Evaluation of a new single-parameter volumetric flow cytometer (CyFlow(green)) for enumeration of absolute CD4+ T lymphocytes in human immunodeficiency virus type 1-infected Thai patients

Use of the standard dual-platform flow cytometric method for determination of CD4(+) T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4(+) T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlow(green) (Partec), a single-parameter SP volumetric FCM. The performance of CyFlow(green) was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4(+) and CD8(+) T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlow(green) were compared with those obtained with the bead-based three-color TruCOUNT system (R(2)=0.96; mean bias, -69.1 cells/microl; 95% confidence interval [CI], -225.7 to+87.5 cells/microl) and the FACSCount system (R(2)=0.97; mean bias, -40.0 cells/microl; 95% CI, -165.1 to+85.1 cells/microl). The correlation of the CD4(+) T-lymphocyte counts obtained by the two bead-based systems was high (R(2)=0.98). Interestingly, CyFlow(green) yielded CD4(+) T-lymphocyte counts that were 21.8 and 7.2 cells/microl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4(+) T-lymphocyte counts were <250 CD4(+) T-lymphocyte counts/microl range or 17.3 and 5.8 cells/microl less, respectively, when CD4(+) T-lymphocyte counts were <200 cells/microl. The single-parameter CyFlow(green) volumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings.     

Affordable CD4+-T-Cell Counting by Flow Cytometry: CD45 Gating for Volumetric Analysis

The flow cytometers that are currently supported by industry provide accurate CD4⁺-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between −29 and +46 cells/mm³ with very little bias for CD4 counts (in favor of the Trio method: +8 CD4⁺ lymphocytes/mm³; 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between −118 and +98 cells/mm³, again with a negligible bias (−10 CD4⁺ lymphocytes/mm³). In the volumetric method using CD45/CD8, the strongly CD8⁺ cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8⁺ cells/mm³; 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.     

Reference Values of CD4 T Lymphocytes in Human Immunodeficiency Virus-Negative Adult Nigerians

A cross-sectional study that involved secondary analysis of data collected from 681 pregnant women and 183 miners (94 men and 89 women; ratio of men to women, 1:0.95) in Jos, Nigeria, was carried out to determine the reference ranges for CD4⁺-cell counts in healthy HIV-negative adult Nigerians. The main results of interest were CD4⁺-cell counts and odds ratios (ORs) of low CD4⁺-cell counts, defined as below 350 cells per μl. CD4⁺-cell counts were similar in men and nonpregnant women, with a mean (standard deviation) of 828 (203) cells per μl, but pregnant women had a lower value of 771 (250) cells per μl. None of the factors assessed was related to the odds of having a low CD4⁺-cell count among men and nonpregnant women, but age, age of marriage, and alcohol usage were significant predictors in pregnant women. Compared to pregnant women less than 20 years old, older women had significantly lower odds of a low CD4⁺-cell count (ORs were 0.06 for women aged 20 to 29 years and 0.22 for those aged 30 to 39 years). When compared with those pregnant women who were married before 20 years of age, those who married at 20 to 29 years and 30 to 39 years had odds ratios of 6.41 and 9.40, respectively. Previous alcohol use was also associated with low CD4⁺-cell counts (OR, 5.15). The 95% confidence interval for CD4⁺-cell counts in healthy adult Nigerians is 547 to 1,327 cells per μl, and this is the first time this has been determined.     

Expression of Adhesion Molecules and CD28 on T Lymphocytes during Human Immunodeficiency Virus Infection

