Immune Lymph Node Research Articles

IL-10-Producing Langerhans Cells and Regulatory T Cells Are Responsible for Depressed Contact Hypersensitivity in Grafted Skin

Although skin grafting is a common surgical technique, the immunological state of grafted skin remains unelucidated. An experimental model has shown that the development of murine contact hypersensitivity (CHS) is depressed when mice are sensitized with a hapten through full-thickness grafted skin. We explored the immunological mechanisms underlying this hyposensitization, focusing on the fate of Langerhans cells (LCs). When FITC was applied to grafted skin, FITC-bearing LCs were capable of migrating to the draining lymph nodes. Epidermal cell suspensions isolated from the grafted skin produced a high amount of IL-10 as assessed by real-time PCR. Adoptive transfer of immune lymph node cells from the sensitized mice suppressed the CHS response of recipients in an antigen-specific manner. CD4(+)CD25(+) but not CD4(+)CD25(-) T cells purified from lymph node cells were responsible for this suppression. Finally, we detected high expression of receptor activators of nuclear factor kappa-B ligand (RANKL) in the grafted skin, and found that recombinant RANKL stimulated LCs to produce IL-10. These findings suggest that the hyposensitization of CHS through the grafted skin is not attributable merely to the reduction of LC number but that IL-10-producing LCs exert a downmodulatory effect by inducing regulatory T cells.

Impaired Initiation of Contact Hypersensitivity by FTY720

FTY720 inhibits lymphocyte emigration from lymphoid organs to peripheral blood by binding one of the sphingosine-1-phosphate (S1P) receptors, S1P(1). We investigated the effects of FTY720 in relation to murine contact hypersensitivity (CHS). CHS was impaired by FTY720 when administered during the sensitization but not the elicitation phase. Consistently, adoptive transfer of immunized lymph node cells from mice treated with FTY720 during the sensitization phase was virtually incapable of inducing CHS response in recipients. FTY720 decreased the number of blood CD44(-)-naive T cells markedly and that of CD44(+) memory T cells modestly. Among memory T cells, the CD62L(-) effector memory subset was more resistant to FTY720 than the CD62L(+) central memory subset. Accordingly, the level of S1P chemotactic response was high in naive T cells, marginal in effector memory T cells, and very low in central memory T cells. Consistently, the S1P(1) mRNA expression level was much lower in memory T cells than in naive T cells. These findings demonstrate that S1P-S1P(1) signaling is essential for recirculation of naive T cells. FTY720 seems to decrease the incidence of interactions between antigen-loaded dendritic cells and circulating naive T-cell clones in the lymph nodes, thereby depressing the sensitization of naive T cells in CHS.

Classical Complement Pathway in Experimental Autoimmune Myasthenia Gravis Pathogenesis

Mice deficient for complement factors C3, C4, or C5 are resistant to experimental autoimmune myasthenia gravis (EAMG). Acetylcholine receptor (AChR) immune lymph node cells (LNC) of C3 deficient mice produce less interleukin 6 (IL-6), and EAMG-resistant IL-6 deficient mice have less serum C3. Increased serum C1q-circulating immune complex (CIC) levels correlated with EAMG disease severity in RIIIS/J mice. The CIC promotes EAMG severity by stimulating the production of LNC IL-6, serum C1q, and C3 via FCgammaR interaction. Therefore, EAMG/MG could be treated by blocking the activation of classical complement pathway (CCP) and/or IL-6. Anti-C1q antibody administration before and following AChR immunization suppressed EAMG by reducing LNC IL-6 production and neuromuscular junction deposits of IgG, C3, and C5b-C9 complexes. Treatment with low dose (10 microg) of anti-C1q antibody twice a week for 4 weeks in mice with ongoing clinical EAMG reduced the clinical severity of disease and LNC IL-6 production. Therefore, inhibitors of CCP factors C1q, C2, or C4 could treat MG and would preserve the alternate complement pathway activation. Our goal is to tailor MG therapy using anti-C2/C4 reagents in combination, with or without anti-cytokine (e.g., anti-IL-6) reagents.

