CD8+ lymphocytes in pregnancy and HIV infection: characterization of CD8+ subpopulations and CD8+ noncytotoxic antiviral activity.

The distribution and function of lymphocytes vary in different clinical states. The object of this study was to characterize the CD8+ lymphocyte subpopulations and CD8+ anti-HIV suppressor activity in HIV-infected and uninfected pregnant and nonpregnant women. The total percentage of CD8+ lymphocytes was not altered by pregnancy but the percentage of activated CD8+ T cells increased during pregnancy and decreased postpartum. HIV infection in pregnant women resulted in both an increased percentage of CD8+ lymphocytes and a marked increase in activated and memory CD8+ lymphocyte subsets, which did not change in the postpartum period. Most HIV-infected women had CD8+-mediated noncytotoxic antiviral activity. However, the activity was not correlated with alterations in CD8+ lymphocyte subsets. This study provides baseline information on changes in CD8 immunologic parameters during pregnancy and HIV infection for further studies that employ antiretroviral therapeutic regimens capable of impacting the immune response.     

Loss of reactivity of vaccine-induced CD4 T cells in immunized monkeys after SIV/HIV challenge

BACKGROUND: Immunization protocols involving priming with DNA and boosting with recombinant live virus vectors such as recombinant modified Vaccinia Ankara (rMVA) are considered as vaccine candidates against HIV. Such protocols improve the outcome of simian/human immunodeficiency virus (SHIV) pathogenic challenge in Rhesus monkeys. OBJECTIVES: To investigate the fate of vaccine-induced T cells after a mucosal SHIV challenge. METHODS: We immunized Rhesus monkeys (Macaca mulatta) by DNA priming followed by rMVA boost. After intrarectal challenge with SHIV 89.6P, immunized animals demonstrated early control of viral replication and stable CD4 T-cell counts. We monitored T-cell responses by measuring IFN-gamma secretion and proliferation. RESULTS: Immunization induced strong and sustained SHIV-specific CD4 and CD8 T-cell responses. CD8 T-cell responses were recalled during acute infection, whereas none of the vaccine-induced SHIV-specific CD4 T-cell responses were recalled. Moreover, most of the CD4 T-cell responses became undetectable in peripheral blood or lymph nodes even after in-vitro peptide stimulation. In contrast, we persistently detected CD4 T-cell responses specific for control recall antigens in infected animals. CONCLUSION: SHIV 89.6P challenge results in a lack of reactivity of vaccine-induced SHIV-specific CD4 T cells. These results may have important implications in the AIDS vaccine field, especially for the evaluation of new vaccine candidates, both in preventive and therapeutic trials.     

In vitro activity of CD4+ and CD8+ T lymphocytes from mice immunized with a synthetic malaria peptide.

In previous work, a T-helper epitope was mapped within the circumsporozoite protein of the murine malaria parasite Plasmodium yoelii. A 21-mer synthetic peptide corresponding to this epitope (amino acid positions 59-79; referred to as Py1) induced a specific T-cell proliferation in BALB/c and C57BL/6 mice and provided help for the production of antibodies to peptides from the repetitive region, (Gln-Gly-Pro-Gly-Ala-Pro)n, of the P. yoelii circumsporozoite protein when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Experiments were then designed to study the in vitro antiparasite efficacy of T cells elicited in vivo by peptide immunization. T-cell activity was evaluated on cultured hepatic stages of P. yoelii. Peptide immunizations led to the preferential activation of CD8+ T cells in BALB/c mice and of both CD4+ and CD8+ T cells in C57BL/6 mice. Parasite elimination was mediated directly by these cells and did not seem to be dependent on lymphokine secretion. These data suggest that peptide-primed CD4+ T cells as well as CD8+ T cells could be cytolytic for the hepatic phase of malaria parasites. The fact that the same peptide could activate different lymphocyte populations, depending on the strain of mouse, highlights the importance of a better understanding of the fine mechanisms behind the immune responses to synthetic peptides being tested for malaria vaccine development.     