Adhesion molecules, which play a major role in lymphocyte circulation, have not been well characterized in human immunodeficiency virus (HIV) infection. T-lymphocyte populations, including CD3, CD4, CD28, and adhesion molecules (L selectin, LFA-1, VLA-4, and ICAM-1) were measured by flow cytometry in a cross-sectional study of 100 HIV-infected and 49 HIV-seronegative adults. HIV-infected adults had lower numbers of CD3⁺ lymphocytes expressing L selectin (P < 0.0001) and VLA-4 (P < 0.01) and higher numbers of CD3⁺ lymphocytes expressing LFA-1^bright (P < 0.002) than did HIV-negative adults. By CD4⁺-lymphocyte count category (>500, 200 to 500, or <200 cells/μl), HIV-infected adults with more advanced disease had lower percentages of CD3⁺ lymphocytes expressing L selectin and VLA-4 and higher percentages of CD3⁺ lymphocytes expressing LFA-1. The percentages of CD3⁺ CD28⁺ lymphocytes and of CD3⁺ L selectin⁺ lymphocytes were positively correlated (Spearman coefficient = 0.86; P < 0.0001), and the percentage of CD3⁺ CD28⁺ lymphocytes and the CD3⁺ LFA-1^bright lymphocyte/CD3⁺ LFA-1^dim lymphocyte ratio were negatively correlated (Spearman coefficient = −0.92; P <0.00001). The results of this study suggest that HIV infection is associated with altered expression of adhesion molecules.     

Association of T cell dysfunction with the presence of IgG autoantibodies on CD4+ lymphocytes in haemophilia patients; results of a 10-year study

HIV induces progressive dysfunction followed by numerical depletion of CD4⁺ lymphocytes. IgG autoantibodies and gp120-containing immune complexes have been implicated in the pathogenesis of AIDS. We carried out a longitudinal study in 19 HIV^– and 72 HIV⁺ haemophilia patients over a 10-year period in order to investigate a possible relationship between the occurrence of autoantibodies and CD4⁺ lymphocyte changes. IgM, IgG, C3d and gp120 on the surface of CD4⁺ lymphocytes were determined in heparinized whole blood with flow cytometry and double-fluorescence. The in vitro response of autoantibody-coated cells was tested in cell cultures with concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PWM), anti-CD3 MoAb or pooled allogeneic stimulator cells (MLC). After a 10-year follow up, 12 of 71 HIV⁺ and 16 of 19 HIV^– haemophilia patients showed no evidence of immunoglobulins on circulating CD4⁺ lymphocytes. HIV^– haemophilia patients without autoantibodies had CD4⁺and CD8⁺ cell counts in the normal range (957 ± 642/μl and 636 ± 405/μl) and normal T cell responses in vitro (mean relative response (RR) ≥ 0.7). In contrast, HIV⁺ haemophilia patients showed immunological abnormalities which were associated with the autoantibody and immune complex load of CD4⁺ blood lymphocytes. HIV⁺ patients without autoantibodies had a mean CD4⁺ lymphocyte count of 372 ± 274/μl, a mean CD8⁺ lymphocyte count of 737 ± 435/μl, and normal T lymphocyte stimulation in vitro (mean RR ≥ 0.7). HIV⁺ patients with complement-fixing IgM on CD4⁺ lymphocytes had somewhat lower CD4⁺ (255 ± 246/μl, P= NS) and CD8⁺ (706 ± 468/μl, P= NS) lymphocyte numbers, and also normal T lymphocyte stimulation (mean RR ≥ 0.7) in vitro. However, patients with complement-fixing IgG autoantibodies showed a strong decrease of CD4⁺ (150 ± 146/μl, P< 0.02) and CD8⁺ (360 ± 300/μl, P< 0.02) lymphocytes and impaired CD4⁺ lymphocyte stimulation in vitro with a mean RR of 0.5 ± 0.5 for Con A (P= NS), 0.7 ± 0.8 for PHA (P< 0.03), 0.4 ± 0.4 for PWM (P= NS), 0.8 ± 1.2 for anti-CD3 MoAb (P< 0.04) and 0.7 ± 1.0 for pooled allogeneic stimulator cells (P= 0.05). Patients with gp120-containing immune complexes on CD4⁺ blood lymphocytes demonstrated strongly decreased CD4⁺(25 ± 35/μl, P< 0.0001) and CD8⁺ (213 ± 212/μl, P< 0.006) lymphocyte counts as well as strongly impaired T lymphocyte responses in vitro upon stimulation with PHA (RR 0.2 ± 0.1, P < 0.02), PWM (RR 0.2 ± 0.2, P= 0.05), anti-CD3 MoAb (RR 0.1 ± 0.1, P< 0.04), and allogeneic stimulator cells (RR 0.2 ± 0.1, P< 0.02). These data led us to speculate that autoantibody formation against CD4⁺ lymphocytes is an important mechanism in the pathogenesis of AIDS. We hypothesize that autoantibodies against circulating CD4⁺ lymphocytes inhibit CD4⁺ cell function, especially the release of cytokines, and induce CD4⁺ cell depletion. The reduction and dysfunction of CD4⁺ lymphocytes may be responsible for the CD8⁺ cell depletion observed in HIV⁺ patients.     

