Resistance of BALB/c mice to Leishmania major infection is associated with a decrease in the precursor frequency of antigen-specific CD4+ cells secreting interleukin-4

BALB/c mice are highly susceptible to infection with the protozoanparasite Leishmania major and develop a chronic fatal disease.They can, however, be manipulated to resist disease and thishas been shown to correlate with increased expression of IFN_–mRNA and the absence of IL-4 mRNA in the draining lymph nodesand spleens of these animals. Here we show that anti-IL-4 oranti-CD4 treatment of BALB/c mice resulted in a reduction inthe size of the lesion and in the number of parasites in thedraining lymph nodes compared with untreated mice. The precursorfrequency of CD4⁺ T cells proliferating in response to Leishmaniaantigens in vitro in the treated animals was not significantlydifferent from untreated animals. Analysis of the lymphokinessecreted by the clonal progeny of these cells showed that theprecursor frequency of IL-4 secreting clones was at least 10-foldlower in animals treated with either mAb. However, there wasno reciprocal increase in the precursor frequencies of IFN_–secreting clones. Comparisons of the total number of precursorsof specific CD4⁺ cells secreting IFN_– showed that anti-CD4-treatedanimals, which are resistant to disease, had considerably fewerfor the first 6 weeks than untreated mice with chronic disease.Protection of BALB/c mice was therefore associated with a reductionin the numbers of precursors of cells secreting IL-4 withouta concomitant increase in the number of precursors of IFN_–secreting cells.     

Establishment of resistance to Leishmania major infection in susceptible BALB/c mice requires parasite-specific CD8+ T cells

Although CD4⁺ T cells are generally accepted to be responsiblefor the determination of resistance to infection in experimentalmurine cutaneous leishmanlasis, a contribution of CD8⁺ lymphocytesto immunity can be demonstrated under certain well-defined conditions.Normally highly susceptible BALB/c mice can be rendered resistantto infection with Leishmania major promastigotes by a singleinjection of monoclonal anti-CD4 antibodies at the beginningof infection. Mice treated in such a way can heal their primarycutaneous lesions and acquire immunity to subsequent challengeinfection. Both the resolution of the primary infection andthe induced state of immunityto reinfection in these mice isshown to be dependent upon the anti-leishmanial effector functionsof CD8⁺ T cells. Furthermore, in contrast to control infectedBALB/c mice, which are unable to mount a delayed-type hypersensitivity(DTH) response to viable parasites, mice cured as a result oftreatment with anti-CD4 antibodies in vivo exhibit a strongDTH response, which can be significantly reduced by injectionof either anti-CD4 or anti-CD8 monoclonal antibodies prior toantigenic challenge with viable promastigotes. Moreover, increasednumbers of specific CD8⁺ T cells, able to transferLeishmania-specificDTH responses, were found in lymphold organs of BALB/c micerendered resistant to infection by immunointervention with anti-CD4monoclonal antibodies at the beginning of infection. Neutralizationin vivo of interleukin 4 during the course of infection in BALB/cmice also enables these otherwise susceptible mice to resolvetheir cutaneous lesions and to decrease the parasite burdenin infected tissues. CD8⁺ T cells are required for both of thesebeneficial effects. Taken together, these results indicate thatin the immune BALB/c mouse, as in the normally resistant CBAmouse, CD8⁺ lymphocytes are involved in the elimination of L.major and in the establishment and maintenance of immunity againstinfection with this parasite.     

Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression

BALB/c mice are susceptible to cutaneous lelshmanlasls uponinfection with Leishmania major while C57BL/6 are not. Thereis a major promastigote surface protease (PSP or gp63) whichis available in both native and recomblnant forms, and for whichthe primary amlno acid sequence is known. Immunization withPSP has been shown to offer some protection against challengewith the live organism. Therefore, we attempted to develop apeptide vaccine with PSP peptldes. In the first experiments,recall prollferatlve responses to PSP were measured using aset of 15mer peptldes spanning the entire PSP molecule whichallowed designation of major determinant regions in BALB/c,C57BL/6, and CBA mice. Several of these determinants were promiscuousand shared almost the identical core amlno acid residues inthe different strains. Immunization with major determinant peptldeswas recalled vigorously with L. major soluble antigen as wellas with PSP. The response to peptide was almost entirely T_h1as measured by a localized ELISA assay for single-cell productionof IFN-. A similar assay for IL-5, which overcomes problemsof sensitivity and inhibition by lymphoklnes produced by T_h1cells, Indicates very little production of T_h1 cells even byBALB/c. It was found that if a major responsive peak was examinedby recall with overlapping peptldes, the highest, central peptidegave a mainly Th1 response while the boundary, less efficientpeptldes gave more of a T_h2 response. Possible reasons for thiswere discussed. These results point to the importance of selectingthe exactly appropriate peptide in considering a vacclnogenthat might protect susceptible individuals. Even the choiceof a somewhat immunogenlc peptide within the determinant envelopemight actually exacerbate infection by steering the responsein a T_h2 direction.     

Protective effect of isoprinosine in genetically susceptible BALB/c mice infected with Leishmania major.

The effects of an immunopotentiating drug Inosine Pranobex (isoprinosine) were investigated in an experimental cutaneous leishmaniasis model. The highly susceptible BALB/c mice treated orally with isoprinosine developed significantly delayed onset of disease when infected with Leishmania major compared to untreated mice. The drug itself is not toxic to the parasite up to millimolar levels in vitro. The increase in resistance to L. major infection is accompanied by a marked decrease in the CD4+/CD8+ ratio and the leishmanial antigen-specific proliferative response of the spleen cells of isoprinosine-treated mice compared to untreated mice. There was a significant increase in the production of IFN-gamma but a decrease in the secretion of IL-3 and IL-4 by the spleen cells of isoprinosine-treated mice in response to concanavalin A with or without L. major infection compared to untreated controls. There was, however, no significant difference in the level of IL-2 production by the spleen cells between mice with or without isoprinosine treatment. These data are consistent with the interpretation that isoprinosine potentiates the resistance to leishmanial infection by up-regulating the host-protective Th1 cells and down-regulating the disease-promoting Th2 cells or, alternatively, by increasing CD8+ T-cell function.     

幽门螺杆菌全菌抗原口服免疫BLAB／c小鼠的免疫应答机理研究

目的 分析幽门螺杆菌全菌抗原的口服免疫应答反应。方法 ELISA分析免疫小鼠血清、唾液、粪便提取物特异抗体水平,ELISPOT分析胃粘膜、派伊尔小结（PP）抗原行异性抗体分泌细胞（ASC）,RT－PCR分析PP T细胞因子mRNA表达水平。结果 ①口服免疫可诱导强烈的血清IgG反应和唾液、粪便提取物特sIgA反应;②胃粘膜、PP结产生大量抗原特异性抗体分泌细胞（ASC）,尤以sIgA-ASC型居多,PP结抗原特异性形成细胞（ASC）数量与特异抗体水平密切相关;③加佐剂免疫组小鼠PP T细胞,体外抗原刺激下,早期高表达IFN－γ晚期高表达IL－4。结论 全菌抗原和粘膜佐剂免疫可诱导H.pylori特异的系统、粘膜免疫应答,局部sIgA可能在抗H.pylori感染中具有重要作用,肠粘膜免疫主要诱导部位PP早期表现为TH1型优势应答,晚期则转为TH2型优势应答。     

卡介苗联合Poly I:C接种对新生期小鼠脾脏T细胞的影响

目的 研究卡介苗(BCG)联合Poly I:C新生期接种对小鼠脾脏T细胞功能亚群发育的影响.方法 新生清洁级BALB/c小鼠32只,分4组:对照组、BCG组、Poly I:C组、BCG和Poly I:C联合接种组,新生期接种4周后取脾细胞用流式细胞仪检测CD3+ CD8+ IFN-γ+、CD3+ CD8- IFN-γ+、CD3+ CD8+ IL-4+ 、CD3+ CD8- IL-4+、CIM+ Foxp3+ T细胞的比例,其分别代表Tc1、TH1、Tc2、TH2和调节性T细胞(Treg)亚群.结果 BCG组、Poly I:C组、BCG和Poly I:C联合接种组TH1、Tc1细胞比例明显高于对照组(P＜0.05或P＜0.01),3个接种组间比较差异无统计学意义;Tc2、TH2和Treg比例各组间比较差异没有统计学意义;3个接种组TH1/TH2和总IFN-γ/IL-4比值明显高于对照组(P＜0.05或P＜0.01),联合接种组TH1/TH2比值高于BCG组(P＜0.05),Tc1/Tc2比值各组间差异没有统计学意义;CD4+ Foxp3+ T细胞比例各组间比较差异没有统计学意义.结论 新生期BCG和Poly I:C接种均能促进TH1、Tc1细胞亚群的发育,提高TH1/TH2比值,其联合接种在TH1/TH2水平可能具有一定的协同作用,但不能影响CD4+ Foxp3+ Treg细胞的比例.     

