Higher Percentage Of CD4 Research Articles

Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2rγnull Mice Engrafted with Mobilized Human Hematopoietic Stem Cells.

We have developed, characterized, and validated a new genetic stock of IL-2r common γ (gamma) chain deficient NOD/LtSz-scid (NOD-scid IL2rγnull) mice that support high levels of human hematopoietic stem cell (HSC) engraftment and multilineage differentiation. Histology, flow cytometry, and functional assays document a severe depletion of lymphocytes and NK cells in NOD-scid IL2rγnull mice. These mice survive beyond 16 months of age and untreated as well as sub-lethally irradiated NOD-scid IL2rγnull mice are resistant to the development of lymphomas and are “non-leaky” throughout life. Intravenous injection of sub-lethally irradiated NOD-scid IL2rγnull mice with 7 x 105 human mobilized CD34+ stem cells leads to high levels of multilineage engraftment. At 10 weeks after engraftment, percentages of human hematopoietic CD45+ cells are six-fold higher in the bone marrow of NOD-scid IL2rγnull mice as compared to NOD-scid controls. Human CD45+ cells include immature and mature B cells, NK cells, myeloid cells, plasmacytoid dendritic cells and HSCs. Spleens from engrafted NOD-scid IL2rγnull mice contain high percentages of immature and mature B cells but low percentages of T cells. Treatment with human Fc-IL7 fusion protein leads to a high percentage of human CD4+CD8+ immature thymocytes and high percentages of CD4+CD8− and CD4−CD8+ mature human T cells in the spleen and blood. Validation of de novo human T cell development was carried out by quantifying T cell receptor excision circles in thymocytes and by analyses of TCRβ repertoire diversity. Human T cell function was evidenced by proliferative responses to PHA and streptococcal superantigen. NOD-scid IL2rγnull mice engrafted with human HSC generate differentiated functional human T and B cells and provide an in vivo model of multilineage human hematopoietic cell engraftment.

ZAP-70 Versus CD38 Expression as the Prognostic Factors in B-Cell Chronic Lymphocytic Leukemia.

B-CLL is a heterogeneous disease with a highly variable clinical course. The mutational status of immunoglobulin heavy-chain variable region (IgVH) genes in the leukemic cells seems to be of great importance in assessing the prognosis in B-CLL. Recent studies have shown that both CD38 and ZAP-70 might be surrogate markers for IgVH mutation identifying patients with a more aggressive clinical course. The aim of our study was to estimate the clinical value of both ZAP-70 and CD38 as B-CLL prognostic factors.The analysis with use of flow cytometry technique was performed at diagnosis in the group of 100 B-CLL patients. 39/100 persons were defined as ZAP-70 positive and 61/100 persons as ZAP-70 negative (based on 20% cut off value in the CD19+/CD5+ population). ZAP-70+ patients had significantly higher lymphocytosis (p=0.003) and LDH level (p=0.009), while hemoglobin (p=0.049) and platelet levels (p=0.002) were significantly lower than in ZAP-70− patients. The proportion of CD19+/CD5+ cells expressing ZAP-70 correlated significantly with stage of disease (p=0.035). Patients with Rai stage III–IV (32/100 patients) had higher percentage of CD19+/CD5+/ZAP-70+ cells than patients with stage 0-II (68/100 patients) (24.81±17.40% and 14.89±13.54%, respectively, p=0.008). The mean treatment-free interval was longer in the ZAP-70 negative (8.46±4.63 months) than in the ZAP-70 positive patients (1.16±0.28 months). Furthermore, patients requiring treatment (57/100) had significantly higher percentage of CD19+/CD5+ cells expressing ZAP-70 protein than untreated patients (20.18±15.09 and 13.44±11.84, respectively, p=0.034). In the midst of patients requiring treatment, percentage of CD19+/CD5+/ZAP-70+ cells was significantly lower in patients achieving remission after first line therapy (26/57) than in patients with progressive disease (31/57) (p=0.004).According to CD38 analysis the patients with ≥ 20% of CD38 expression were considered positive (CD38+) and those < 20% were considered negative (CD38−). Patients CD38 positive, like the ZAP-70 positive patients, had more advanced Rai stage of disease, significantly higher lymphocytosis and LDH serum level. Patients requiring treatment had higher percentage of CD19+/CD5+CD38+ cells than untreated patients, however no statistical significance was detected. Percentage of CD19+/CD5+CD38+ was not a dicriminating factor between patients with good response to the first line therapy and those with progressive disease.The obtained results indicate that both ZAP-70 and CD38 expression may be prognostic markers identifying a subgroup of B-CLL patients with aggressive clinical presentation. However, the ZAP-70 expression seems to have more prognostic power, especially in predicting response to the first line therapy.

