Impact of regulatory T cell therapy on immune cell composition and fetal survival rate in abortion prone mice.

CONTEXT: Implantation of fertilised eggs and survival of a semi-allogenic embryo rely on the interactions between the cells and molecules preparing the uterus. We investigated the effect of regulatory T cell (Treg) therapy on the mechanism of local immune tolerance of mice prone to spontaneous abortion. METHODS: Naive T cells were stimulated in vitro with 17ß-oestradiol (E2), progesterone (P4) and TGF-ß1 for 96h to generate induced Tregs (iTreg). The iTregs were injected into DBA/2-mated pregnant CBA/J female mice (abortion prone model). On day 14 of pregnancy, mice were killed and decidual and placental tissues were collected for cellular composition analysis. RESULTS: Abortion prone mice (PBS treated) showed significantly lower survival rates (P <0.0001), increased CD3+ CD8+ (P <0.05), lower IDO+ (P <0.05) and increased natural killer cells (uNK) cell numbers (P <0.001) in the uterus, as well increased NK cells in the placenta (P <0.05) than in normal pregnant mice (CBA/J×BALB/c). Adoptive transfer of iTregs increased fetal survival in abortion-prone mice (P <0.01) and histopathological evaluation revealed a significantly decreased number of uNK cells in the uterus of TGF-ß1-, E2- and P4-iTregs (P<0.05, P<0.0001 and P<0.05, respectively) than in the PBS treated group. In the placenta, we found significantly lower numbers of uNK cells from TGF-ß1-, E2- and P4-iTregs than in the PBS treated group (P <0.05, P <0.05 and P <0.01, respectively). CONCLUSIONS: We propose that modulation of uterine NK cell activity through immunotherapy using Treg cells should be given more attention as an immunological strategy in the treatment of recurrent miscarriage.

Adoptive cell therapy with induced regulatory T cells normalises the abortion rate in abortion-prone mice.

Ovarian hormones drive invivo generation of regulatory T cells (Tregs) during pregnancy. Little is known about the therapeutic potential of invitro hormone-derived Tregs in pregnancy loss. We investigated the effects of hormone-induced Tregs in a murine model of abortion. CD4+CD25- T cells were isolated from the spleens of CBA/J mice and stimulated with either 17ß-oestradiol (E2), progesterone (P4) or transforming growth factor-ß1 (TGFB1) plus retinoic acid (RA) for 4 days to generate induced Tregs (iTregs). On Days 1-4 of gestation, DBA/2-mated pregnant CBA/J female mice (abortion prone) were injected intravenously with iTregs or Tregs isolated from normal BALB/c-mated pregnant CBA/J mice (np-Tregs). On Day 14, the number of resorbed fetuses was assessed. Serum interferon (IFN)-γ and uterine forkhead box p3 (Foxp3) expression was analysed by ELISA and immunohistochemistry respectively. Using a 3H-thymidine incorporation assay, isolated CD4+CD25+ Tregs induced by the different treatments suppressed the proliferation of CD4+CD25- T cells. Adoptive transfer of iTregs (from all induction groups) significantly decreased fetal resorption in abortion-prone mice. There were no significant changes in serum IFN-γ concentrations after the adoptive transfer of iTregs or np-Tregs. Immunohistochemistry revealed significantly higher Foxp3 expression in gravid uteri from mice injected with np-Tregs and P4-induced iTregs than in the phosphate-buffered saline-treated group. The findings of this study indicate a potential therapeutic benefit of invitro-induced Tregs in patients with recurrent abortion.

Expression profiling of plac1 in murine cancer cell lines

AIM: Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2% to 42.5%. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization.