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-β concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-γ, TGF-β and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.     

Vaccination of BALB/c mice with Leishmania major amastigote-specific cysteine proteinase

Cellular immune mechanisms resulting in interferon-gamma (IFN-gamma) production are essential for protection against cutaneous leishmaniasis. Antigens of the intracellular amastigote form of the parasite, found in mammalian hosts, are likely to be good candidates for the induction of T cell response and protection from development of leishmaniasis. We purified a stage-specific antigen from amastigote soluble antigen (A-SLA) of Leishmania major by immunoaffinity chromatography. The purified protein was characterized as a cysteine proteinase with enzymatic activity which is inhibited by E-64, and it was named the amastigote cysteine proteinase (ACP). BALB/c mice were immunized by two intraperitoneal injections, at a month interval, of 5 microg of ACP or A-SLA in Freund's complete adjuvant (FCA). Animals were challenged 4 weeks later with 106 L. major promastigotes and examined 4 months after the last injection. The immunized animals developed significantly smaller or no lesions compared with controls. Spleen cells from immunized mice showed a significant proliferative response and produced a high level of IFN-gamma in response to ACP, suggesting the induction of Th1 cells after immunization. These results make 24-kD ACP a possible component for an eventual cocktail vaccine against L. major infection.     

Reconstitution of C.B-17 scid mice with BALB/c T cells initiates a T helper type-1 response and renders them capable of healing Leishmania major infection

C.B-17 scid mice, which were found to be very susceptible to infection with Leishmania major, were reconstituted with various doses of T cells, T plus B cells or unfractionated spleen cells from nonhealer BALB/c mice. All reconstitution protocols, except for the transfer of very high numbers of BALB/c spleen cells, led to a spontaneously healing infection and resistance to reinfection, rather than the lethal, nonhealing infection typical of BALB/c mice. These healing responses were associated with a strong T helper 1 (Th1)-like response characterized by delayed-type hypersensitivity (DTH) responsiveness, but no elevation of serum IgE, and by the production of high levels of interferon-γ (IFN-γ), but no interleukin-4 (IL-4) by lymph node and spleen cells after restimulation with antigen in vitro. The development of this Th1 response from BALB/c Th cells requires IFN-γ during the initial infection period. Treatment of scid mice with a single injection of neutralizing anti-IFN-γ antibody prior to infection and reconstitution prevented healing and permitted the development of a Th-2 like response as indicated by elevated serum IgE, but no DTH, and by the production of IL-4, but very little IFN-γ, after antigen stimulation in vitro. As few as 10⁴ transferred T cells led to a Th1-like response, suggesting that the IFN-γ is of host rather than donor origin. The transfer of very high numbers (7.5 x 10⁷) of BALB/c spleen cells overcame the effects of the IFN-y and led to the nonhealing infection and cytokine pattern characteristic of BALB/c mice. The enrichment or depletion of B cells from the transferred T cells had no measureable effect upon the development of a healing response in reconstituted scid mice.     

Coexistence of antigen-specific TH1 and TH2 cells in genetically susceptible BALB/c mice infected with Leishmania major

CD4-positive T cell clones with specificity for the protozoan parasite Leishmania major (L. major) of both the protective TH1 and the disease-exacerbating TH2 subtype were isolated from a diseased L. major-infected mouse of the susceptible BALB/c strain. In addition, TH2 cells were isolated from the lesion-draining lymph nodes of an animal clinically healed nine months after sublethal irradiation and subsequent infection. Our data support the notion that the differential outcome of the disease in non-irradiated versus irradiated BALB/c mice reflects the regulation of the two CD4+ T cell subsets. These data also argue against the possibilities that: 1) TH2 cells and their precursors are totally eliminated by irradiation and that 2) TH2 cells are capable of completely hindering the expansion of TH1 cells in diseased animals.     