Rat Bone Marrow Mesenchymal Stem Cells Cotransplanted with BM Improve Hematopietic Reconstitution and Immunoreconstitution with Alleviating Graft-versus-Host Disease.

Prolonged immunodeficiency after allogeneic bone marrow transplantation (BMT) causes significant morbidity and mortality from infection, while graft versus host disease (GVHD) after allogeneic BMT and immunosuppression therapies against GVHD further deteriorate this process. Adult bone marrow mesenchymal stem cells (MSC) have recently been shown to inhibit T-cell proliferation and reduce GVHD after allo-BMT. In this study, we characterized the effect of MSC on immunoreconstitution and hematopoietic-reconstitution after bone marrow transplantation. BMT model from Fischer344 rats (RT-1Al) to WF rats (RT-1Au) was established for this experiment. Effects of MSCs on hematopoietic reconstitution, immunoreconstitution and GVHD were studied by survival rate, peripheral blood counts, histological analysis and FACS at day +30 after transplantation. Immune function recovery were assessed by lymphocyte proliferation reaction stimulated with ConA and LPS and allogeneic mixed-lymphocyte reaction. At day 30 postransplant, compared with BMT groups, we observed that cotransplantation of MSC and bone marrow promoted the recovery of peripheral blood white blood cells (5.47±1.11x109/L vs7.12±1.70x109/L, p<0.05), lymphocytes and platelets. Accordingly, it was noticed that cotransplantation of MSC with BM not only improved recovery of bone marrow cellularity, but also enhanced B lymphopoiesis (4.66±1.03x109/L vs 6.05±1.39x109/L, p<0.05) and megakaryocytopoiesis (402.50±63.70 x109/L vs 594.33±121.09x109/L, p<0.05). MSC was also shown to improve thymic and spenic architecture reconstitution. Histological analysis showed that near normal thymus architecture and normal spenic architecture, with well-developed red and white pulp and intact lymphoid follicles in the cotransplantation group, while loss of demarcation between cortex and medulla in the thymus and lymphocytic depletion of spenic arteriolar sheaths in BM transplantation rats.The total number of thymus cells and spleen cells were also increased in cotransplantation group compared to only BM transplantation group. Most notable, the higher percentage of CD3+CD4+ was observed in the spleens of cotransplantation group (11.47±3.68% vs 19.14±4.03%, respectively), while no difference in the percentage of CD3+CD8+ between two groups (10.61±3.37% vs 12.13±2.27%, respectively). So the ratio of CD4+/CD8+ cells increased and inclined to normal in cotransplantation groups compared to only BM transplantation group.There are no difference in the percentage of natural killer (NK) cells and monocytes between BMT group and BMT with MSC group. The immuno-inhibitory effect of MSC on reducing GVHD in recipients of an MHC-mismatched BMT was also evident in our study, nonetheless, splenic cells from recipients of MSC cotransplantation group displayed improved non-specific proliferation capability (against ConA LPS stimulation) and alloreactivity (against the third party splenocytes) compared to BM transplantation group. In conclusion, rat bone marrow mesenchymal stem cells cotransplanted with BM improves hematopietic reconstitution and immunoreconstitution with alleviating graft-versus-host disease.

Interaction of Human Mesenchymal Stem Cells with Alloantigen-Induced T Lymphocyte Activation Favors the Differentiation of CD4+ T Cell Subsets Expressing CD25 and/or CTLA4 Molecules.

