potassium dichromate and cobalt chloride) were examined using the local lymph node assay in CBA/N mice, F344 rats and Hartley guinea pigs. The effect of various species sera on lymph node cell (LNC) proliferation was also investigated. Exposure to potassium dichromate and cobalt chloride induced significant LNC proliferative responses in the three species. The LNC responses to potassium dichromate in the rats were higher than those in the mice and guinea pigs. Mice exhibited the highest response to cobalt chloride among the three species, whereas, exposure to nickel sulfate failed to induce a marked LNC proliferation. Increased draining lymph node weights and LNC numbers were also observed following exposure to the metal salts. However, these parameters were less sensitive compared with the LNC proliferative response. There was a large difference in the lymph node weight between individual guinea pigs. The [ methyl- 3thymidine incorporation into LNC of each species cultured in the presence of the homologous serum in vitro was lower than in the presence or absence of fetal calf serum. However, there was no significant difference in stimulation indices among the different culture conditions. The local lymph node assay may be performed in rats as well as in mice for the detection of metal allergens.

A nonspecific inhibitor of contact sensitivity elaborated by macrophages: Genetic restriction in its production but not in its action

It is known that macrophages armed with haptene-specific T suppressor factor (TsF) and then exposed to antigen (haptenized spleen cells) liberate a nonspecific inhibitor of the transfer of contact sensitivity (CS). This is called macrophage suppressor factor (MSF). This paper shows that MSF is only released when the source of the TsF and the haptenized spleen cells share the same I-J subregion. This is based on the comparison of B10.A(3R) and B10.A(5R) mice. In contrast, the action of MSF is antigen nonspecific and genetically unrestricted. In these respects it resembles the antigen-nonspecific inhibitor (nsTsF-1) made by the T acceptor cell when armed with TsF. However, it differs from nsTsF-1 in acting directly on the I-A − population which transfers contact sensitivity and not indirectly via I-A + T cells. In vitro, MSF fails to inhibit the proliferative response of lymph node cells to specific antigen and their production of IL-3 activity, IFN- t , and IL-2. This indicates that MSF is not a global inhibitor of T cell activity. The finding that MSF inhibits systemic passive transfer of contact sensitivity, but has no effect on local passive transfer strongly supports the view that MSF affects the arrival of certain cells critical for the development of the reaction to the skin challenge site.

Immunogenicity of free synthetic peptides corresponding to T helper epitopes of the influenza HA 1 subunit. Induction of virus cross reacting CD4+ T lymphocytes in mice.

Four linear synthetic peptides corresponding to residues 12-29, 50-67, 121-138 and 131-147 of the HA 1 subunit of H3 subtype influenza virus (NT/60/68) were tested for their capacity to elicit in vivo peptide-specific CD4+ T cells cross reacting with whole virus. By studying the in vitro peptide proliferative response of lymph node cells from mice sensitized in vivo with free peptides emulsified in complete or incomplete Freund adjuvant, it was found that region 12-29 could be recognized by CD4+ T lymphocytes in the context of H-2k and H-2b, region 50-67 in association with H-2b and region 121-138 in the context of H-2d MHC molecules. Outbred OF 1 mice could recognize regions 50-67 and 121-138. Peptides 50-67 and 121-138 are of potential interest for synthetic vaccine design since they induced in BALB/c (peptide 121-138) and OF 1 (both peptides) mice a CD4+ T cell population that cross reacted with whole virus. The region 50-67 is of particular interest since only few substitutions have been found in this area in natural variants of the H3 virus subtype.