Temporal regulation of interleukin-12p70 (IL-12p70) and IL-12-related cytokines in splenic dendritic cell subsets during Leishmania donovani infection

Dendritic cells (DC) play an essential role in initiating and directing T-cell responses, in part by production of interleukin-12p70 (IL-12p70), IL-23, and IL-27. However, comparative studies on the capacity for cytokine production of DC subsets are rare. Here, we compare splenic CD8α⁺, CD4⁺, and double-negative (DN) DC, isolated 5 h to 28 days after Leishmania donovani infection, for (i) production of IL-12p70, (ii) accumulation of IL-12/23p40, IL-12p35, IL-23p19, and IL-27p28 mRNAs, and (iii) their capacity to direct CD4⁺ T-cell differentiation. At 5 h, conventional DC (cDC) accumulated mRNA for IL-12/23p40 (CD8α>CD4>DN), IL-23p19 (CD4>CD8α>DN), and IL-27p28 (CD8α>CD4>DN), in an infection dose-dependent manner. IL-12p70 was restricted to CD8α⁺ cDC, reflecting the subset-specific accumulation of IL-12p35 mRNA. In contrast, cDC from mice infected for 14 to 28 days accumulated little mRNA for IL-12p40 and IL-12p19, though IL-27p28 mRNA remained detectable (CD8α>DN>CD4). IL-12p70 secretion by CD8α⁺ cDC was also absent, reflecting deficient IL-12/23p40, rather than IL-12p35, mRNA accumulation. The capacity of CD8α⁺ cDC isolated early after infection to direct Th1 cell differentiation was mediated through IL-12/23p40, whereas this ability in CD4⁺ and DN cDC was independent of IL-12/23p40 and did not result from overexpression of Delta 4 Notch-like ligand. However, DN cDC produced gamma interferon (IFN-γ) and also contained a rare population of CD11c^hi DX5⁺ IFN-γ-producing cells. Our data illustrate the extensive diversity in, and temporal regulation of, splenic cDC subsets during infection and suggest caution in interpreting data obtained with unfractionated or minimally purified DC.     

Expression of Regeneration and Tolerance Factor Correlates Directly with Human Immunodeficiency Virus Infection and Inversely with Hepatitis C Virus Infection

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3⁺ T cells of HIV-seropositive (HIV⁺) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV⁺ HCV-seropositive (HCV⁺), HIV- and HCV-seropositive (HIV⁺ HCV⁺), and HIV- and HCV-seronegative (HIV⁻ HCV⁻) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV⁺ individuals is upregulated and that its expression on T cells from HCV⁺ individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3⁺ T cells was seen in both HIV⁺ and HIV⁺ HCV⁺ individuals compared to HIV⁻ HCV⁻ individuals. HCV⁺ individuals had lower levels of RTF expression than HIV⁻ HCV⁻ individuals (P < 0.005 for CD4⁺; P < 0.0005 for CD8⁺). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV⁺ > HIV⁺ HCV⁺ > HIV⁻ HCV⁻ > HCV⁺. The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.     