Immunization with a Toll-like receptor 7 and/or 8 agonist vaccine adjuvant increases protective immunity against Leishmania major in BALB/c mice

Activation of Toll-like receptors (TLRs) on antigen-presenting cells of the innate immune system initiates, amplifies, and directs the antigen-specific acquired immune response. Ligands that stimulate TLRs therefore represent potential vaccine adjuvants. In the present study, we determined whether imiquimod and its related compound R848, which are TLR7 and/or TLR8 agonists, represent potential vaccine adjuvants when delivered topically, subcutaneously, or intramuscularly. Using the Leishmania major infection model in BALB/c mice, vaccination with crude Leishmania antigen was not protective against subsequent challenge infection unless it was administered with R848 or a topical application of imiquimod containing cream on the skin. Subcutaneous vaccination with these adjuvants mediated a TH1 response against L. major antigen, which appeared to suppress the TH2 response following a challenge infection. Protective immunity was generated following subcutaneous vaccination but not intramuscular vaccination. These observations suggest that topically administered imiquimod or subcutaneously injected R848 represent potential vaccine adjuvants to enhance the TH1 response, which can be used with existing or new vaccine formulations.     

Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.     

BALB/c小鼠滴鼻免疫灭活SARS病毒对全身性免疫的影响

目的　了解灭活SARS病毒滴鼻免疫小鼠对全身性免疫的影响。方法　将 5周龄雌性BALB c小鼠随机分为两组 ,每组 10只 ,免疫组小鼠每只免疫 5 0 μg的灭活病毒悬液 ,对照组注射等体积PBS。分别在免疫的 8d和 14d处死小鼠 ,分离脾细胞和外周血淋巴细胞 ,流式细胞仪观察其淋巴细胞表型 ;间接ELISA法测定其血清中抗SARS病毒特异性抗体IgM和IgG。结果　第 8天脾脏的CD4 T细胞的构成比增加 (P =0 .0 2 7)。外周血淋巴细胞表型在第 8天即开始发生改变 ,在第 14天变化明显 ,与对照组差异有显著性 ,CD4 ,CD8 ,CD3 T细胞构成比和T B比值显著降低 (P <0 .0 5、0 .0 1、0 .0 1、0 .0 1) ,同时CD4 5R B2 2 0 细胞 (B细胞 )构成比显著增加 (P =0 .0 0 0 )。但是此时血清中特异性抗体未能被检测到。结论　在未应用佐剂的情况下 ,小鼠滴鼻接种灭活SARS病毒虽然可以导致诱导机体免疫细胞表型的变化 ,却未能诱导抗体的产生。     

T cells that react to the immunodominant Leishmania major LACK antigen prevent early dissemination of the parasite in susceptible BALB/c mice

Susceptibility of BALB/c mice to Leishmania major depends on the early production of IL-4 by CD4(+) T cells which react to the parasite LACK antigen. Here, we show that LACK-specific cells are rapidly recruited to the site of infection and favor the early dissemination of L. major to the internal organs.     

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4⁺ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β^–/– × TCR δ^–/–) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4⁺ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.     

Modulation of the allergic immune response in BALB/c mice by subcutaneous injection of high doses of the dominant T cell epitope from the major birch pollen allergen Bet v 1