Experimental evidence and preliminary clinical studies have demonstrated that human mesenchymal stem cells (MSCs) display important immune modulatory function of potential relevant interest in the setting of allogeneic hematopoietic stem cell (HSC) transplantation. Effectiveness of MSCs in controlling severe GVHD seems to be related to the immune-regulatory role they play in suppressing alloantigen-specific T-cell activation. Aim of the present study was to extend the analysis of the mechanisms responsible for the immune regulatory effect of interaction between MSCs and alloantigen-specific immune response elicited in vitro in primary and in secondary mixed lymphocyte culture (MLC). At difference with most previously reported studies, we decided to employ non-irradiated MSCs, reasoning that irradiation might impair, beside the proliferative capacity, also the differentiation capability of MSCs and, consequently, alter their interaction pattern with lymphocyte subsets. MSC were added to primary MLC at different doses (MLC-responder-PBMC:MSC ratios = 1:1 and 10:1). Dendritic cell (DC) differentiation, lymphocyte proliferation, alloantigen-specific cytotoxic activity and differentiation of CD4+ T-cell subsets expressing CD25 and/or CTLA4 antigens were assessed in primary and secondary MLC, comparing the effect observed using third-party MSCs with that obtained employing autologous to the MLC-responder (autologous) MSCs. Results demonstrated that human MSCs: (1) strongly inhibit alloantigen-induced DC1 differentiation; (2) down-regulate, in a dose-dependent manner, alloantigen-induced lymphocyte expansion, especially that of CD8+ T cells and of NK lymphocytes; (3) favor the differentiation of CD4+ T cells co-expressing CD25 and/or CTLA4, a phenotype associated with regulatory/suppressive function of immune response; (4) cause a dose-dependent reduction of alloantigen-specific cytotoxic capacity mediated by either cytotoxic T lymphocytes or NK cells; (5) exert more effective suppressive activity on MLC-induced T-cell activation when they are allogeneic rather than autologous with respect to responder cells. In particular, higher percentages of CD4+ and of CD4+CD25+ T cells co-expressing CTLA4+ were detected when third-party, rather than autologous, MSCs were added to MLC. These data suggest that T-cell recognition of alloantigens expressed by MSCs may further facilitate the preferential differentiation of activated CD4+ T cells expressing CTLA4, a glycoprotein, known to deliver an inhibitory signal to T cells and to mediate apoptosis of previously activated T lymphocytes. Several studies previously demonstrated that MSCs exert inhibitory effect on lymphocyte activation through the release of soluble factors. Our data suggest that the preferential differentiation of CD4+CD25+ regulatory T-cell subsets may be favored by other mechanisms of MSC-mediated inhibition of alloantigen-induced effector cell activation and expansion, and, in turn, these CD4+CD25+ cells contribute to propagate and extend suppressor activity. Altogether, our results provide immunological support to the use of MSCs for prevention of immune complications related to both HSC and solid organ transplantation and to the theory that MSCs are “universal” suppressors of immune reactivity.

An Immune Basis for Lung Parenchymal Destruction in Chronic Obstructive Pulmonary Disease and Emphysema

ABSTRACTBackgroundChronic obstructive pulmonary disease and emphysema are a frequent result of long-term smoking, but the exact mechanisms, specifically which types of cells are associated with the lung destruction, are unclear.Methods and FindingsWe studied different subsets of lymphocytes taken from portions of human lungs removed surgically to find out which lymphocytes were the most frequent, which cell-surface markers these lymphocytes expressed, and whether the lymphocytes secreted any specific factors that could be associated with disease. We found that loss of lung function in patients with chronic obstructive pulmonary disease and emphysema was associated with a high percentage of CD4+ and CD8+ T lymphocytes that expressed chemokine receptors CCR5 and CXCR3 (both markers of T helper 1 cells), but not CCR3 or CCR4 (markers of T helper 2 cells). Lung lymphocytes in patients with chronic obstructive pulmonary disease and emphysema secrete more interferon gamma—often associated with T helper 1 cells—and interferon-inducible protein 10 and monokine induced by interferon, both of which bind to CXCR3 and are involved in attracting T helper 1 cells. In response to interferon-inducible protein 10 and monokine induced by interferon, but not interferon gamma, lung macrophages secreted macrophage metalloelastase (matrix metalloproteinase-12), a potent elastin-degrading enzyme that causes tissue destruction and which has been linked to emphysema.ConclusionsThese data suggest that Th1 lymphoctytes in the lungs of people with smoking-related damage drive progression of emphysema through CXCR3 ligands, interferon-inducible protein 10, and monokine induced by interferon.