Frequency and expression of inhibitory markers of CD4(+) CD25(+) FOXP3(+) regulatory T cells in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is the most symptomatic primary antibody deficiency associated with recurrent infections and chronic inflammatory diseases as well as autoimmunity. CD4(+) CD25(+) FOXP3(+) regulatory T cells (Tregs) are critical T cell subsets for maintaining self-tolerance and regulation of immune response to antigens thus play a pivotal role in preventing autoimmunity. Thirty-seven CVID patients and 18 age-/sex-matched controls were enrolled. Peripheral blood mononuclear cells (PBMCs) were obtained from both groups, and the percentage of Tregs was calculated using flow cytometry method. The mRNA expression of Tregs' surface markers cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor receptor (GITR), which are associated with Tregs' inhibitory function, was compared between patients and controls by quantitative real-time PCR TaqMan method. The results revealed that the frequency of Tregs was significantly lower in CVID patients than normal individuals (P < 0.001). In addition, CVID patients with autoimmunity were found to have markedly reduced proportion of Tregs compared to those cases without autoimmune diseases (P = 0.023). A significant difference was seen in factor forkhead box P3 (FOXP3) expression between CVID patients and controls (P < 0.001). The mRNAs of CTLA-4 and GITR genes were expressed at lower levels in CVID patients compared to control group (P = 0.005 and <0.001, respectively). Our findings showed reduced proportion of Tregs in CVID patients together with downregulation of FOXP3 protein and diminished expression of inhibitory Tregs' markers. It could be concluded that all of these changes may be responsible for cellular immune dysregulation observed in these patients especially those with autoimmune manifestation.

Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells.

ROR1 is a receptor tyrosine kinase (RTK) recently identified to be overexpressed at the gene and protein levels in chronic lymphocytic leukemia (CLL). Monoclonal antibodies (MAbs) against RTKs have been successfully applied for therapy of solid tumors. We generated five MAbs against the Ig (n = 1), cysteine-rich (CRD) (n = 2) and kringle (KNG) (n = 2) domains, respectively, of the extracellular part of ROR1. All CLL patients (n = 20) expressed ROR1 on the surface of the leukemic cells. A significantly higher frequency of ROR1 expression was found in patients with progressive versus non-progressive disease, and in those with unmutated versus mutated IgVH genes. All five MAbs alone induced apoptosis in the absence of complement or added effector cells (Annexin-V and MTT, as well as cleavage of poly-(ADP ribose)-polymerase, caspase-8 and caspase-9) of CLL cells but not of normal B cells. Most effective were MAbs against CRD and KNG, significantly superior to rituximab (P < 0.005). Cross-linking of anti-ROR1 MAbs using the F(ab')(2) fragments of anti-Fc antibodies significantly augmented apoptosis. Two of the MAbs induced complement-dependent cytotoxicity (CDC) similar to that of rituximab and one anti-ROR1 MAb (KNG) (IgG1) showed killing activity by antibody-dependent cellular cytotoxicity. The identified ROR1 epitopes may provide a basis for generating human ROR1 MAbs for therapy.

Detectability of Her2 positive tumors using monoclonal antibody conjugated iron oxide nanoparticles in MRI.

Detecting an imaging signal from a small number of cells is vital when a disease needs to be diagnosed in an early stage of development. Molecular and genetic information from cancer cell types provide a guide for specific imaging based on gene expression and their activities on the cell membrane. Various physical and biological parameters affect the capability of an imaging system to provide an efficient procedure for biomarker imaging. Iron oxide based magnetic nanoparticles conjugated to breast cancer monoclonal antibody (Her2) were used as a specific contrast agent for detection of the tumor cells in nude mice models. All processes for the nanoparticle synthesis, antibody development, and conjugation strategies were designed and evaluated in the current work. The final engineered product was found to be without precipitate containing 20 microg antibody/mg magnetic nanoparticles at 10 mg Fe/ml solution. This contrast agent has a high affinity for the BT474 breast cancer cells. MRI images of nude mice bearing tumor cells confirm this specific biomarker based imaging protocol.

Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice.

OBJECTIVES: The presence of vitamin D receptor (VDR) and the identification of localized vitamin D3 synthesis in placenta and decidua implicate the importance of vitamin D3 in reproductive function. There is, however, no data on the expression profile of VDR in the mouse placenta and endometrium throughout the pregnancy period. STUDY DESIGN: In the present work expression of VDR in reproductive tissues of pregnant mice at different gestational phases has been addressed. Expression of VDR was determined by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. RESULTS: The results showed that VDR mRNA and protein were expressed in decidua, placenta and ovary throughout the pregnancy. VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy. Additionally, VDR expression level in decidua rose significantly as pregnancy progressed from early to mid stages. VDR expression in decidua of pregnant mice was higher in comparison to endometrium of non-pregnant mice. Immunohistochemical analysis revealed that VDR protein is consistently expressed by luminal and glandular epithelial cells of decidua, giant cells, glycogen rich cells and labyrinth cells of placenta and by almost all follicular cell types of ovary. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. CONCLUSION: It seems that expression of VDR in reproductive organs is finely tuned during pregnancy indicating its eminent role in reproductive biology.