P19/Arf and p53 suppress sentinel lymph node lymphangiogenesis and carcinoma metastasis

The ability of tumor cells to metastasize is increasingly viewed as an interaction between the primary tumor and host tissues. Deletion of the p19/Arf or p53 tumor suppressor genes accelerates malignant progression and metastatic spread of 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced squamous cell carcinomas, providing a model system to address mechanisms of metastasis. Here, we show that benign pre-metastatic papillomas from wild-type mice trigger lymphangiogenesis within draining lymph nodes, whereas there is no growth of primary tumor lymphatic vessels. Lymph node lymphangiogenesis is greatly accelerated in papilloma-bearing p19/Arf- or p53-deficient mice, which coincides with the greater propensity of these tumors to progress to carcinomas and to metastasize. The extent of accumulation of B cells within the tumor-draining lymph nodes of wild-type mice predicted the level of lymph node lymphangiogenesis and metastatic potential. Arf or p53 deficiency strongly accelerated lymph node immune cell accumulation, in a manner that was associated with the extent of lymph node lymphatic sinus growth. This immune cell accumulation and lymph node lymphangiogenesis phenotype identifies host anti-tumor responses that could drive metastatic spread of cancers via the lymphatics.

Gene Expression Model of Survival and Transformation in Follicular Lymphoma (FL): A Study by the LLMPP.

Background: FL is a common NHL that has a broad spectrum of clinical outcomes. Over time some pts will transform to an aggressive histology (Tly) associated with inferior survival. In 2004, the LLMPP constructed a model that was predictive of overall survival (OS) based on the gene expression profiles (GEP) of 191 specimens taken from pts with untreated FL. The genes associated with survival were derived from the non-neoplastic immune response (IR) cells. However the risk of developing Tly was not addressed in this study. Thus we re-analyzed the GEP with updated clinical data. Our goal was to validate our previous model with extended follow-up and to create a model that would predict the risk of developing TLy.Methods: 170 of 191 previously untreated FL pts had updated clinical information but only 142 had transformation outcome. Transformation was defined as biopsy proven DLBCL or clinically based on the presence of at least one of the following: hypercalcemia, a sudden rise in LDH >twice baseline, unusual extranodal growth or rapid discordant nodal growth. Raw CEL files from Affymetrix U133A arrays were pre-processed and normalized using Bioconductor's GCRMA package. Models were developed using SignS package (http://signs/bioinfo.cnio.es/), with 10 times cross-validation. All gene lists produced in these analyses were then re-tested for association with outcome using Bioconductor's Globaltest package. Over Representation Analysis of signature components was performed using Dchip.Results: The median OS of these patients was 8 yrs. A new 7-component survival model (85 genes) was developed that was significantly associated with survival (p= 2.9×10−13). In Globaltest, these gene lists were associated with survival at a level of (p=2.6×10−5). The previous model using IR-1 and IR-2 signatures was associated with survival at a level of p=2.6×10−4. Although there is little overlap between the 2 models, the new model confirms the importance of IR genes and extracellular matrix genes as being prognostically important. Interestingly, one component containing 10 genes on chromosome 6q was associated with a superior survival (p<1×107). 27% developed Tly over a median follow-up time of 11.2 yrs (69% biopsy proven). Our transformation model included 53 genes divided into 3 components (p=0.001). The Globaltest analysis for association of these genes with transformation was significant (p=0.018). 54 genes overlapped between the survival genes and transformation genes that were present in >1 cross validation run. These were significantly enriched in genes important in immune response like T cell and macrophage activation.Conclusion: Our survival model is stable and confirms the importance of key genes involved in the immune response and lymph node remodeling. It also introduces new genes that are potentially important for survival. Our transformation model may shed light on the mechanisms involved in the progression of FL to DLBCL but it is less stable and less reliable than our survival model at predicting outcome.