Comparison of Neutralizing and Hemagglutination-Inhibiting Antibody Responses to Influenza A Virus Vaccination of Human Immunodeficiency Virus-Infected Individuals

A neutralization enzyme immunoassay (N-EIA) was used to determine the neutralizing serum antibody titers to influenza A/Taiwan/1/86 (H1N1) and Beijing/353/89 (H3N2) viruses after vaccination of 51 human immunodeficiency virus (HIV) type 1-infected individuals and 10 healthy noninfected controls against influenza virus infection. Overall, the N-EIA titers correlated well with the hemagglutination-inhibition (HAI) titers that were observed in the same samples in a previous study (F. P. Kroon, J. T. van Dissel, J. C. de Jong, and R. van Furth, AIDS 8:469–476,1994). The N-EIA appeared to be more sensitive than the HAI test. Significantly more fourfold or higher rises in N-EIA titer and higher mean N-EIA titers occurred in HIV-infected individuals with ≥200 CD4⁺ cells per μl than in those with <200 CD4⁺ cells per μl.     

CD4+ T-cell dependence of primary CD8+ T-cell response against vaccinia virus depends upon route of infection and viral dose

CD4⁺ T-cell help (CD4 help) plays a pivotal role in CD8⁺ T-cell responses against viral infections. However, the role in primary CD8⁺ T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8⁺ T-cell responses to vaccinia virus (VACV) in MHC class II^−/− mice. CD4 help deficiency diminished the generation of VACV-specific CD8⁺ T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8⁺ T cells in i.p. infected MHC II^−/− mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II^−/− mice when a small viral dose was used. These data suggested that primary CD8⁺ T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8⁺ T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8⁺ T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8⁺ T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8⁺ T cells than i.p. infection.     

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA⁺) and O-acetyl-negative (OA⁻) forms. This study investigates the impact of OA status (OA⁺ versus OA⁻) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA⁺ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA⁻ antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA⁺ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA⁻ antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA⁺ antigen than against the OA⁻ antigen. These sera were also distinguished by the inability of fluid-phase OA⁻ antigen to compete for antibody binding to OA⁺ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA⁺ and OA⁻ target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA⁺ versus OA⁻ W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA⁺ versus anti-OA⁻ W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.     

Distribution of Lymphocyte Subsets in Healthy Human Immunodeficiency Virus-Negative Adult Ethiopians from Two Geographic Locales

Immunological values for 562 factory workers from Wonji, Ethiopia, a sugar estate 114 km southeast of the capital city, Addis Ababa, Ethiopia, were compared to values for 218 subjects from Akaki, Ethiopia, a suburb of Addis Ababa, for whom partial data were previously published. The following markers were measured: lymphocytes, T cells, B cells, NK cells, CD4⁺ T cells, and CD8⁺ T cells. A more in depth comparison was also made between Akaki and Wonji subjects. For this purpose, various differentiation and activation marker (CD45RA, CD27, HLA-DR, and CD38) expressions on CD4⁺ and CD8⁺ T cells were studied in 60 male, human immunodeficiency virus-negative subjects (30 from each site). Data were also compared with Dutch blood donor control values. The results confirmed that Ethiopians have significantly decreased CD4⁺ T-cell counts and highly activated immune status, independent of the geographic locale studied. They also showed that male subjects from Akaki have significantly higher CD8⁺ T-cell counts, resulting in a proportional increase in each of the CD8⁺ T-cell compartments studied: naïve (CD45RA⁺CD27⁺), memory (CD45RA⁻CD27⁺), cytotoxic effector (CD45RA⁺CD27⁻), memory/effector (CD45RA⁻CD27⁻), activated (HLA-DR⁺CD38⁺), and resting (HLA-DR⁻CD38⁻). No expansion of a specific functional subset was observed. Endemic infection or higher immune activation is thus not a likely cause of the higher CD8 counts in the Akaki subjects. The data confirm and extend earlier observations and suggest that, although most lymphocyte subsets are comparable between the two geographical locales, there are also differences. Thus, care should be taken in extrapolating immunological reference values from one population group to another.     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

