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.     

Nematode infection induces Th2 cell-associated immune responses in LEC mutant rats with helper T cell immunodeficiency

Long-Evans Cinnamon (LEC) rats have maturational arrest of CD4⁺8⁻ T cells from CD4⁺8⁺ cells in the thymus. Despite this, CD4⁺8⁻ T cells are always present in peripheral lymphoid organs of LEC rats, suggesting that these CD4⁺8⁻ T cells are generated by an uncommon pathway. We investigated the role of LEC rat peripheral CD4⁺8⁻ T cells in Th2-associated responses to infection with the nematode Nippostrongylus brasiliensis . After infection, the numbers of CD4⁺8⁻ TCRαβ⁺ T cells significantly increased in mesenteric lymph nodes (MLN) and the spleen, while those in the thymus were still negligible. Infection also induced significant up-regulation of IL-4 gene expression in LEC rat MLN cells. Total serum IgE levels in LEC rats were markedly increased two weeks after infection. Mucosal mast cell responses in the gut and lungs of LEC rats were induced as prominently as in control Long-Evans Agouti (LEA) rats. Faecal egg count data indicated that LEC rats rejected nematodes faster than LEA rats. These results suggested that Th2-associated responses can be induced by nematode infection in LEC rats probably through the extrathymic recruitment and proliferation of CD4⁺8⁻ TCRαβ⁺ T cells .     

CD3−CD4−CD8−CD56−CD19−CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4⁺ T cells. We found that IL-4 was produced by CD3⁻CD4⁻CD8⁻CD56⁻CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells in AIDS patients. Moreover, CD3⁻CD4⁻ CD8⁻CD56⁻CD19⁻CD14⁻ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4⁺ T helper cells, C3⁻CD4⁻CD8⁻CD56⁻ CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

Expression of CD8β and alteration of cell surface phenotype in adult T-cell leukaemia cells

Typical adult T-cell leukaemia (ATL) cells have a CD4⁺CD8⁻ cell surface phenotype, but atypical phenotypes such as CD4⁺CD8⁺ and CD4⁻CD8⁺ have also been reported. The CD8 molecule is composed of α and β chains and commonly used monoclonal antibodies against CD8 molecule detect only CD8α. Since it has been reported that CD8α can be induced in mature CD4⁺ T cells by cell activation, but not CD8β, we studied whether ATL cells which express CD8α may also express CD8β. We found some cases of CD8α⁺ ATL were also positive for CD8β. Furthermore, we experienced a case whose ATL cell surface phenotype changed from CD4⁺CD8α⁺CD8β⁺ to CD4⁻CD8α⁺CD8β⁺ and finally to CD4⁺CD8α⁻CD8β⁻. Southern blot analysis revealed that the monoclonal integration of human T lymphotropic virus type I (HTLV-I) was identical throughout the course of the study, indicating that a single clone had demonstrated the alterations. These data suggest that peripheral CD4⁺CD8⁺ ATL cells can express not only CD8α, but also CD8β and that a single ATL cell clone has the potential to change its surface phenotype in vivo as well as in vitro .     

CD3+ CD8+ CD56− clonal large granular lymphocyte leukaemia and HIV infection

High-grade malignant lymphomas associated with HIV infection are usually derived from B lymphocytes. Although a broad spectrum of T-cell-derived malignancies has been described, no case of monoclonal T large granular lymphocyte leukaemia has been reported to date. We report a case of clonal T-LGL (CD3⁺, CD4⁻, CD8⁺, CD56⁻, CD57⁺) in an HIV-infected, HTLV1/2-negative individual. Large granular lymphocytes are thought to represent activated cytotoxic T lymphocytes. HIV infection, as previously reported for HTLV1/2, may represent a pathway of antigen activation and lead to clonal expansion of T large granular lymphocytes.     