B Cell-Driven Lymphangiogenesis in Inflamed Lymph Nodes Enhances Dendritic Cell Mobilization

Dendritic cell (DC) migration from the periphery to lymph nodes is regulated by the pattern of genes expressed by DCs themselves and by signals within the surrounding peripheral environment. Here, we report that DC mobilization can also be regulated by signals initiated within the downstream lymph nodes, particularly when lymph nodes enlarge as a consequence of immunization. Lymph node B lymphocytes orchestrate expansion of the lymphatic network within the immunized lymph node. This expanded network in turn supports increased DC migration from the periphery. These results reveal unique relationships between B cells, lymphatic vessels, and migratory DCs. Knowledge that DC migration from the periphery is augmented by B cell-dependent signals reveals new potential strategies to increase DC migration during vaccination.

Nadifloxacin downmodulates antigen-presenting functions of epidermal Langerhans cells and keratinocytes

Nadifloxacin is an anti-microbial quinolone derivative widely used for the treatment of acne as a topical agent. This drug has been suggested to have not only anti-bacterial but also anti-inflammatory actions, which may have a beneficial effect on some aspects of inflammatory acne. To further clarify its abilities to modulate skin immunity, we investigated whether nadifloxacin affects the hapten- and superantigen-presenting capacities of epidermal Langerhans cells (LC) and keratinocytes, respectively. Immune lymph node CD4+ T cells from trinitrophenyl-sensitized BALB/c mice were cocultured with LC-enriched epidermal cells (LC-EC) that were freshly isolated from syngeneic mice and derivatized with trinitrophenyl hapten in the presence or absence of nadifloxacin. Alternatively, LC-EC were preincubated with nadifloxacin (NDFX), modified with the hapten, and cultured with immune T cells. The effects of nadifloxacin on the surface molecule expression in LC and keratinocytes were also tested by flow cytometry and cellular ELISA. LC-EC cultured with nadifloxacin at 10 microg/ml or more significantly suppressed the antigen-presenting function of LC for T cells. The ability of MHC class II+ keratinocytes to present a superantigen to T cells was suppressed by preincubation of keratinocytes with 30 microg/ml or more of nadifloxacin. These functional reductions in LC and keratinocytes reflected the decreased expression of MHC class II and/or costimulatory molecules. Nadifloxacin downmodulates cutaneous immunity by interfering with the antigen-presenting ability of epidermal cells.

H2 complex controls CD4/CD8 ratio, recurrent responsiveness to repeated stimulations, and resistance to activation-induced apoptosis during T cell response to mycobacterial antigens

Genetic variation in the major histocompatibility complex (MHC) influences susceptibility and immune responses to Mycobacterium tuberculosis in mice and humans, but connections among the severity of tuberculosis (TB), dynamic changes in T cell responses to mycobacteria, and MHC genetic polymorphisms are poorly characterized. The overall effect of the MHC genes on TB susceptibility and cellular responses to mycobacteria is moderate; thus, such studies provide reliable results only if congenic mouse strains bearing a variety of H2 haplotypes on an identical genetic background are analyzed. Using a panel of H2-congenic strains on the B10 background, we demonstrate that T cells from mice of three different strains, which are resistant to TB infection, readily respond by proliferation to repeated stimulations with mycobacterial sonicate, whereas T cells from three susceptible mouse strains die after the second stimulation with antigen. This difference is specific, as T cells from TB-susceptible and -resistant mouse strains do not differ in response to irrelevant antigens. The CD4/CD8 ratio in immune lymph nodes correlates strongly and inversely with TB susceptibility, being significantly lower in resistant mice as a result of an increased content of CD8+ cells. These differences between the two sets of mouse strains correlate with an elevated level of activation-induced T cell apoptosis in TB-susceptible mice and a higher proportion of activated CD44+ CD62 ligand- T cells in TB-resistant mice. These results may shed some light on the nature of the cellular basis of MHC-linked differences in susceptibility to TB.

