Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen.

Paramyosin (Sm97), a 97-kDa myofibrillar protein identified by the unusually monospecific antibody response induced by intradermal vaccination of mice with a complex soluble worm antigen preparation (SWAP) of adult Schistosoma mansoni administered with bacillus Calmette-Guérin (BCG), was purified and tested for its capacity to protect mice against challenge infection. When administered intradermally with BCG at total doses of only 4-40 micrograms per mouse, both the native molecule and a recombinant expression product containing approximately 50% of the whole protein were found to confer significant resistance (26-33%) against challenge infection, while 2 mg of unfractionated SWAP was required to induce similar levels of protection. In addition, paramyosin was shown to stimulate T lymphocytes from vaccinated mice to produce lymphokines [e.g., gamma interferon (IFN-gamma)] that activate macrophages to kill schistosomula. Neither schistosome myosin nor a heterologous paramyosin from a different invertebrate genus were protective, indicating a requirement for specific epitopes in the immunization. That the protection induced by paramyosin involves a T-cell-mediated mechanism was supported by the failure of anti-paramyosin antibodies to passively transfer significant resistance to infection to recipient mice. Lymphocytes from mice vaccinated with paramyosin were found to produce IFN-gamma in response to living schistosomula, suggesting that during challenge infection of vaccinated hosts, paramyosin (a nonsurface antigen) may elicit a protective T-cell response as a consequence of its release from migrating parasite larvae. Paramyosin-depleted SWAP was also found to be protective as well as stimulatory for T lymphocytes from SWAP-vaccinated mice, indicating that other antigens in this preparation may have immunoprophylactic potential. In summary, these results (i) suggest that the induction of T-cell-dependent cell-mediated immunity against soluble nonsurface antigens may be an effective strategy for immunization against multicellular parasites and (ii) in the case of schistosomes, identify paramyosin as a candidate vaccine immunogen in this category.     

Haemophagocytic lymphohistiocytosis in association with granular lymphocyte proliferative disorders in early childhood: characteristic bone marrow morphology

Five paediatric cases of haemophagocytic lymphohistiocytosis (HLH) which showed proliferation of granular atypical lymphocytoid cells in bone marrow are reported. All cases were girls aged 8 months to 4 years who had marked hepatosplenomegaly. Marker analysis on peripheral blood mononuclear cells revealed an increase in the CD3⁺HLADR⁺ subset in three cases and the CD³⁻CD56⁺ subset in one case. An Epstein-Barr virus genome was detected in three cases, and monoclonality was confirmed in two cases. A characteristic morphology of large granular lymphocytes (LGL) was identified, with elongated bizarre features that resembled horsetail-, tadpole-, cucumber- or shooting star-type configurations on the bone marrow smear. Serum concentrations of soluble interleukin-2 receptor and interferon-gamma were elevated in all cases. All five cases required multi-agent chemotherapy which resulted in two complete remissions, two partial remissions and one no response. Refinement of treatment is required for these paediatric GLPD cases which probably comprise a specific high-risk subgroup among secondary HLH patients which had previously escaped notice.     

Alloreactive CD4+ T lymphocytes can exert cytotoxicity to chronic myeloid leukaemia cells processing and presenting exogenous antigen

Existing evidence supports that CD4⁺ T lymphocytes play a role in the graft-versus-leukaemia (GVL) reaction after allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML), not only as initiators of the immune response but also as effectors of GVL. In BMT between HLA-identical pairs this CD4-mediated GVL would require CML cells to process and present antigens through MHC class II molecules. To investigate whether CML cells are capable of processing and presenting antigens, and suitable targets for CD4⁺ T-cell-mediated cytotoxicity, we generated HLA-DR1-restricted CD4⁺ cytotoxic T-cell clones that specifically recognized tuberculous purified protein derivative (PPD). We have shown that CML cells and B lymphoblastoid cell line (B-LCL) cells but not PHA-blasts from patients with CML processed exogenous antigen, PPD, and induced proliferative and cytotoxic CD4⁺ T-cell responses. Antigen presentation was blocked by antibodies to HLA-DR but not to MHC class I and by treatment with chloroquine and brefeldin. This indicates that CML cells use a classic MHC class II antigen processing pathway to present PPD antigens to CD4⁺ T cells. Cytotoxicity to CML was shown by antibody blocking studies to be mediated mainly through fas antigen. These findings indicate that donor CD4⁺ T cells alone are sufficient to mediate GVL effects following allogeneic BMT for CML.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