Comparison of decidual leukocytes following spontaneous vaginal delivery and elective cesarean section in uncomplicated human term pregnancy

The aim of this study was to quantify and compare leukocyte populations in term decidua basalis and parietalis obtained after spontaneous vaginal delivery (SVD) or elective cesarean section (CS) without labor. Decidua basalis and parietalis samples were obtained from placentas after SVD (n=20) and after CS (n=30). Following mechanical disaggregation, leukocytes were purified and stained with monoclonal antibodies. Percentages of leukocyte subclasses within the CD45+ cell fraction and activated T cells were determined by flow cytometry. No differences were found in the percentages of CD45+ cells or CD56brightCD16− uterine natural killer (NK) cells between decidua basalis from SVD and CS or between decidua parietalis from SVD and CS. In decidua basalis and parietalis from SVD, a significantly higher number of CD56dimCD16+ NK cells was found compared to CS. In decidua basalis from SVD, there was a significantly lower percentage of CD14+ cells and higher percentage of CD19+ cells compared to CS. The percentage of CD3+ T cells expressing CD25 or human leukocyte antigen (HLA)-DR was significantly decreased in decidua basalis and parietalis from SVD compared to CS. Comparison of decidua collected after SVD or CS suggests that labor is associated with dynamic changes in the distribution of decidual leukocytes, specifically NK and T cell subpopulations. In particular, the disappearance of the CD4+CD25+ T cell population, which possibly contains a subpopulation of regulatory T cells, may contribute to the initiation of labor. Further investigation into factors affecting decidual leukocytes may expand our understanding of the immunological events at the maternal–fetal interface.

Comparison of decidual leukocytes following spontaneous vaginal delivery and elective cesarean section in uncomplicated human term pregnancy*1

The aim of this study was to quantify and compare leukocyte populations in term decidua basalis and parietalis obtained after spontaneous vaginal delivery (SVD) or elective cesarean section (CS) without labor. Decidua basalis and parietalis samples were obtained from placentas after SVD (n=20) and after CS (n=30). Following mechanical disaggregation, leukocytes were purified and stained with monoclonal antibodies. Percentages of leukocyte subclasses within the CD45+ cell fraction and activated T cells were determined by flow cytometry. No differences were found in the percentages of CD45+ cells or CD56brightCD16− uterine natural killer (NK) cells between decidua basalis from SVD and CS or between decidua parietalis from SVD and CS. In decidua basalis and parietalis from SVD, a significantly higher number of CD56dimCD16+ NK cells was found compared to CS. In decidua basalis from SVD, there was a significantly lower percentage of CD14+ cells and higher percentage of CD19+ cells compared to CS. The percentage of CD3+ T cells expressing CD25 or human leukocyte antigen (HLA)-DR was significantly decreased in decidua basalis and parietalis from SVD compared to CS. Comparison of decidua collected after SVD or CS suggests that labor is associated with dynamic changes in the distribution of decidual leukocytes, specifically NK and T cell subpopulations. In particular, the disappearance of the CD4+CD25+ T cell population, which possibly contains a subpopulation of regulatory T cells, may contribute to the initiation of labor. Further investigation into factors affecting decidual leukocytes may expand our understanding of the immunological events at the maternal–fetal interface.

CD4+ and CD8+ T-lymphocyte subsets in Zambian children.

Age-related changes in lymphocyte subsets in HIV-uninfected Zambian children are described. The total lymphocyte count and numbers of CD4+ and CD8+ T-lymphocytes declined with increasing age, while the percentage of CD4+ and CD8+ T-lymphocytes changed little during childhood. Girls between the ages of 12 and 71 months had a higher percentage of CD4+ T-lymphocytes and a higher CD4:CD8 ratio than did boys of a similar age.

Expression of adhesion molecules on milk and blood lymphocytes from periparturient dairy cattle with Johne's disease.

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.