Establishment of murine model of allergic photocontact dermatitis to ketoprofen and characterization of pathogenic T cells

Ketoprofen is well known to evoke the allergic type of photocontact dermatitis when it is applied to the skin and irradiated with ultraviolet A (UVA) light. We aimed to establish a murine model of this photosensitivity and to characterize pathogenic T cells concerned with the sensitivity. Various strains of mice were sensitized on two consecutive days by application of ketoprofen to the shaved abdomen and irradiation of the skin with UVA. Five days later, they were elicited with ketoprofen plus UVA on the earlobes. Immune lymph node cells and epidermal cells from the challenged sites were analyzed by RT-PCR. Mice were successfully sensitized and challenged with 4% and 2% ketoprofen, respective, plus UVA at 20J/cm2. The responses in H-2k mice were higher than those in the other strains examined. Immune lymph node CD4+ or CD8+ cells from ketoprofen-photosensitized H-2k mice were transferred i.v. to naïve syngeneic recipients. Mice receiving CD4+ but not CD8+ cells exhibited ketoprofen photosensitivity, but transference of both CD4+ and CD8+ cell populations was more effective. Lymph node cells from photosensitized mice expressed high levels of mRNA for Th2 cytokine (IL-4) and Th2 chemokine receptor (CCR4) as well as Th1 cytokine (IFN-gamma) and Th1 chemokine receptor (CXCR3), as assessed by RT-PCR. In addition, epidermal cells from challenged earlobes expressed increased levels of both Th1 (TARC) and Th2 (Mig) chemokines. It is considered that not only Th1 but also Th2 cells participate in the pathogenesis of murine photocontact dermatitis to ketoprofen.

Identification of a CD4+CD25+ T cell subset committed in vivo to suppress antigen-specific T cell responses without additional stimulation.

Naturally occurring CD4+ regulatory T cells can be identified on the basis of expression of CD25 and suppression of T cell responses in vitro after TCR triggering. Here, we demonstrate that a CD134+ subset of CD4+CD25+ T cells in naive rats suppresses antigen-specific T cell responses in vitro without additional TCR stimulation. In contrast, CD4+CD25+CD134- regulatory T cells and total CD4+CD25+ regulatory T cells have suppressive activity only during simultaneous activation of responder and regulatory T cells or after in vitro pre-activation. Furthermore CD4+CD25+CD134+ T cells have a more activated phenotype than CD4+CD25+CD134- T cells, as based on the expression of CD62L, CD45RC, and MHC class II. We propose that the CD134+ regulatory T cells contain an in vivo activated and highly suppressive regulatory T cell subset. CD4+CD25+CD134+ T cells can be found in several compartments of the immune system, including spleen, lymph nodes, and blood. Interestingly though, the relative amounts of these cells within the CD4+ population and their CD134 expression levels are highest in mucosa-draining lymph nodes and lowest in blood. This suggests that the presence of CD4+CD25+CD134+ T cells indicates sites of active immune suppression.

Influence of the mouse Bcg, Tbc-1 and xid genes on resistance and immune responses to tuberculosis infection and efficacy of bacille Calmette-Guérin (BCG) vaccination.

We have studied the role of three mouse distinct non-H-2 genes (Bcg, Tbc-1, xid) in several phenomena of antituberculosis immunity and resistance. On the basis of median survival time (MST) of mice following infection with virulent Mycobacterium tuberculosis H37Rv, Bcg gene did not control resistance to the lethal dose of H37Rv infection in non-vaccinated and Myco. bovis (BCG)-vaccinated mice. However, Bcgr allele, in comparison with Bcgs allele, determined more effective suppression of an early multiplication in spleens of H37Rv mycobacteria after a low dose (5x10(4) colony-forming units (CFU)) injection. CBA/N mice, which are not protected efficiently against tuberculous challenge by BCG vaccination, were characterized by a decreased in vitro proliferation of immune lymph node cells, both spontaneous and stimulated with mycobacterial antigens. The decreased proliferation was due to immunosuppression caused by interactions between responding T cells and CBA/N antigen-presenting cells (APC). We have confirmed that the defective response to BCG-vaccination in CBA/N mice is linked with the X-chromosome and thus is presumably determined by the xid gene itself. I/St mice (Tbc-1s), supersusceptible to H37Rv infection, were not able to restrict the growth of H37Rv mycobacteria in spleens, even following infection with a low dose (5x10(4)), but restricted the growth of Myco.bovis BCG more effectively than Bcgs mice.