Correlation of CD4+ T-Cell Counts Estimated by an Immunocapture Technique (Capcellia) with Viral Loads in Human Immunodeficiency Virus-Seropositive Individuals

As antiretroviral therapy becomes more affordable, valid, reliable, and inexpensive laboratory tests are also needed to monitor the progression of disease in people with human immunodeficiency virus (HIV) infection. The CD4⁺ T-cell counts estimated by Capcellia, an immunocapture method, and flow cytometry were compared and were correlated with HIV type 1 (HIV-1) load. There was a significant negative correlation between the HIV-1 load and CD4⁺ T-cell counts estimated by flow cytometry (r = −0.63, P = <0.001) as well as between the HIV-1 load and CD4⁺ T-cell counts estimated by Capcellia (r = −0.61, P = <0.001). Capcellia is a cost-effective, user-friendly assay that correlated well with HIV-1 load determinations for individuals both with and without treatment.     

Reciprocal Regulation of Th1- and Th2-Cytokine-Producing T Cells during Clearance of Parasitemia in Plasmodium falciparum Malaria

Flow cytometry for the intracellular detection of T-cell cytokines was performed for 15 Gabonese patients during acute uncomplicated Plasmodium falciparum malaria. A striking expansion of CD4⁺ and CD8⁺ T cells producing gamma interferon (IFN-γ) was found during drug-induced clearance of parasitemia, paralleled by a decrease of interleukin-2 (IL-2) production. The frequency of IL-4- and IL-13-producing CD4⁺ cells gradually decreased, whereas the frequency of T cells producing IL-2⁺–IFN-γ⁺, IL-4⁻–IL-5⁺, and IL-4⁺–IL-5⁺ cytokines as well as IL-4⁺–IFN-γ⁺ and IL-13⁺–IFN-γ⁺ cytokines was not significantly altered. The capacity for IL-10 production within the CD4⁺ subset increased due to an expansion of both IL-10⁺–IFN-γ⁻ and IL-10⁺–IFN-γ⁺ cytokine-expressing cells. Thus, a more pronounced Th2-driven immune response during acute untreated P. falciparum infection with a shift towards Th1 responsiveness induced by parasite clearance is suggested.     

Immunohematological Reference Ranges for Adult Ethiopians

A cross-sectional survey was carried out with 485 healthy working adult Ethiopians who are participating in a cohort study on the progression of human immunodeficiency virus type 1 (HIV-1) infection to establish hematological reference ranges for adult HIV-negative Ethiopians. In addition, enumeration of absolute numbers and percentages of leukocyte subsets was performed for 142 randomly selected HIV-negative individuals. Immunological results were compared to those of 1,356 healthy HIV-negative Dutch blood donor controls. Immunohematological mean values, medians, and 95th percentile reference ranges were established. Mean values were as follows: leukocyte (WBC) counts, 6.1 × 10⁹/liter (both genders); erythrocyte counts, 5.1 × 10¹²/liter (males) and 4.5 × 10¹²/liter (females); hemoglobin, 16.1 (male) and 14.3 (female) g/dl; hematocrit, 48.3% (male) and 42.0% (female); platelets, 205 × 10⁹/liter (both genders); monocytes, 343/μl; granulocytes, 3,057/μl; lymphocytes, 1,857/μl; CD4 T cells, 775/μl; CD8 T cells, 747/μl; CD4/CD8 T-cell ratio, 1.2; T cells, 1,555/μl; B cells, 191/μl; and NK cells, 250/μl. The major conclusions follow. (i) The WBC and platelet values of healthy HIV-negative Ethiopians are lower than the adopted reference values of Ethiopia. (ii) The absolute CD4 T-cell counts of healthy HIV-negative Ethiopians are considerably lower than those of the Dutch controls, while the opposite is true for the absolute CD8 T-cell counts. This results in a significantly reduced CD4/CD8 T-cell ratio for healthy Ethiopians, compared to the ratio for Dutch controls.     