Summary. In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117⁺ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients.
Our results show that most of normal BM CD117⁺ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117⁺ cells are CD34⁺, these cells displaying a different FSC/SSC distribution when compared to the CD117⁺/CD34⁻ cells. No CD117⁺/CD15⁺ and CD117⁺/CD10⁺ cells were detected and very few CD117⁺ cells (<1 × 10⁻³) expressing the HLADR⁻/CD34⁻, CD33⁺/HLADR⁻ and CD34⁺/HLADR⁻ phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117⁺/CD15⁺ blast cells and eight had the CD117⁺ phenotypes detected at low frequencies (<1 × 10⁻³) in normal BM.
In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117⁺/CD15⁺ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.     

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa -inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10–35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression on CD4⁺ and CD8⁺ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4⁺ and CD8⁺ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4⁺ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8⁺ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals ( P  < 0.0001), coinciding with an increase in CD8⁺ T cell numbers in these cultures. The data show that factors besides IL-2, and probably an imbalance in the percentages of CD4⁺ and CD8⁺ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa -inoculated rhesus monkeys .     

日本血吸虫成虫抗原合并蚯蚓抗原对小鼠胸腺淋巴细胞及亚群的影响

ＢＡＬＢ／ｃ小鼠经可溶性日本血吸虫成虫抗原（ＳＷＡＰ）或／和可溶性蚯蚓抗原（ＳＥＷＰ）免疫后,观察、计数其胸腺Ｔ细胞总数、活性Ｔ细胞、Ｌ３Ｔ４细胞以及Ｌｙｔ２细胞。结果显示,ＳＷＡＰ免疫组Ｌ３Ｔ４细胞明显增加（Ｐ＜０．０５）;ＳＥＷＰ免疫组４个指标则均无明显变化（Ｐ＞０．０５）;ＳＷＡＰ与ＳＥＷＰ混合制剂免疫组Ｔ细胞总数、活性Ｔ细胞以及Ｌ３Ｔ４细胞均显增加（Ｐ＜０．０５）。相关分析表明：ＣＤ＋４Ｔ细胞（即Ｌ３Ｔ４细胞）在ＳＷＡＰ所诱导的保护性免疫力中可能起重要作用,而ＳＥＷＰ存在细胞免疫以外的其它可能保护性免疫机制,但ＳＥＷＰ与ＳＷＡＰ有明显的协同作用     

SLEx expression of normal CD 34 positive bone marrow haemopoietic progenitor cells

Summary. We investigated sialylated Lewis x (sLe^x) antigen expression on CD 34 positive (CD 34⁺) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD 34⁺ bone marrow progenitors strongly expressed the sLe^x antigen. This contradicts previous publications which reported sLe^x expression on malignant blast cells but not on normal CD 34⁺ progenitor cells.     

Protective CD4+ T-cell lines raised against Plasmodium chabaudi show characteristics of either Th1 or Th2 cells

Splenic T-lymphocyte lines were established in vitro from Plasmodium chabaudi-infected NIH mice on days 16 and 20 of a primary infection, and from mice after two or three infections. Each line responded specifically to stimulation with a lysed soluble extract of P. chabaudi-infected erythrocytes (pRBC), and displayed a CD4⁺ (L3T4⁺) surface phenotype. Both the day 16 and 20 cell lines, when stimulated in vitro, secreted interleukin-2 (IL-2) and γ-interferon (IFN-γ), indicative of their belonging to the T helper 1 (Th1) CD4⁺ subset. In contrast, both lines derived from reinfected mice secreted interleukin-4 (IL-4) and provided helper activity in antibody production to P. chabaudi in vitro, and thereby had the characteristics of Th2 cells. All four T-cell lines provided significant protection to naïve mice infected with P. chabaudi. In immuno-compromised mice, the day 16 T-cell line was as protective as in naïve mice whereas the cell line from mice infected twice required the additional transfer of mature naïve splenic B cells to provide protection comparable to that seen in the immunocompetent naïve recipients. The results establish that protective immunity to P. chabaudi may be associated with the induction of CD4⁺ Tcells of either the Th1 or Th2 subset which confer protection against this malaria parasite by mechanisms independent of, and dependent upon, B-cell involvement, respectively.     