Production and Purification of Anti-Rhombomys opimus Immunoglobulins.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is an increasing public health problem in some endemic regions. Horseradish peroxidase (HRP) conjugated rabbit anti-Rhombomys opimus (R. opimus) Ig is needed for immunoblotting and ELISA tests used to explore the immune response of the rodents against the sand fly saliva. In this study, the production of HRP conjugated rabbit anti-R. opimus Ig was conducted for the first time. METHODS: Rhombomys opimus Ig was purified from serum by protein G affinity chromatography column and injected into rabbit to produce anti-R. opimus Ig antibody. The titration of antibody against R. opimus Ig in rabbit serum was checked using indirect ELISA. Rabbit anti-R. opimus Ig was purified by Sepharose-4B-R. opimus Ig column. Reactivity of this antibody was assessed by indirect ELISA and was conjugated to HRP by periodate method. RESULTS: Approximately 3.5 mg Ig was purified from 1 ml R. opimus serum using protein G affinity chromatography column. The molecular weight of purified R. opimus Ig was estimated about 150 kDa by SDS-PAGE. Nearly 2.3 mg rabbit anti-R. opimus Ig was purified from 1 ml immunized rabbit serum. The purified antibody was conjugated to HRP and the optimum titer of HRP conjugated rabbit anti-R. opimus Ig was determined as 1:8000 using direct ELISA. CONCLUSION: HRP conjugated rabbit anti-Gerbil IgG has been produced by a few companies, but to our knowledge HRP conjugated rabbit anti-R. opimus Ig is not commercially available. Production of HRP conjugated rabbit anti-R. opimus Ig is considerably helpful for immunological studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries.

Indoleamine 2,3-dioxygenase is expressed in the endometrium of cycling mice throughout the oestrous cycle.

Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan catabolism, is thought to be required to prevent the rejection of the allogenic fetus by maternal T cells and to protect against intra- and extra-cellular pathogens. Consequently, we studied the expression of IDO in the endometrium of female Balb/c mice during the oestrous cycle. At each phase, the endometrium was peeled away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR. The presence of IDO protein was confirmed in each phase by Western blotting and immunohistochemistry. Our results showed that IDO is expressed in the endometrium of cycling mice during all the phases of oestrous cycle. The expression of IDO was highest at the oestrus and lowest at the dioestrus. By means of Western blotting and immunohistochemistry, we obtained evidence that IDO protein is synthesised in the endometrium of cycling mice throughout the oestrous cycle. In accordance with RT-PCR results, IDO protein was predominant at the oestrus phase. IDO protein was mainly localised in the glandular and luminal epithelial cells. Our results support the concept of IDO providing a mechanism of innate immunity to protect from ascending infections of the female reproductive tract. In addition, considering the fact that mating only occurs during the oestrus phase, the high expression of IDO in this phase is likely to be a mechanism that induces immunological tolerance of the fetus.

Analysis of the immunoglobulin heavy chain variable region gene expression in Iranian patients with chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) results from clonal expansion of phenotypically mature but functionally immature B-lymphocytes. The incidence of this type of leukemia is low in Asian countries, whereas it is the most frequent type of leukemia in the West. Previous investigations mainly conducted in Western populations have demonstrated non-random rearrangement of certain immunoglobulin variable region heavy (VH) and/or light (VL) chain genes in different groups of B-CLL patients. Little is known about the profile of VH gene expression in Asian patients. In the present study, we determined the frequency of VH gene family usage in 59 Iranian patients with B-CLL. VH gene family of patients was determined by reverse transcriptase-polymerase chain reaction using VH1-VH7 family specific primers. The most frequently expressed VH gene family was found to be VH3 (45.8%) followed by VH4 (32.2%), VH1 (18.6%), VH5 (1.7%) and VH6 (1.7%), with no expression of VH2 and VH7 gene families. The results indicate a lower representation of the VH1 and VH2 gene families and a higher representation of the VH4 gene family in Iranian B-CLL patients compared to Western patients, suggesting involvement of ethnic and/or environmental factors in B-CLL disease initiation.