Suplatast tosilate inhibits eosinophil production and recruitment into the skin in murine contact sensitivity

Antiallergic drugs and antihistamines have been widely used for controlling mucosal allergic diseases in which eosinophilia is prominent. Although H1-receptor antagonists are effective for treating histamine-induced wheal and itch in urticaria, the effects of antihistamines and antiallergic agents on other eosinophilic skin diseases remain to be determined. We investigated the effects of oral administration of antiallergic drugs and antihistamines, such as suplatast tosilate, emedastine difumarate, and azelastine hydrochloride, on a novel murine model of eosinophilia in contact sensitivity to picryl chloride. Among the drugs tested, only suplatast tosilate remarkably inhibited blood and tissue eosinophilia and the ear swelling responses. The inhibitory effects on eosinophilia seemed to be mediated by the suppression of IL-5 production in spleen cells during eosinophil development, while the effects on the ear swelling response seemed to be mediated by suppression of IL-4 production in immune lymph node cells in the efferent phase. Suplatast tosilate may effectively treat eosinophilic skin diseases in which Th2-cell-derived cytokines are predominant.

Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor alpha expression in hematopoietic cells.

It is widely accepted that females have superior immune responses than males, but the ways by which sex hormones may enhance T cell responses are still poorly understood. In the present study, we analyzed the effect of estrogens on CD4 T cell activation and differentiation after immunization with exogenous antigens. We show that administration of low doses of 17beta-estradiol (E2) to castrated female mice results in a striking increase of antigen-specific CD4 T cell responses and in the selective development of IFN-gamma-producing cells. Quantitative assessment of the frequency of T cells bearing a public TCR beta chain CDR3 motif demonstrated that the clonal size of primary antigen-specific CD4 T cells was dramatically increased in immune lymph nodes from E2-treated mice. By using mice with disrupted estrogen receptor (ER) alpha or beta genes, we show that ERalpha, but not ERbeta, was necessary for the enhanced E2-driven Th1 cell responsiveness. Furthermore, ERalpha expression in hematopoietic cells was essential, since E2 effects on Th1 responses were only observed in mice reconstituted with bone marrow cells from ERalpha+/+, but not ERalpha-deficient mice. These results demonstrate that estrogen administration promotes strong antigen-specific Th1 cell responses in a mechanism that requires functional expression of ERalpha in hematopoietic cells.

Splenic T cells from mice receiving intracameral antigen suppress in-vitro antigen-induced proliferation and interferon-? production by sensitized lymph node cells

Purpose: To characterize the immunoregulatory mechanisms in vitro of spleen cells that are activated by intracameral injection of antigen (AC-spleen cells). Methods: AC-spleen cell regulation of in-vitro antigen-induced proliferation and interferon-? production by lymph node cells from TNP-BSA-immunized mice was quantified by co-culture of the lymph node cells with TNP-BSA and AC-spleen cells induced by intracameral TNP-BSA. Cytokine production was quantified by ELISA. Results: AC-spleen cells produced significantly more IL-4 and IL-10 than spleen cells from TNP-BSA-immunized mice or naive spleen cells; unlike spleen cells from immunized mice, AC-spleen cells did not produce IFN-?. AC-splenic CD4 + , CD8 + , CD4 - /CD8 - (DN) T cells differentially suppressed antigen-induced proliferation and IFN-? production by immunized lymph node cells by a mechanism dependent on IL-10 and antigen. Cultures of lymph node cells, antigen, and AC-splenic T cells contained increased amounts of IL-10 and/or TGFß2. Conclusions: The differential, cytokinedependent immunoregulatory effects of CD4 + and CD8 + AC-spleen cells observed in vitro parallel their effects in vivo. We suggest that the suppression of antigen-induced lymphocyte proliferation and IFN-? production by AC-spleen cells provides a useful in-vitro assay of the immunoregulatory activity of cell populations that are induced by the injection of antigen into the anterior chamber.