CD8+ T-cell interaction with HCV replicon cells: evidence for both cytokine- and cell-mediated antiviral activity

The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8(+) T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with (51)Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-gamma), anti-tumor necrosis factor alpha (TNF-alpha), and anti-FasL. Cytolytic activity was measured by (51)Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8(+) cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8(+) antiviral effect was markedly reduced by blocking either IFN-gamma or FasL but was unaffected by blocking TNF-alpha. In conclusion, HCV-specific CD8(+) cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes.     

Simian immunodeficiency virus infection of CD4+CD8+ T cells in a macaque with an unusually high peripheral CD4+CD8+ T lymphocyte count

We assessed the possible role in vivo CD4(+) CD8(+) T cells as a viral reservoir for simian immunodeficiency virus (SIV), in a macaque with 50% CD4(+) CD8(+) T cells in peripheral blood. During primary infection (day 14) of this rhesus macaque with the pathogenic SIVmac251 strain, proviruses were detected at similar frequencies in CD4(+) CD8(+) T cells (1/10) and CD4(+) T cells (1/10) and at a lower frequency in CD8(+) T cells (1/800). On day 235, no viral DNA was detected in CD8(+) cells, despite the persistent high viral load, indicating that CD8(+) cells do not constitute a reservoir during the chronic phase of SIV infection. Infection induced early lymphopenia of CD4(+), CD4(+) CD8(+), and CD8(+) cells; only the CD8(+) cell population returned to initial levels and expanded further. We found that CD4(+) CD8(+) T cells expressed the costimulatory CD28 molecule less and were more prone to die in vitro after phytohemagglutinin/interleukin 2 stimulation than were CD4(+) T cells. Taken together, massive death of CD4(+) CD8(+) T cells during acute stages of SIV infection may explain why CD8(+) T cells did not represent a major reservoir for SIV at the onset of infection.     

慢性乙型肝炎患者外周血CD8+CD28+T淋巴细胞百分比的变化及其临床意义探讨

目的 探讨不同病程阶段的慢性乙型肝炎患者外周血CD8⁺CD28⁺T淋巴细胞百分比的变化,以及CD8⁺CD28⁺T淋巴细胞百分比变化与血清HBsAg水平的关系。方法 2018年4月~2018年8月我院诊治的慢性乙型肝炎患者88例,其中免疫耐受期20例,免疫清除期28例,非活动期20例,再活动期20例,另选择健康人20例。使用流式细胞术检测外周血CD8⁺CD28⁺T淋巴细胞百分比。结果 健康人与免疫耐受期患者外周血CD8⁺CD28⁺T淋巴细胞百分比分别为（26.1±3.5）%和（26.3±3.4）%,差异无统计学意义（P>0.05）;免疫清除期患者CD8⁺CD28⁺T淋巴细胞百分比为（40.1±4.7）%,显著高于健康人（P<0.05）;非活动期和再活动期患者外周血CD8⁺CD28⁺T淋巴细胞百分比分别为（20.3±2.2）%和（26.1±2.2）%,显著低于健康人（P<0.05）;外周血HBsAg低、中、高三组人群外周血CD8⁺CD28⁺T淋巴细胞百分比分别为（24.0±7.5）%、（28.4±8.9）%和（33.2±8.5）%,各组间差异有统计学意义（P<0.05）。结论 不同病程阶段的慢性乙型肝炎患者外周血CD8⁺CD28⁺T淋巴细胞百分比存在明显差异,可能与病毒长期刺激机体免疫系统,导致免疫系统功能失调有关,而这种失调可能参与了慢性乙型肝炎的发病过程。     

Severe bactrim-induced neutropenia with reversal of CD4+/CD8+ lymphocyte ratio: response to recombinant human granulocyte-colony stimulating factor (R-metHUG-CSF)

A patient presented with severe bactrim-induced neutropenia with a reversed CD4⁺/CD8⁺ lymphocyte ratio. R-metHUG-CSF at 300 μg daily produced a dramatic neutrophil response and the therapy was discontinued after 2 weeks.     

Chimpanzees immunized with recombinant soluble CD4 develop anti-self CD4 antibody responses with anti-human immunodeficiency virus activity.

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.     

RSV-induced bronchial epithelial cell PD-L1 expression inhibits CD8+ T cell nonspecific antiviral activity

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8(+) T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8(+) T cells, we demonstrated that RSV-infected BECs increased CD8(+) T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8(+) T cells enhanced CD8(+) T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.     