T-cell form of chronic lymphocytic leukaemia: a reaffirmation of its existence

Early in the 1980s three categories of T-cell chronic lymphocytic leukaemia were recognized: CD4⁺ CD8⁻ knobby type, CD4⁻ CD8⁺ azurophilic type and CD4⁺ CD8⁻ adult T-cell leukaemia (ATL) type. Both azurophilic and ATL types were later shown to be distinctive disorders, whereas the knobby type has been largely neglected and even considered non-existent by some authors. In this report we describe two patients with leukaemia of CD3⁺ CD4⁺ CD8⁻ post-thymic T lymphocytes presenting with marked lymphocytosis, generalized lymphadenopathy and hepatosplenomegaly. We believe that CLL of post-thymic T-lymphocytes is a distinct entity, and merits a separate designation from other T-cell leukaemias.     

Influence of melatonin therapy and orchiectomy on T cell subsets in male Wistar rats infected with Trypanosoma cruzi

Abstract:  Gonadal steroids exert an important influence on the host immune response during infection. Changes resulting from the absence or replacement of gonadal hormones may represent a distinct evolution of a particular parasite. Taking into account the greater susceptibility of males to parasites, the magnitude of the immune response seems to depend on the interaction of many hormones that will act synergistically with other immune cells. The aims of this research were to evaluate the effects of the luck of male sex hormones due to orchiectomy, and the influence of oral administration of melatonin on the immune response of male Wistar rats infected with the Y strain of Trypanosoma cruzi . The percentage of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ lymphocyte T cell subsets were evaluated using flow cytometry and the measurement of IL-2 and IL-12. For all parameters examined, a synergistic action of melatonin and orchiectomy on the host's immune response was observed, promoting an effective response against the parasite during the acute phase of infection. These results offer insight into other possibilities for possibly controlling T. cruzi proliferation through melatonin therapy and also the stimulatory effects on host's immune response triggered by the absence of male gonadal steroids during the acute phase of infection.     

In situ characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens

We characterized the leucocyte subpopulations after infection with Eimeria tenella in both naive and immune chickens. Immunocytochemical staining was used to characterize the cells in situ , so that the interaction between host and parasite could be studied. More leucocytes were detected in the lamina propria of immune chickens, and leucocytes infiltrated the ceca more rapidly than in naive chickens, but the infiltration was less pronounced than in naive chickens. In naive chickens, most infiltrated leucocytes were macrophages and T cells. Two days after inoculation the number of CD4⁺ cells had increased greatly. In immune chickens, mainly T cells (CD4⁺ and CD8⁺) infiltrated the lamina propria, and in contrast to naive chickens, the number of CD8⁺ cells exceeded the number of CD4⁺ cells. Furthermore, we characterized which cells contained a parasite and which cells were detected next to the parasites, because these cells are probably involved in the arrested development of the parasites. In naive chickens, sporozoites were significantly more often located within or next to macrophages than in immune chickens. In immune chickens, sporozoites were significantly more often located within or next to CD3⁺, CD8⁺, and TCR2⁺ cells. In conclusion, the marked increase of CD4⁺ cells after primary infection suggests that these cells are involved in the induction of the immune response, whereas the increase of CD8⁺ cells after challenge infection suggests that these cells act as effector cells.     

In pregnant mice, the infection of Toxoplasma gondii causes the decrease of CD4+CD25+ -regulatory T cells

Acute maternal infection with Toxoplasma gondii during pregnancy is associated with adverse pregnancy outcomes. Although previous reports have indicated that T. gondii may result in abortion without direct transmission of the parasite to the foetus, the molecular mechanism remains unclear. CD4⁺CD25⁺-regulatory T cells are known to be involved in maternal tolerance toward the foetus-bearing alloantigens. With a model of pregnant mice infected with T. gondii , we found that Foxp3 mRNA expression levels in both splenocytes and placenta were reduced markedly during the process of infection. Furthermore, the numbers of splenic CD4⁺CD25⁺-regulatory T cells and placental Foxp3⁺ cells decreased synchronously in the infected mice, and the reduction of splenic CD4⁺CD25⁺-regulatory T cells were associated with apoptosis induced by the infection. Additionally, injection of pregnant mice with excretory–secretory antigens (ESA) of T. gondii also resulted in foetal loss, which could be partly prevented by adoptive transfer of CD4⁺CD25⁺-regulatory T cells from normal pregnant mice. These data suggest that foetal loss caused by T. gondii can be independent of vertical infection and that the decrease of CD4⁺CD25⁺-regulatory T cells during infection may represent a previously unrecognized mechanism for the pathogenesis of abortion caused by this parasite.     

Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation

The main objective of the present study was to determine the role of CD34⁺ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34⁺ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34⁺ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 10⁶ CD34⁺CD38⁻ cells for neutrophil engraftment, and 2.0 × 10⁶ CD34⁺CD38⁺ cells for platelet recovery. It is suggested that the analysis of CD34⁺ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34⁺ cells are collected.     

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.     

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 .     

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.     

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10⁺CD19⁺ cells and CD20⁻CD22⁺ cells in the CD34⁺ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34⁺CD10⁺CD19⁺ and CD34⁺CD20⁻CD22⁺ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.     

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 .     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

Identification of early plasma cells in peripheral blood and their clinical significance

In the peripheral blood (PB) we detected so-called early plasma cells that might already be committed to entering the bone marrow (BM). By two-colour staining with FITC–anti-CD38 antibody, their intensity (CD38⁺⁺) of expression of CD38 antigen was between that of germinal centre (GC) B cells (low expression (CD38⁺)) and that of BM plasma cells (high expression (CD38⁺⁺⁺)), and their phenotype was CD38⁺⁺ CD19⁺ CD10⁻ CD20⁻ CD21⁺ CD24⁻ CD39⁺ CD5⁻ VLA-4⁺ VLA-5⁻ MPC-1⁻ without expression of surface membrane IgM (SmIgM). Morphological and immunological examination of the sorted cells confirmed that they were plasmacytoid cells with expression of cytoplasmic IgG (cIgG). Variations of these early plasma cells were examined in various diseases. In active systemic lupus erythematosus, bacterial septicaemia and liver cirrhosis, early plasma cell levels were significantly increased in PB, and after subsidence of such inflammation (inactive states) these cells returned to normal levels. In contrast, normal early plasma cells were significantly suppressed in myelomas, whilst normal or slightly increased numbers of early plasma cells was found in benign monoclonal gammopathy (BMG). In addition, the number of normal early plasma cells returned to a normal level in myeloma cases with complete responses. Therefore, early plasma cells were identified phenotypically, and an increase and decrease in these cells in PB may reflect mobilization and suppression, respectively, of activated B cells into BM plasma cells.     

Foxp3 expression on normal and leukemic CD4+CD25+ T cells implicated in human T-cell leukemia virus type-1 is inconsistent with Treg cells

Foxp3 is a master gene of Treg cells, a novel subset of CD4⁺ T cells primarily expressing CD25. We describe here different features in Foxp3 expression profile between normal and leukemic CD4⁺CD25⁺ T cells, using peripheral blood samples from healthy controls (HCs), human T-cell leukemia virus type-1 (HTLV-1)-infected asymptomatic carriers (ACs), patients with adult T-cell leukemia (ATL), and various hematopoietic cell lines. The majority of CD4⁺CD25⁺ T cells in HCs were positive for Foxp3, but not all CD4⁺CD25⁺ T cells in ACs were positive, indicating that Foxp3 expression is not always linked to CD25 expression in normal T cells. Leukemic (ATL) T cells constitutively expressing CD25 were characteristic of heterogeneous Foxp3 expression, such as intra- and inter-case heterogeneity in intensity, inconsistency with CD25 expression, and a discrepancy in the mRNA and its protein expression. Surprisingly, a discernible amount of Foxp3 mRNA was detectable even in most cell lines without CD25 expression, a small fraction of which was positive for the Foxp3 proteins. The subcellular localization of Foxp3 in HTLV-1-infected cell lines was mainly cytoplasmic, different from that of primary ATL cells. These findings indicate that Foxp3 has two facets: essential Treg identity and molecular mimicry secondary to tumorigenesis. Conclusively, Foxp3 in normal T cells, but not mRNA, is basically potent at discriminating a subset of Treg cells from CD25⁺ T-cell populations, whereas the modulation of Foxp3 expression in leukemic T cells could be implicated in oncogenesis and has a potentially useful clinical role.