Chemokines modulate experimental autoimmune thyroiditis through attraction of autoreactive or regulatory T cells.

A critical event in the pathogenesis of experimental autoimmune thyroiditis (EAT) is the entry of thyroid-specific T lymphocytes into the thyroid gland. To investigate the role of soluble mediators in that infiltration, we have assayed the expression of various chemokines in diseased thyroid glands and in cytokine-treated cultures of normal thyroid epithelial cells. MCP-1 (monocyte chemotactic protein-1) and RANTES are produced during EAT and induced in vitro by IFN-gamma, IL-10, TNF-alpha, and IL-1beta. In vitro chemotaxis experiments using immune lymph node (LN) cells showed that RANTES attracted mTg-specific responder LN cells, whereas MCP-1 attracted mTg-specific CD4(+), CD25(+) regulator cells that secreted IL-10. The in vivo transfer of LN T cells attracted in vitro either by RANTES or by MCP-1 confirmed their opposite effects on the course of EAT.

Beneficial effect of the immunosuppressant LF 15-0195 on passively induced rat experimental autoimmune encephalomyelitis

EXPERIMENTAL allergic encephalomyelitis (EAE), an inflammatory autoimmune disease of the central nervous system (CNS), serves as an experimental model for multiple sclerosis. EAE is a T-cell-dependent paralytic disease that can be induced in experimental animals by injection of myelin-derived antigens emulsified in Complete Freund’s Adjuvant (CFA) or following adoptive transfer of T-cell lines or clones specific for various myelin proteins. The clinical course of EAE varies with the animal model and the protocol. In most models, acute EAE is followed by permanent remission and concomitant resistance to further disease induction or by a transient remission that is followed by subsequent relapses and remissions. A large body of evidence correlates the induction phase of EAE to a Th1-type immune response, whereas the remission phase as well as the subsequent resistance is characterized by an up-regulation of the anti-inflammatory cytokines IL-4, IL-10, and TGF. Deoxyspergualin (DSG) is a synthetic analogue of the anti-tumor antibiotic spergualin, a natural product isolated from Bacillus laterosporus. DSG is claimed to have potent immunosuppressive effects and several advantages compared to available immunosuppressive drugs. However, preclinical and clinical use of DSG is limited by its chemical and metabolic instability. Several analogues of DSG have been recently prepared through organic synthesis, such as LF 15-0195, an analogue that is more potent than DSG due to improved activity and increased metabolic resistance to oxidative metabolism in vivo. This new immunosuppressant is currently in clinical development for autoimmune diseases. The present study was undertaken to analyze the capacity of LF 15-0195 to inhibit the pathogenicity of differentiated effector T cells. We tested (i) the effect of LF 15-0195 administration in vivo on development of EAE induced by adoptive transfer of anti-myelin basic protein (MBP) pathogenic CD4 T cells and (ii) the effect of stimulation of immune lymph node cells in the presence of LF 15-0195 in vitro on their ability to transfer EAE. We showed that LF 15-0195, both in vitro and in vivo, reduced the pathogenicity of autoreactive effector anti-MBP CD4 T cells. This beneficial effect could not be explained by inhibition of the expansion of pathogenic CD4 T cells or by the inhibition of their production of effector cytokines. MATERIALS AND METHODS Animals

Gamma interferon inhibits production of Anti-OspA borreliacidal antibody in vitro.

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-gamma). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-gamma was not affected by the time of treatment. In addition, treatment with rIFN-gamma inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-gamma marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-gamma does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.