Broad influenza-specific CD8+ T-cell responses in humanized mice vaccinated with influenza virus vaccines

The development of novel human vaccines would be greatly facilitated by the development of in vivo models that permit preclinical analysis of human immune responses. Here, we show that nonobese diabetic severe combined immunodeficiency (NOD/SCID) beta(2) microglobulin(-/-) mice, engrafted with human CD34+ hematopoietic progenitors and further reconstituted with T cells, can mount specific immune responses against influenza virus vaccines. Live attenuated trivalent influenza virus vaccine induces expansion of CD8+ T cells specific to influenza matrix protein (FluM1) and nonstructural protein 1 in blood, spleen, and lungs. On ex vivo exposure to influenza antigens, antigen-specific CD8+ T cells produce IFN-gamma and express cell-surface CD107a. FluM1-specific CD8+ T cells can be also expanded in mice vaccinated with inactivated trivalent influenza virus vaccine. Expansion of antigen-specific CD8+ T cells is dependent on reconstitution of the human myeloid compartment. Thus, this humanized mouse model permits preclinical testing of vaccines designed to induce cellular immunity, including those against influenza virus. Furthermore, this work sets the stage for systematic analysis of the in vivo functions of human DCs. This, in turn, will allow a new approach to the rational design and preclinical testing of vaccines that cannot be tested in human volunteers.     

Allo-activated CD4+ and CD8+ lymphocyte subsets: new ultrastructural findings based on computer-assisted image analysis

Primary allo-stimulation induces substantial and differentiated modifications in the development of cytoplasmic organelles of peripheral T-lymphocyte subsets. The present computer-assisted image analysis provides evidence that the CD4+ subset undergoes significant increase in organelle density after stimulation, while the CD8+ subset exhibits a post-stimulation organelle development proportional to the cell size. Each immunological subset contains two distinct patterns of cytoplasmic organization, here defined as type I (containing a few cytoplasmic organelles) and type II (with well-developed synthesis and storage organelles). On the basis of these findings cytologic parameters could be ascribed to a heterogeneous proliferation of functionally distinct T-cell clones in CD4+ and CD8+ subsets.     

Phenotypic abnormalities in CD8+ T lymphocyte subsets in patients with rheumatoid arthritis.

We analyzed the cell surface phenotype of CD8+ cells in both peripheral blood and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Utilizing the monoclonal antibodies anti-CD45RA, anti-CD29 and anti-S6F1-, one can define both suppressor effector (CD45RA+CD29-S6F1-) and killer effector (CD45RA-CD29+S6F1+) cells within the CD8 population. In patients with OA, normal proportions of CD8+CD45RA+, CD8+CD29+ and CD8+S6F1+ cells were found in both peripheral blood and SF. The peripheral blood of patients with RA, in contrast, showed a decreased percentage of CD8+CD45RA+ cells (13.4 +/- 2.6) (p less than 0.05), but a normal percentage of CD8+CD29+ and CD8+S6F1+ cells. In the SF of patients with RA, we observed a more dramatic decrease in CD8+CD45RA+ suppressor effector cells (6.4 +/- 5.0) (p less than 0.001), a significant increase in killer effector cells as measured by both CD8 + CD29+ (35.5 +/- 9.9) (p less than 0.001) and CD8 + S6F1+ cells (28.2 +/- 11.4) (p less than 0.01). These changes may contribute to the immunologic abnormalities previously noted in this disease and may provide some insight into the pathophysiologic mechanisms of RA.     

CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro

HIV infection is characterized by CD4(+) T cell depletion and progressive immune dysfunction; particularly impacted are HIV-specific T cell responses. An important component of immune-mediated control of HIV replication, killing of infected cells, appears to be impaired, in part due to poor cytolytic activity of HIV-specific cytotoxic T cells (CTL). In vitro, several functions of HIV-specific T cells, such as cytokine production, can be enhanced by the depletion of the immunosuppressive CD25(+) FoxP3(+) CD4(+) regulatory (Treg) cell subset. However, the effect of CD25(+) Treg cells on virus-specific cytolytic activity in the context of HIV or any human viral infection has not been investigated. The present study demonstrates that CD25(+) Treg cells isolated from the peripheral blood of HIV-infected subjects significantly suppress HIV Gag-specific cytolytic activity in vitro. In addition, CD25(+) Treg cells suppress effector function (coexpression of TNF-alpha and IFN-gamma) of HIV-specific CD8(+) T cells that proliferate in response to HIV antigen. Finally, the secretion of HIV-inhibitory CC-chemokines by HIV-specific and nonspecific CD8(+) T cells is significantly reduced in the presence of CD25(+) Treg cells. These data suggest that CD25(+) Treg-mediated suppression of the antiviral activity of HIV-specific CD8(+) T cells could impact the ability of HIV-infected individuals to control HIV replication in vivo.     