Different Trypanosoma cruzi strains promote neuromyopathic damage mediated by distinct T lymphocyte subsets.

The proliferative response of CD4 and CD8 T lymphocytes obtained from C3H/HeN mice chronically infected with Trypanosoma cruzi strains that differ in virulence, tropism and immunogenicity, was assayed against skeletal muscle, sciatic nerve and spinal cord homogenates. Although both CD4 and CD8 T lymphocytes from mice infected with the RA strain strongly proliferated against the nervous system, no response against skeletal muscle antigens was detected. CD4 and CD8 T lymphocytes from mice infected with the K-98 clone (from CA-I strain) showed low proliferative response against all the antigens assayed. To determine whether the proliferation patterns showed correlation with T cell-mediated neuromuscular damage, passive cell transfer studies were performed. Fifteen days after transfer of CD4 T cells from RA-infected donors (CD4-RA), normal syngeneic recipients displayed exclusively nervous tissue damage, such as perineural, endoneural and/or meningeal inflammatory infiltrates, with predominance of CD4 T cells. Fifteen days after transfer of CD4 T lymphocytes from mice infected with K-98 (CD4-K98), recipients showed inflammatory infiltrates only in skeletal muscle, where CD4 T lymphocytes and macrophages were predominant cells. Recipients of CD8 T cells from RA-infected mice (CD8-RA) showed lesions in both spinal cord and sciatic nerves. Higher percentages of CD8 T cells were observed in comparison with the recipients of CD4-RA or CD4-K98. In contrast, CD8 T cells from K-98-infected donors (CD8-K98) did not induce tissue damage. These results provide evidence that mice infected with T. cruzi populations that differ in their biological characteristics show diverse immune mechanisms that may be involved in the pathogenesis of peripheral nervous system damage.

Effect of age on immune parameters and the immune response of dogs to vaccines: a cross-sectional study.

The evaluation of anti-aging intervention strategies in dogs would benefit from reliable quantitative biomarkers of aging. In the present study, the expression of various immune parameters was measured in young and old dogs to identify potential biomarkers of aging. The second goal of the study was to determine the effect of age on the immune response to vaccines. The immune function, including the antibody response to vaccines, was determined in 32 young adult (3.15+/-0.8 years of age) and 33 old dogs (12.1+/-1.3 years of age) of various breeds. Old dogs had a significantly lower lymphocyte proliferative response and a lower percentage of CD4+ T cells and CD45R+/CD4+ T cells, and a higher percentage of CD8+ T cells and a higher concentration of serum and salivary IgA. The most significant differences (P<0.001) occurred in the lymphocyte proliferative responses to ConA and PHA, the CD4:CD8 ratio, and the percentage of CD45R+/CD4+ T cells suggesting that these parameters are potential biomarkers of aging. There was no difference in the percentage of total T and B lymphocytes and the concentration of serum IgM and IgG. Both groups of dogs had protective titers against distemper virus, parvovirus and rabies virus before annual revaccination. The pre-vaccination titer against rabies virus was higher in the old dogs than in the young dogs, and there were no differences in post-vaccination titers against any of the viruses. This suggests that annual vaccination protocols provide adequate protection for old dogs.

The potential palliative role and possible immune modulatory effects of low-dose total body irradiation in relapsed or chemo-resistant non-Hodgkin's lymphoma

In a group of 35 patients with relapsed and/or chemo-resistant non-Hodgkin's lymphoma (NHL), low-dose total body irradiation (LTBI) (+involved-field radiotherapy to bulky sites) achieved a complete remission rate of 29%, 2-years progression-free survival of 32% and a median progression-free survival of 12 months. The 2-year survival was 42% and the median survival was 17 months. Immuno-staining and flow cytometry of peripheral blood in 14 patients showed that LTBI leads to a significant increase in the percentage of CD4+ cells with a consequent significant increase in the CD4+/CD8+ ratio. High lymphocytic percent and a high percentage of CD4+ cells before LTBI were significantly correlated with longer response duration and overall survival. These data may suggest that the palliative potential of LTBI should be investigated as an alternative to chemotherapy in NHL patients. The pre-treatment percentage of lymphocytes and CD4+ cells may be used as predictors for response to LTBI.

CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression.

Active suppression by CD4+CD25+ regulatory T cells plays an important role in the down-regulation of the response of T cells to foreign and self antigens. Experimental tumor models in mice revealed that regulatory T cells inhibit antitumor immune responses. The purpose of the current study was to demonstrate the possible involvement of CD4+CD25+ regulatory T cells in immune system impairment in patients with gastrointestinal malignancies. The phenotypes of lymphocytes, particularly those of CD4+CD25+ T cells, were analyzed in peripheral blood in 149 patients with gastrointestinal malignancies and in ascites in 7 patients with peritoneal dissemination. In addition, cytokine production after in vitro stimulation was examined in CD4+CD25+ and CD4+CD25- T cells isolated from patients with malignant disease. Compared with healthy volunteers, patients with gastrointestinal malignancies had a higher proportion of CD4+CD25+ T cells in peripheral blood, due to the presence of a drastically smaller number of CD4+CD25- T cells. Among patients with gastric carcinoma, those with higher percentages of CD4+CD25+ T cells had a poorer prognosis than did those with lower percentages. CD4+CD25+ T cells also were present in greater proportions in ascites from patients who had advanced-stage disease with peritoneal dissemination. Isolated CD4+CD25+ T cells from patients with malignant disease produced interleukin (IL)-4 and IL-10 but not IL-2 or interferon-gamma; these cells also inhibited cytokine production by CD4+CD25- T cells after in vitro stimulation. The relative increase in CD4+CD25+ regulatory T cells may be related to immunosuppression and tumor progression in patients with gastrointestinal malignancies. This finding suggests that the use of immunomodulatory therapy to treat patients with gastrointestinal malignancies may be an effective strategy.

Marked elevation of human circulating CD4+CD25+ regulatory T cells in sepsis-induced immunoparalysis.

Immunoparalysis has recently emerged as a possible cause explaining the failure of clinical trials in septic shock. Because human peripheral blood CD4+CD25+ T cells have been characterized as suppressor T cells, we hypothesized they might be increased in sepsis-induced immunoparalysis. Prospective, observational, clinical study. Adult intensive care units in a university hospital. Patients with septic shock (n = 16) and healthy individuals (n = 36). None. In patients with septic shock (mortality rate at 28 days, 56%; mean admission Simplified Acute Physiology Score II, 47), we first illustrated immunoparalysis by showing a severe diminished monocytic human leukocyte antigen (HLA)-DR expression. Afterward, compared with control values, we found in these patients a marked elevation of circulating CD4+CD25+ T cells that were also CD45RO+ and CD69- and overexpressed CTLA-4. Importantly, nonsurvivors (n = 9) presented prolonged lower monocytic HLA-DR expression and higher percentage of CD4+CD25+ T-suppressor T cells. These data support the concept that the persistence of a pronounced immunoparalysis after septic shock is associated with a poor outcome. Whether CD4+CD25+ T cells directly participate in sepsis-induced immunoparalysis remains to be investigated.

IL-2 therapy and thymic production of naive CD4 T cells in HIV-infected patients with severe CD4 lymphopenia

IL-2 therapy increases memory and naive CD4 T cells in HIV-infected patients, but its effect on thymopoiesis is unknown. To investigate this effect, we quantified T-cell receptor rearrangement excision circles (TREC) in CD4 T cells from lymphopenic AIDS patients treated with highly active antiretroviral therapy and IL-2. CD4 cell subsets were evaluated by flow cytometry using anti-CD45RO/RA, CD62L, Ki67 and CD95 monoclonal antibodies. The proportion of recent thymic emigrant had been quantified by a real-time polymerase chain reaction assay for signal joint TREC in peripheral blood mononuclear and purified CD4 T cells. At initiation of IL-2, TREC copies/microl of blood were correlated with naive T cell numbers and age. Both naive and TREC numbers/microl significantly increased over time in all patients, with a wide range of TREC increases. Higher percentages of CD4+CD45RO-negative cells positive for the Ki67 cell-cycle marker were found in patients with a low TREC increase, but remained stable under IL-2. TREC and naive cell recovery were correlated; they also correlated with the numbers of TREC and naive cells at the start of IL-2, and with age, suggesting a thymic origin for naive T-cell recovery. A mathematical model showing the linear recovery of naive cells and TREC under IL-2 also strongly suggested that a naive T-cell increase reflects thymic export and involves little net death and proliferation. Although we cannot rule out a mechanism of altered proliferation or death rate, the thymus plays an important role in the long-term recovery of naive T cells under IL-2 therapy.