The distribution of Entamoeba histolytica and Entamoeba dispar in northern, central, and southern Iran.

The present study was carried out from August 1999 through February 2002 in order to determine the prevalence of Entamoeba histolytica and Entamoeba dispar in three different climatic regions of Iran by using a PCR-RFLP method. A total of 16,592 stool samples were randomly collected from different age-groups in central, northern, and southern Iran in both urban and rural areas. The samples were examined by direct and formalin-ether concentration methods. A total of 226 samples were positive for E. histolytica/E. dispar cysts. Of these, 101 isolates were cultured and maintained successfully in Robinson's medium and were identified by the PCR-RFLP method. The study showed that 92.1% of isolates were E. dispar and 7.9% were E. histolytica or mixed infections. The ratio of E. histolytica to E. dispar was higher in southern regions (tropical and subtropical) than in the other two regions. This study demonstrated that E. dispar is the predominant species found among "cyst passers" in Iran.

Identification of cross-reactive and restricted epitopes localized on human chorionic gonadotropin beta-subunit by monoclonal antibodies.

Human chorionic gonadotropin (hCG) belongs to the family of glycoprotein hormones. All members of the family are composed of an identical alpha subunit and structurally related beta subunit which confers biological specificity. Specific quantification and functional analysis of hCG require the use of monoclonal antibodies recognizing different epitopes of hCGbeta. This study describes the production and characterization of monoclonal antibodies (MAbs) to hCGbeta with no cross-reactivity to other glycoprotein hormones. Spleen cells from Balb/c mice immunized with hCG were fused with mouse SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of highly purified and recombinant glycoprotein hormones, their subunits and peptides representing the C-terminal end of hCGbeta (hCGbeta-CTP) by ELISA and immunoblotting. The affinity constant (K(aff)) was also determined by ELISA. Three murine hybridomas designated G5M1, B12M2 and F4M3 were obtained that secrete MAbs specific for hCGbeta. The G5M1 MAb reacts only with hCGbeta, hCGbeta-CTP and intact hCG with no detectable cross-reaction with hCGalpha or any of the other glycoprotein hormones. The specificity of B12M2 MAb is very similar to G5M1, but it does not react with hCGbeta-CTP. The F4M3 MAb also has similar specificity to G5M1 and B12M2, but it strongly cross-reacts with hLH. The affinity constant (Kaff) of G5M1, B12M2 and F4M3 was found to be 4.28 x 10(9), 5.2 x 10(8), and 1.97 x 10(9) M(-1), respectively. Our results indicate that G5M1 and B12M2 MAbs are specific for hCG and recognize epitopes restricted to hCGbeta, but F4M3 recognizes a common epitope expressed both on hCGbeta and hLHbeta.

Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII.

Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.

Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR.

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.

Analysis of T-cell receptor beta chain variable gene segment usage in healthy adult responders and nonresponders to recombinant hepatitis B vaccine.

One to 10 per cent of healthy adult individuals do not produce protective levels of anti-hepatitis B surface (HBs) antibodies, following a standard vaccination protocol. Lack of an HBs antigen (Ag)-specific T-cell repertoire is amongst the possible defects, which may lead to humoral unresponsiveness and is the main objective of this study. We analysed TcR BV (T-cell receptor beta chain variable) gene usage in T lymphocytes from nine healthy adult responders and six nonresponders to recombinant HB vaccine, before and after booster vaccination. CD4+ and CD8+ T-cell populations were isolated from peripheral blood mononuclear cells by magnetic beads, and the expression of TcR BV genes in each population was investigated by reverse transcription polymerase chain reaction and hybridization with specific probe. When the usage of each TcR BV gene within CD4+ and CD8+ T cells of the responders was compared with that of nonresponders, statistically significant difference (P < 0.01) was noted for BV5S2-3 gene family in CD4+ T cells of nonresponders. Furthermore, individual vaccinees were shown to overexpress several TcR BV genes. To characterize the T-cell repertoire and determine their clonal nature, analysis of CDR3 length polymorphism was performed. Our results show that T-cell response to HBsAg is generally oligoclonal and involves multiple BV families. Furthermore, overexpressed individual TcR BV genes and CDR3 length distributions in response to HBsAg are subject-dependent. In conclusion, our results are not in line with the notion that defective TcR repertoire may be an explanation for unresponsiveness to recombinant HBsAg vaccine.