Dominant expression of CXCR3 is associated with induced expression of IP-10 at hapten-challenged sites of murine contact hypersensitivity: a possible role for interferon-γ-producing CD8 + T cells in IP-10 expression

Murine contact hypersensitivity is elicited as a consequence of immunologic reactions triggered by skin-applied antigen, interactions among Langerhans cells, T cells, keratinocytes and mast cells, and a variety of chemokines generated by cellular interactions. In this study, we sensitized and challenged BALB/c mice with hapten, dinitrofluorobenzene or picryl chloride, and examined the expressions of mRNA for chemokines and their receptors by reverse transcription-polymerase chain reaction in the skin of elicited earlobes. CXC chemokines, IP-10 and Mig, were transcribed 24–48 h after challenge. This was associated with the expression of their agonistic receptor CXCR3, while mRNA for TARC and MDC, and their receptor CCR4 were not detected. Since CXCR3 and CCR4 are expressed preferentially on types 1 (Th1/Tc1) and 2 (Th2) T cells, respectively, the results suggested that the former type of T cells predominantly infiltrate at the elicited sites. Immune lymph node cells of the sensitized mice also expressed mRNA for CXCR3 but not CCR4 with concomitant transcription of interferon-γ (IFN-γ) but not interleukin-4 subsequent to challenge. The percentage of lymph node CD8 + T cells was increased from 16% in naive mice to 30–50% in hapten-challenged mice, and in the immune lymph nodes, CD8 + cells were the major source of IFN-γ compared to CD4 + cells. Since IFN-γ is known to stimulate keratinocytes to produce IP-10 and Mig, it is suggested that these IFN-γ-producing CD8 + T cells enhance the production of these chemokines, thereby functioning as not only the effector cells but also the cytokine source to sustain the challenge reaction.

The effect of cyclosporin A, FK506 and rapamycin on the murine contact sensitivity reaction.

We have evaluated the effects of three potent immunosuppressive agents, cyclosporin A (CsA), FK506 and rapamycin, on the murine contact sensitivity (CS) reaction to the hapten trinitrochlorobenzene. Development of CS reaction requires participation of three distinct T cell subsets: alphabeta+, CD4+ T lymphocytes, which are the classical effector cell of the CS reaction, gammadelta+ T lymphocytes, and alphabeta+, double-negative (CD4- CD8-) T lymphocytes that express the B220 molecule and produce IL-4. We found that all three drugs inhibit the development of the CS reaction, but they affect different target cells. In fact, rapamycin and FK-506 block both alphabeta+, CD4+ and gammadelta+ T lymphocytes, while CsA inhibits only the alphabeta+, CD4+ T lymphocyte. None of the three drugs exerted any inhibitory activity on the alphabeta+, double-negative (CD4- CD8-) T lymphocytes. Hapten-immune lymph node cells from mice treated in vivo with CsA or FK506 failed to proliferate and to produce IL-2 when re-exposed to the specific antigen in vitro. In contrast, immune lymph node cells from mice that had been treated in vivo with rapamycin gave optimal antigen-specific proliferation and IL-2 production in vitro. The implications of these observations are discussed in relation to the use of these immunosuppressive agents for prevention of allograft rejection.

Essential role of TGF-β in the natural resistance to experimental allergic encephalomyelitis in rats

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease induced in susceptible rat strains by a single immunization with myelin basic protein (MBP). The Lewis (LEW) strain is susceptible to disease induction while the Brown Norway (BN) strain is resistant. This resistance involves non-MHC genes since congenic BN-1L rats, with LEW MHC on a BN-derived background, are also resistant. In the present study we show that, upon immunization with MBP, the non-MHC-encoded resistance to develop clinical EAE in BN-1L rats is associated with a decreased production of IFN-gamma. This may be due to a difference between LEW and BN-1L rats in their ability to produce regulatory cytokines such as IL-4, IL-10 and TGF-beta. In comparison to LEW rats, immune lymph node cells from BN-1L rats express an increased amount of IL-4 mRNA but produce less IL-10. Furthermore, the sera from BN-1L rats contain higher amounts of active TGF-beta1. Therefore, we have investigated the involvement of IL-4 and TGF-beta in the resistance of BN-1L rats to develop EAE using neutralizing mAb. Neutralization of TGF-beta, but not IL-4, renders BN-1L rats susceptible to clinical EAE without affecting the proliferation or the cytokine repertoire of immune lymph node cells. With respect to the origin of the endogenous TGF-beta production, we excluded the involvement of CD8 T cells and discuss a possible role of platelets and of CD4 T cells exhibiting the CD45RC(low) phenotype.