Preliminary observations on the efficacy of a recombinant multistage Plasmodium falciparum vaccine in Aotus nancymai monkeys

A vaccine trial was conducted to determine the efficacy of a multicomponent candidate vaccine, FALVAC-1, against Plasmodium falciparum in Aotus nancymai monkeys. After two immunizations, animals were challenged intravenously with parasites of the Vietnam Oak Knoll (FVO) strain of P. falciparum. The primary outcome was to determine the protective response of the monkeys to immunization with the FALVAC-1 antigen produced in baculovirus when combined with different adjuvants (alum, QS-21, ASO2a, CRL1005/oil, and CRL1005/saline) as compared with FALVAC-1 with FCA/FIA and antigen alone. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other 5 antigen-adjuvant combinations were able to provide discernable levels of protection based on log(parasitemia) and log(cumulative parasitemia) to Day 11.     

CD4+ T cells and monocytes elicited by immunization of rhesus monkeys with antigen-pulsed dendritic cells control SIV replication

Most HIV infections occur by transmission across mucosal surfaces, where dendritic cells (DCs) are the first cells to encounter the virus. Dendritic cells are specialized antigen-presenting cells critical for eliciting T cell-mediated immune responses. Delayed-type hypersensitivity (DTH) is a cellular immune response in some viral infections and it is mediated by CD4+ and/or CD8+ T cells. We hypothesized that a DTH response to HIV induced by antigen-pulsed DCs would protect against a mucosal exposure to the virus. In a small pilot experiment, six rhesus monkeys were immunized with autologous, antigen-pulsed DCs by the intradermal route and five of the monkeys were boosted with a second dose of DCs at 3 months. Antibody responses to SIV were detected in two of six vaccinated monkeys, lymphocyte-proliferative responses were detected in five of the six monkeys and cytotoxic T lymphocyte (CTL) responses were detected in four of the six monkeys. Using a novel in vitro assay of SIV replication in DCs cocultured with autologous CD4+ T cells and monocytes, suppression of viral replication was detected from five of the six monkeys at multiple time points before and after SIV challenge. Macaques were orally challenged with SIVmac239 at 1-3 months after the booster inoculation. Peak viral loads were similar to those of four naive animals but, compared with naive monkeys, declined at 6 months to levels 1 log(10) or more lower in monkeys that had been vaccinated and that had > or = 50% suppression of SIV replication in DCs. Optimizing this immunization strategy may result in a strong antiviral DTH response that could better control a mucosal lentiviral infection.     

In vivo antiviral activity of recombinant type alpha interferon A in monkeys with infections due to simian varicella virus

Recombinant type alpha interferon A (rIFN-alpha-A) administered to African green monkeys (Cercopithecus aethiops) intramuscularly in a dose of 3 X 10(6) units/kg of body weight resulted in substantial blood levels of interferon. Peak levels of greater than 1,000 units/ml of serum appeared at 1 and 2 hr after inoculation and interferon was detectable for as long as 12 hr after inoculation. Injection of rIFN-alpha-A at a dose of 10(6) units/kg twice daily for eight days effectively inhibited simian varicella virus infection of the African green monkey. Antiviral activity was demonstrated in monkeys with prophylactic treatment begun 4 hr prior to virus inoculation or with therapeutic treatment deferred until 44 hr after virus inoculation. No adverse effects of treatment were observed.     

Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6

The recently failed clinical efficacy trial of an acquired immunodeficiency syndrome (AIDS) vaccine that elicits antiviral CD8⁺ T‐cell responses has emphasized the challenge of producing an effective vaccine against human immunodeficiency virus (HIV). In the simian immunodeficiency virus (SIV)/ rhesus monkey model of AIDS, live‐attenuated lentivirus ‘vaccines’ provide the best protection from uncontrolled viral replication and clinical disease after pathogenic SIV challenge. This review summarizes a recent series of studies in which we show that after vaginal SIV challenge of rhesus macaques immunized with an attenuated lentivirus protection from uncontrolled viral replication is primarily mediated by CD8⁺ T cells in the vaginal mucosa. Immunization with a chimeric simian/human immunodeficiency virus (SHIV) results in a systemic infection that induces a moderate population of SIV‐specific CD8⁺ and CD4⁺ T cells with cytolytic potential in the vaginal mucosa. Depletion of CD8⁺ T cells at the time of SIV challenge completely abrogates the protection mediated by prior infection with attenuated SHIV. Further after vaginal SIV challenge, the only significant expansion of SIV‐specific T cells occurs in the vagina in these animals. No significant expansion of T‐cell responses was observed in systemic lymphoid tissues. Thus, the presence of SIV‐specific CD8⁺ T cells in the vagina on the day of vaginal SIV challenge and a modest expansion of local effector T cells is sufficient to stop uncontrolled SIV replication. It seems that T‐cell based vaccine strategies that can elicit mucosal effector CD8⁺ T‐cell populations and avoid inducing systemic T‐cell proliferation upon exposure to HIV have the greatest potential for mimicking the success of live‐attenuated lentiviral vaccines.     

Cellular proliferative responses in squirrel monkeys immunized with recombinant and synthetic Plasmodium vivax circumsporozoite peptides

The role of circulating peripheral blood momonuclear cells (PBMC) in mediating protective immunity was examined during an immunization trial in Saimiri monkeys. Three engineered constructs representing different but overlapping regions of the circumsporozoite (CS) protein of Plasmodium vivax were used to immunize the Saimiri monkeys. Monkeys were randomly placed into three immunization groups: rPvCS2, rPvCS3, and LCV3 (representing three different but overlapping portions of the P. vivax CS protein) and two control groups: an alum adjuvant control group and an unimmunized control group. Collections of PBMC were made throughout the study at weeks 0, 2, 8, challenge (week 16), and two weeks after challenge. Proliferative responses to all immunogens and pokeweed mitogen were measured in all monkeys. Fourteen of 18 monkeys immunized with either rPvCS2 or rPvCS3 responded on the day of challenge to the appropriate immunogen with a stimulation index less than 2. Immunization with LCV3, which represents the repeat region only, elicited a specific response in only one monkey. However monkeys in both control groups also responded to rPvCS2 and rPvCS3, regardless of immunization, suggesting the presence of epitopes in rPvCS2 and rPvCS3 capable of associating with differing MHC antigens. Furthermore, the frequency of these cells in the periphery was increased by immunization, as demonstrated by a greater number of responding monkeys in the rPvCS2 and rPvCS3 immunized groups.     

Evaluation of a quantitative determination of CD4 and CD8 molecules as an alternative to CD4+ and CD8+ T lymphocyte counts in Africans

In the developed word, monitoring HIV-infected patients is routinely determined by CD4+ T lymphocyte absolute counts. The reference procedure, flow cytometry, is expensive, requires sophisticated instrumentation and operators with specific training. Due to these limitations, CD4 counting is often unavailable in developing countries. The Capcellia assay is an enzyme-linked immunoassay for quantitative determination of CD4 and CD8 molecules. We evaluated this method in West Africa on blood samples collected from 39 HIV-uninfected and 44 HIV-infected adult subjects. CD4 concentration ranges were determined according to the clinical stages of the disease. We then studied the relationship between the two methods in the HIV-infected patients. The Spearman's rank correlation was 0.61 (95% confidence interval: 0.38-0.76, P < 0.0001). Nevertheless, determination of limits of agreement revealed discrepancies between the two methods, especially for CD4 counts > 0.4 x 10(9)/l, which are discussed. We conclude that the Capcellia assay is a convenient means to determine the immunodepression level where flow cytometric instrumentation is unavailable, and can be complementary to CD4 T lymphocyte enumeration.