Morphological and cytochemical characteristics of purified murine splenic dendritic cells.

Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult. In this study, we purified dendritic cells from mouse spleen by a three-step enrichment method and evaluated morphological and cytochemical characteristics of isolated cells. We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) and Non Specific Esterase (NSE) activity. In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the purity of isolated cells, we recommend that a combination of morphological and cytochemical studies be used.

Diminished Th1 and Th2 cytokine production in healthy adult nonresponders to recombinant hepatitis B vaccine.

Vaccination of healthy adults with recombinant hepatitis B (rHB) vaccine fails to induce a protective antibody response in a proportion of individuals. Imbalanced T-helper (Th)1/Th2 response has been attributed to the lack of specific antibody response to rHB vaccine. In this study, in vitro production of interleukin (IL)-2, interferon (IFN)-gamma and IL-10 was investigated in Iranian healthy adults vaccinated with rHB vaccine. Peripheral blood mononuclear cells (PBMC) were isolated from 18 high responders and eight nonresponders and stimulated with rHB antigen or phytohaemaglutinin (PHA) mitogen. The cytokines were quantitated in culture supernatants by sandwich enzyme-linked immunosorbent assay (ELISA). Our results demonstrated a significant decrease in the production of IL-2, IFN-gamma and IL-10 (P < 0.005) in response to rHB antigen. The levels of all cytokines induced by PHA were similarly represented in both groups of vaccinees. These findings suggest that unresponsiveness to rHB vaccine may be owing to inadequate Th1- and Th2-like cytokine production.

Dendritic cells in patients with non-progressive B-chronic lymphocytic leukaemia have a normal functional capability but abnormal cytokine pattern.

Dendritic cells (DC) are attractive candidates for use in vaccine-based immunotherapy. We have analysed the functional capability of DC generated in vitro from blood CD14(+) cells of chronic lymphocytic leukaemia (CLL) patients and healthy donors by culturing for 10 d with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4) and tumour necrosis factor-alpha (TNF-alpha). Two distinct DC populations were identified in patients as well as in controls. The majority of DC expressed CD11c and a minority also CD123. Most of the DC generated from both patients and controls exhibited a mature phenotype indicated by CD83 and major histocompatibility complex (MHC) class II expression, as well as by a characteristic morphology. Less than 1% of DC exhibited CD14. CLL DC had a similar expression of accessory molecules (CD54, CD80 and CD86) as control DC. The mean fluorescence intensity of CD80 and MHC class I molecules was significantly higher on CLL DC than on control DC (P < 0.05). At the gene level (real-time polymerase chain reaction) the expression of IL-10 was higher in CLL (P = 0.028) than in control DC. IL-1 beta and IL-12p(35) transcripts were also more abundant in CLL than in control DC but did not reach statistical significance. The expression of IL-4 and TNF-alpha was similar to that of control DC. The interferon gamma (IFN-gamma) gene expression level in CLL DC was decreased compared with control DC. DC of CLL patients had a similar capacity to stimulate in mixed leucocyte reaction as well as to present a recall antigen (PPD) as control DC. Thus, DC of CLL patients seem to have a normal function and may serve as antigen preserving cells for presentation of tumour antigens in a therapeutic vaccination approach. The mechanisms behind the observed increase in some surface molecules and the abnormal cytokine profile of CLL DC is not clear but might indicate pre-activation of DC in vivo, which may have a regulatory role in the pathobiology of CLL.

Autologous T lymphocytes may specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia.

This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that granulocyte-macrophage colony-stimulating factor (GM-CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were gamma-interferon (IFN), interleukin (IL)-2 and tumour necrosis factor (TNF)-alpha, but not IL-4. This profile suggests a type 1 anti-B-CLL T-cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T-cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T-cell DNA synthesis proliferation. The proliferative T-cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T-cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T-cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti-CLL T-cell immunity is warranted that may facilitate the development of effective anti-tumour vaccines in CLL.