CD8α-Deficient Mice Are Highly Susceptible to 5-Fluorouracil-Induced Lethality

Intestinal intraepithelial lymphocytes (i-IEL) expressing CD8α are located in the intestine and may confer protection against invasion of intestinal microflora. We found that mice rendered deficient in CD8α molecules by homologous recombination were susceptible to 5-fluorouracil (5-FU)-induced lethality accompanied by translocation of members of the enterobacteria. The number of i-IEL was greatly reduced on day 6 after 5-FU administration in both CD8α^+/− mice and CD8α^−/− mice, whereas the recovery of the level of i-IEL thereafter was significantly impaired in CD8α^−/− mice compared with that in CD8α^+/− mice. The ability of i-IEL to produce gamma interferon in response to immobilized T-cell receptor (TCR) αβ or TCR γδ monoclonal antibodies was significantly lower in CD8α^−/− mice than in CD8α^+/− mice. Transfer of CD8⁺ i-IEL conferred significant protection against 5-FU-induced lethality in CD8α^−/− mice. The results suggest that CD8⁺ i-IEL play an important role in protection against 5-FU-induced lethality with translocation of Enterobacteriaceae.     

β2 Integrin deficiency yields unconventional double-negative T cells distinct from mature classical natural killer T cells in mice

Expressed on leucocytes, β₂ integrins (CD11/CD18) are specifically involved in leucocyte function. Using a CD18-deficient (CD18^−/−) mouse model, we here report on their physiological role in lymphocyte differentiation and trafficking. CD18^−/− mice present with a defect in the distribution of lymphocytes with highly reduced numbers of naïve B and T lymphocytes in inguinal and axillary lymph nodes. In contrast, cervical lymph nodes were fourfold enlarged harbouring unconventional T-cell receptor-αβ (TCR-αβ) and TCR-γδ CD3⁺ CD4⁻ CD8⁻ (double-negative; DN) T cells that expanded in situ. Using adoptive transfer experiments, we found that these cells did not home to peripheral lymph nodes of CD18^wt recipients but, like antigen-experienced T or natural killer (NK) T cells, recirculated through non-lymphoid organs. Lacking regulatory functions in vitro, CD18^−/− TCR-αβ DN T cells did not suppress the proliferation of polyclonally activated CD4⁺ or CD8⁺ (single-positive; SP) T cells. Most interestingly, CD18^−/− TCR-αβ DN T cells showed intermediate TCR expression levels, an absent activation through allogeneic major histocompatibility complex and a strong proliferative dependence on interleukin-2, hence, closely resembling NKT cells. However, our data oppose former reports, clearly showing that, because of an absent reactivity with CD1d-αGalCer dimers, these cells are not mature classical NKT cells. Our data indicate that CD18^−/− TCR-αβ DN T cells, like NKT and TCR-γδ T cells, share characteristics of both adaptive and innate immune cells, and may accumulate as a compensatory mechanism to the functional defect of adaptive immunity in CD18^−/− mice.     

Human mesenchymal stromal cells enhance the immunomodulatory function of CD8+CD28? regulatory T cells

One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8⁺CD28⁻ Treg cells and found that the MSCs could not only increase the proportion of CD8⁺CD28⁻ T cells, but also enhance CD8⁺CD28⁻T cells'' ability of hampering naive CD4⁺ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4⁺ T cells and inducing the apoptosis of activated CD4⁺ T cells. Mechanistically, the MSCs affected the functions of the CD8⁺CD28⁻ T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8⁺CD28⁺ T cells to CD8⁺CD28⁻ T cells, but did increase the proportion of CD8⁺CD28⁻ T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8⁺CD28⁻ Treg cells, shedding new light on MSCs-mediated immune regulation.     

An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible to Mycobacterium tuberculosis

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10³ or 10⁵ CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44⁻ CD45RB⁺ cells disappeared within 2 weeks postinfection, while the number of CD44⁺ CD45RB^−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3⁺ CD4⁺ CD8⁻ T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ⁺ IL-4⁻) clone and one Th0-like (IFN-γ⁺ IL-4⁺ IL-10⁺) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.