EFFECT OF CHRONIC HEPATITIS C INFECTION ON LYMPHOCYTE SUBSETS IN RENAL TRANSPLANT PATIENTS

Chronic hepatitis C (HCV) infection is prevalent in renal allograft recipients. Consequently, this group is more likely to develop liver complications and have poorer graft outcome. The interaction between viral and host factors under immunosuppressive therapy is important in highlighting the response to HCV infection after renal transplantation. The aim of this study was to assess changes that occur in the lymphocyte subsets of HCV‐infected kidney recipients, and to determine how these relate to allograft rejection. We investigated 50 patients (33 males and 17 females; mean age 32.0±9.9years; mean time since transplantation: 27.6±26.5months) who were receiving standard immunosuppressive therapy. Data concerning acute or chronic allograft rejection were retrieved from the patients' records. Lymphocyte subsets (CD4, CD8, CD3, CD19, CD16–56), serum albumin levels, and anti‐HCV positivity were evaluated after renal transplantation, and findings in the HCV‐positive and ‐negative groups were compared. Seven of the 50 patients had HCV infection. Anti‐HCV positivity was detected before transplantation in three cases, and these patients received interferon therapy before transplantation. Four patients were found to be HCV positive after transplantation. The anti‐HCV (+) patients had significantly lower serum albumin (P&lt;0.02), lower CD4/CD8 (P&lt;0.02) ratios, and a lower percentage of CD 19 (P&lt;0.001) and a higher percentage of CD8 (P&lt;0.01) cells. Chronic allograft rejection was observed in 71.4% of the HCV‐positive patients (P&lt;0.04), whereas no acute rejection was detected in this group. The findings indicate that HCV infection may alter the immune response in renal transplant recipients, and that the immunogenic potential of the virus may be higher in this patient group because of low serum albumin levels. It appears that this immunomodulatory action and low albumin levels may increase the risk of chronic rejection for HCV‐infected kidney recipients.

Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs)

Although a number of chemokine receptors display coreceptor activities in vitro, chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) remain the major coreceptors used by the human immunodeficiency virus type 1 (HIV-1). In this study, we used an envelope-mediated fusion assay to demonstrate low CCR4 coreceptor activity with some primary HIV-1 and simian immunodeficiency virus-1 (mac316) isolates in vitro. The coreceptor activity was sensitive to CCR4-specific antibodies and to the CCR4-specific chemokine ligand macrophage-derived chemokine (MDC)/chemokine ligand 22 (CCL22). Treatment of peripheral blood mononuclear cells (PBMCs; which express high levels of CCR4) with CCL22 caused down-modulation of endogenous CCR4 but had no significant effect on HIV-1 entry, suggesting that CCR4 may not be used as an entry coreceptor. Despite expression of other minor coreceptors on PBMCs, CCR5 and CXCR4 are preferentially used by HIV-1 isolates, as shown by chemokine-inhibition data. To determine the factors involved in this selective use, we analyzed CCR4 coreceptor activity and compared it with CCR5 use in PBMCs. We used a quantitative fluorescence-activated cell-sorting assay to estimate the numbers of CCR4 and CCR5 antibody-binding sites (ABS) on PBMCs. Although CCR4 was found on a higher percentage of CD4(+) cells, CCR5 ABS was twofold greater than CCR4 ABS on CD4(+) cells. Confocal microscopy revealed strong cell-surface CD4/CCR5 but weak CD4/CCR4 colocalization in PBMCs. Binding studies demonstrated that soluble gp120 had greater affinity to CCR5 than CCR4. The results suggested that coreceptor density, colocalization with CD4, and affinity of the viral gp120 to the coreceptor may determine preferential coreceptor use by HIV-1.