Schistosoma mansoni: genetic non-response to p40, the major protein antigen of the egg, reveals a novel mechanism enhancing IgM production during infection

p40 is the major protein antigen in eggs and miracidia of Schistosoma mansoni. Immunization with recombinant p40 produced in bacteria and with p40 from miracidia reveals a conventional immune response gene effect in which H-2^b mice fail to produce antibody against p40. This is true when either denatured recombinant p40 and non-denatured miracidial p40 are used as immunogens. In contrast, during infection all strains of mice produce antibodies to p40. However, non-responder H-2^b mice produce only IgM to p40 and never any IgG. Thus, H-2^b mice appear to be producing specific IgM to p40 in the absence of MHC-restricted T-cell help. The mechanism revealed in these non-responder mice might play an important role in stimulating the production of IgM 'blocking' antibodies to antigens from schistosomula which cross-react with egg antigens.     

The immune response and immunopathology in infection with Schistosoma mansoni: a key role of major egg antigen Sm-p40

The immune response and related granulomatous inflammation in infection with Schistosoma mansoni are ultimately dependent on SEA-sensitized CD4⁺ Th cells and comprise multiple pathways variously involving the activation and recruitment of different cell populations and the production of different inflammatory cytokines, all under the influence of regulatory genetic factors. The spontaneous down-regulation of granuloma formation (immunomodulation), in turn, is a well-known phenomenon, but the full extent of its precipitating factors is still uncertain. This review describes a pathway leading to immunomodulation that features at its centre the down-regulatory cytokine IL-10. This mechanism is attractive because it offers a cogent correlation between findings in the laboratory and those displayed by patients affected with the disease. The Sm-p40 antigen, a major component of schistosome eggs, elicits a strong CD4⁺ Th cell response in H-2^k mice that correlates with intense granuloma formation; in contrast, its immunogenicity is relatively minor during infection of other mouse strains that develop smaller granulomas. Of great interest is that the Sm-p40 antigen only elicits a Th-1 type cytokine response, a phenotype that remains constant even as the overall response to SEA shifts to a Th-2 type. The Sm-p40 molecule has a dominant epitope that is the target of CD4⁺ Th cells from infected H-2^k mice; indeed, a minimal peptide that bears the epitope binds to I-A^k. The importance of pursuing a systematic elucidation of the major egg antigens, resides in the exciting possibility of specifically desensitizing the CD4⁺ Th cells that mediate granuloma formation, which may achieve meaningful prevention or amelioration of clinical disease .     

Acute myeloid leukaemia expressing the leucocyte integrin CD11b — a new leukaemic syndrome with poor prognosis: result of an ECOG database analysis

While assessing the prognostic implications of immunophenotyping in 382 patients enrolled in treatment protocols of the Eastern Cooperative Oncology Group (ECOG) for de novo adult acute myeloid leukaemia, we identified 95 patients with a unique antigen profile characterized by high expression of the leucocyte integrin CD11b (CD11b⁺ AML). High expression of CD11b was defined as 32% positive blasts based on the retrospectively established prognostic cut-off point for this antigen. Although CD11b is normally expressed by mature monocytes, natural killer cells and granulocytes, leukaemic blasts in CD11b⁺ AML lacked other immunologic monocytic features (e.g. CD14 and CD122, the interleukin-2 receptor β chain) and demonstrated a high degree of immaturity, as reflected by a high incidence of blasts expressing the stem cell factor receptor, CD117, and few blasts positive for the myeloid differentiation antigen CD15. Furthermore, by FAB criteria, only 41% of CD11b⁺ AML cases were classified as M4/M5. Patients with CD11b⁺ AML had a low response rate (54%) when compared with acute monocytic leukaemia (AMOL; 82%, P  = 0.006) or AML overall (68%, P  = 0.031), independent of age, cytogenetic abnormalities and P-glycoprotein expression. Because of its poor prognosis, recognition of CD11b⁺ AML is clinically warranted and, given its morphologic and cytogenetic ambiguity, must be based on the unique antigen profile.     