Several in vitro and in vivo studies indicate that application of high doses of dominant T cell epitopes can induce a state of antigen-specific non-responsiveness (anergy). In the present study, we developed a murine model of an allergic immune response to Bet v 1, the major birch pollen allergen. Mice were sensitized by injection of rBet v 1 and the allergic state was proven by the presence of allergen-specific IgE and positive immediate-type skin tests to Bet v 1. In epitope mapping experiments, an immunodominant T cell epitope of Bet v 1 in BALB/c mice was identified by the use of overlapping peptides. This peptide (BV139) was subsequently employed for treatment. Two tolerization protocols were used: in one approach, the peptide was administered to naive mice before immunization (group BV139-S), in the second, already sensitized mice were treated (S-BV139). The results demonstrated that administering high doses of the dominant T cell epitope of Bet v 1 profoundly diminished T cell proliferation to the peptide in the BV139-S group, and to the peptide as well as to the whole protein in the S-BV139 group. Skin test reactivity to Bet v 1 was reduced in the BV139-S group. However, no differences in terms of specific antibody production between treated and untreated mice could be observed. This study provides evidence that administration of dominant T cell epitopes can down-regulate the allergen-specific T cell response. Proceeding on the assumption that the T lymphocyte response to allergens is crucial for the induction and maintenance of the allergic disease, a modulation of the immune response to allergens by treatment with T cell epitope peptides could represent a promising concept for immunotherapy in the future.     

口服幽门螺杆菌苗免疫小鼠后胃粘膜的免疫应答

目的 观测Hp全菌抗原和粘膜佐剂LT口服免疫Balb/c小鼠后的胃肠粘膜免疫反应。方法 采用ELISPOT法检测了胃粘膜、肠粘膜相关淋巴组织PP结抗原特异性形成细胞（AFC）。结果 抗原免疫组、抗原 佐剂组胃肠粘膜抗原特异性sIgA-AFC、IgG-AFC数量明显增加,尤以sIgA-AFC为甚。并且抗原 佐剂组明显高于单纯抗原组和对照组。结论 口服菌苗可有效诱导胃肠道粘膜免疫应答。局部sIgA可能在抗Hp感染中具有重要作用。     

Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions

 The study of experimental infection of inbred strains of mice with the intracellular protozoan parasite Leishmania major has contributed significantly not only to our understanding of this fascinating host/parasite relationship but also to that of many basic immunological phenomena. Much has been learned about the cognate interaction of antigen-specific T cells and antigen-presenting cells, about cytokine and T cell subset regulation, and the requirements for costimulation. Specifically, the immune response to experimental L. major infection is the paradigm for polarized T helper cell (Th) 1 and Th2 differentiation. In this model system a Th1 response characterized by interleukin (IL)-2 and interferon (IFN)-γ secretion leads to self-curing disease, whereas a Th2 response (IL-4, IL-10) leads to nonhealing disease. Numerous manipulations, including the injection of cytokines and of neutralizing anti-cytokine antibodies, cytokine transgene expression, and more recently cytokine and cytokine receptor gene knockout studies, have all provided intriguing new pieces to the still incomplete mosaic of our understanding of the immune response. Some of these findings were clearly unexpected and are still incompletely understood. For instance, based on earlier neutralizing anti-IL-4 monoclonal antibody injection studies, IL-4 gene-disrupted BALB/c mice were expected to be unable to mount the biased Th2 response typical of the IL-4^+/+ wild-type mice and to be able to control their lesions; quite unexpectedly, the BALB/c IL-4 knockout mice remain unable to heal their L. major infection. Based on these unexpected findings, we reexamine the literature in an attempt to resolve this apparent paradox and to relate the large body of experimental findings in the mouse system to that which is known about natural and experimental infections in the human. Received: 18 February 1997 / Accepted: 31 July 1997     

PELA幽门螺杆菌口服疫苗微球黏膜免疫研究

目的：应用复乳挥发法制备PELA泛影葡胺显影微球与缨门螺杆菌超声上清口服疫苗微球,并进行靶向与黏膜免疫研究。方法：采用CT技术,研究显影微球的靶向,PELA幽门曙杆菌超声上清口服疫苗微球口服免疫小鼠后,运用ELISA法检测血清,唾液,肠粘液的抗体改变情况,ELISPOT法分析派伊尔氏结（PP结）抗原特异性抗体形成细胞（ASC）的数量增减,结果;口粒径在10um以下的微球,首先粘附在胃肠黏膜表面,后投递于PP结;H.pylori疫苗微球免疫后可诱导较高的唾液sIgA水平和肠道sIgA反应,PP结抗原特异性抗体形成细胞（ASC）数量与肠道 sIgA水平密切相关,结论：可生物降解的PELA微球可用于靶向口服疫苗的研究。