Shifts of T4/T8 T Lymphocytes from BAL Fluid and Peripheral Blood by Clinical Grade in Patients with Pulmonary Tuberculosis

We investigated the shifts of T4/T8 lymphocytes from BAL fluid (BALF) and peripheral blood by the clinical grade of pulmonary tuberculosis (TB), which is determined by factors such as extent of pulmonary involvement, fever, and loss of body weight. In order to explore these questions, BALF was collected from 45 patients presenting with active pulmonary TB and 14 healthy control subjects. The percentages for T-lymphocyte subpopulations, including CD4(+), CD8(+), and CD3(+) T cells, were measured using two-color flow cytometry. A higher percentage of CD3(+)CD4(+) T lymphocytes, with a relatively lower percentage of CD3(+)CD8(+) T lymphocytes, was revealed for the patients with a higher grade of pulmonary TB, compared to patients with a lower grade of pulmonary TB, resulting in an increased BALF C4(+)/CD8(+) ratio. By contrast, a higher percentage of CD3(+)CD8(+) T lymphocytes with a relatively low percentage of CD3(+)CD4(+) T lymphocytes was demonstrated for these patients with a higher grade of pulmonary TB, resulting in a decreased peripheral blood CD4(+)/CD8(+) ratio. Our findings suggest that compartmentalization of the CD4(+) T lymphocytes in the infected lungs may occur for patients with higher grades of pulmonary TB.

Murine Coronavirus Spike Glycoprotein Mediates Degree of Viral Spread, Inflammation, and Virus-Induced Immunopathology in the Central Nervous System

The mouse hepatitis virus (MHV) spike glycoprotein is a major determinant of neurovirulence. We investigated how alterations in spike affect neurovirulence using two isogenic recombinant viruses differing exclusively in spike. S4R, containing the MHV-4 spike gene, is dramatically more neurovirulent than SA59R, containing the MHV-A59 spike gene (J. J. Phillips, M. M. Chua, E. Lavi, and S. R. Weiss, 1999, J. Virol. 73, 7752–7760). We examined the contribution of differences in cellular tropism, viral spread, and the immune response to infection to the differential neurovirulence of S4R and SA59R. MHV-4 spike-mediated neurovirulence was associated with extensive viral spread in the brain in both neurons and astrocytes. Infection of primary hippocampal neuron cultures demonstrated that S4R spread more rapidly than SA59R and suggested that spread may occur between cells in close physical contact. In addition, S4R infection induced a massive influx of lymphocytes into the brain, a higher percentage of CD8+ T cells, and a higher frequency of MHV-specific CD8+ T cells relative SA59R infection. Despite this robust and viral-specific immune response to S4R infection, infection of RAG1−/− mice suggested that immune-mediated pathology also contributes to the high neurovirulence of S4R.

Cryptic MBP epitope 1–20 is inducing autoimmune anterior uveitis without EAE in Lewis rats

Lewis rats immunized with myelin basic protein (MBP) developed experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Although several cryptic epitopes of MBP have strong encephalitogenic and uveitogenic properties, the peptide corresponding to the MBP residues 1–20 was uniquely capable of inducing AU without EAE. In this study, we showed that acetylation of the N-terminal amino acid did not produce encephalitogenicity, did not enhance uveitogenicity, and did not improve T cell proliferation in Lewis rats. The cytokine production profile induced by MBP 1–20 immunization was consistent with a Th1 response. In MBP-injected rats and in peptide-injected rats, the frequency of the IFN-γ-secreting cells in MBP 69–89-stimulated T cells was significantly higher than the frequency of IFN-γ-secreting cells in MBP 1–20-stimulated T cells. However, similar numbers of IFN-γ-producing specific cells were found in the eyes of MBP 69–89 and MBP 1–20 immunized rats. In these rats, the iris-infiltrating cells consisted of a much higher percentage of CD4 + T cells expressing L-selectin (CD62L) than did those cells found in the spinal cord. The results demonstrate that MBP 1–20 is immunogenic and uveitogenic, although it induced only weak proliferation and weak Th1 reaction. The fact that T cells with the same specificity have different effects on target organs suggested that, in the eye and spinal cord, a distinct mechanism might mediate the recruitment of cells to these organs.