Major role for CD8+ T cells in the protection against Toxoplasma gondii following dendritic cell vaccination

Toxoplasma gondii is the causative agent of toxoplasmosis, a worldwide zoonosis for which an effective vaccine is needed. Vaccination with pulsed dendritic cells is very efficient but their use in a vaccination protocol is unconceivable. Nevertheless, unravelling the induced effector mechanisms is crucial to design new vaccine strategies. We vaccinated CBA/J mice with parasite extract-pulsed dendritic cells, challenged them with T. gondii cysts and carried out in vivo depletion of CD4⁺ or CD8⁺ T lymphocytes to study the subsequent cellular immune response and protective mechanisms. CD4⁺ lymphocytes were poorly implicated either in spleen and mesenteric lymph node (MLN) cytokine secretion or in mice protection. By contrast, the increasing number of intracerebral cysts and depletion of CD8⁺ cells were strongly correlated, revealing a prominent role for CD8⁺ lymphocytes in the protection of mice. Splenic CD8⁺ lymphocytes induce a strong Th1 response controlled by a Th2 response whereas CD8⁺ cells from MLNs inhibit both Th1 and Th2 responses. CD8⁺ cells are the main effectors following dendritic cell vaccination and Toxoplasma infection while CD4⁺ T cells only play a minor role. This contrasts with T. gondii infection which elicits the generation of CD4⁺ and CD8⁺ T cells that provide protective immunity.     

Germinal centre and marginal zone B cells expand quickly in a second Plasmodium chabaudi malaria infection producing mature plasma cells

Antibodies and B cells are critical in the protective immune response to the blood stage of the malaria parasite, Plasmodium chabaudi. However, little is known about the development of memory B cells and their differentiation into plasma cells during infection or after re-infection. Here we have shown that B cells with phenotypic characteristics of memory cells (CD19 ⁺ IgD⁻ CD38 ⁺ , IgG1 ⁺ ) are generated in a primary Plasmodium chabaudi chabaudi infection of mice. In addition, we observed that germinal centre cells (CD19⁺, GL7⁺, MHCII^hi) and Marginal Zone B cells (CD19⁺CD23⁻IgD⁻) show faster expansion on re-infection than in the primary, though other subsets do not. Interestingly, though both IgM⁻ and IgM⁺ memory cells are produced, IgM⁺ memory cells do not expand on second infection. The second infection quickly produced mature bone marrow plasma cells (intracellular Ig^hi, CD138^hi, CD9⁺, B220⁻), compared to primary infection; which generates a very large population of immature splenic plasma cells (B220+). This analysis suggests that a memory B cell population is generated after a single infection of malaria, which on re-infection responds quickly producing germinal centres and generating long-lived plasma cells making the second encounter with parasite more efficient.     

Setting in motion the immune mechanisms underlying genetically determined resistance and susceptibility to infection with Leishmania major

The murine model of infection with Leishmania major has allowed the demonstration of a causal relationship between, on the one hand, genetically determined resistance to infection and the development of a Th1 CD4⁺ cell response, and on the other hand, genetically determined susceptibility and Th2 cell maturation. Using this murine model of infection, the role of cytokines in directing the functional differentiation pathway of CD4⁺ T cell precursors, has been demonstrated in vivo . Thus, IL-12 and IFN-γ have been shown to favour Th1 cell development and IL-4 is crucial for the differentiation of Th2 responses. Maturation of a Th2 response in susceptible BALB/c mice following infection with L. major is triggered by the IL-4 produced during the first two days after parasite inoculation. This IL-4 rapidly renders parasite specific CD4⁺ T cells precursors unresponsive to IL-12. A restricted population of CD4⁺ T cells expressing the Vβ4Vα8 TCR heterodimer and recognizing a single epitope on the LACK (Leishmania Activated C-Kinase) antigen of L. major is responsible for this rapid production of IL-4, instructing subsequent differentiation towards the Th2 phenotype of CD4⁺ T cells specific for several parasite antigens .     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

日本血吸虫肺期童虫收集方法的实验研究

目的建立一种收集纯净肺期童虫的方法。方法用日本血吸虫尾蚴感染昆明小鼠,分别于感染后第1、2、3、4、5、6、7、10、12、14、21天解剖,从皮肤、肺脏、肠系膜静脉及肝门静脉处收集童虫。结果肺期童虫出现的高峰期为感染后第3天,获得最佳收获肺期童虫的时间,建立了回收大量纯净童虫的方法。