CD4 Immune Responses Research Articles

Production and evaluation of immunologic characteristics of mzNL4-3, a non-infectious HIV-1 clone with a large deletion in the pol-Sequence

Inactivation of integrase (IN) and reverse transcriptase (RT) can revoke the replication of HIV virions, and non-infectious HIV particles are desirable virus-like particle (VLP) vaccine candidates. Here, we produced inactive HIV-1 particles fit for vaccine and virological purposes by introducing a mutation into the pol sequence. Proviral DNA (pNL4-3) was cut at two points in the pol region using the BalI restriction enzyme and then religated. HEK 293T cells were transfected with the resultant plasmid (pmzNL4-3) to produce mutated virions. To confirm a production of VLPs and evaluate their biological activity the p24 load and syncytium formation (MT2 cells) were analyzed. The assay indicated that mzNL4-3 virions were assembled and contained functional envelope glycoproteins (ENV). In addition, mzNL4-3 virions were able to infect neither the MT2 nor HEK 293T cells. Furthermore, the immunogenicity of VLPs was investigated in a mouse model. According to the data on vaccinated mice, the titer of ENV-specific antibodies rose rapidly after a boosting injection. Moreover, lymphoid cells extracted from these mice proliferated after exposure to the antigen. The mzNL4-3 virus particles possessed immunogenic antigens of HIV and can effectively trigger humoral and CD4 immune responses. Non-infectious mzNL4-3 virions may also be used in biomedical experiments for improving the biological safety conditions. Moreover, the mzNL4-3 seems to be a promising candidate for further HIV-1 vaccine investigations.

The Effect of Tumor Necrosis Factor-α-Induced Protein 8 Like-2 on Immune Response of CD4+T Lymphocytes in Mice after Thermal Injury

The status of cellular immunity has been shown to be associated with the occurrence and development of sepsis. Accumulating evidence has demonstrated that tumor necrosis factor-α-induced protein 8 like-2 (TIPE2) plays an important role in maintaining homeostasis of immune function. The present study, with the use of a controlled in vivo approach, demonstrated the effect of TIPE2 on cell-mediated immunity of CD4+ T lymphocytes in thermal injury murine model. One hundred and twenty-eight male mice were randomly allocated into four groups, which were sham burn group (n=48), burn group (n=48), burn with lentivirus-RNAi-TIPE2 transfection group (n=16), burn with negative control transfection group (n=16), and they were sacrificed at the designated time points. CD4+ T lymphocytes were isolated from the spleen using MACS microbeads. Phenotypes were analyzed by flow cytometry analysis, and cytokines were determined using ELISA kits. We found that the expression of TIPE2 was markedly increased in CD4+T lymphocytes in mice at 24,48 and 72 hours postburn. Down-regulation of TIPE2 by lentivirus-RNAi-TIPE2 attenuated the suppressive effect of CD4+T lymphocytes, which was associated with profound elevation of nuclear factor of activated T cell (NF-AT) activity. These results demonstrate that TIPE2 appear to be involved in the immune regulation of CD4+ T lymphocytes, and the decrease in TIPE2 expression on CD4+T lymphocytes in vivo can enhance peripheral T lymphocyte function after thermal injury. These data might provide a valid strategy to prevent the development of immunosuppressive state resulted from major burns.

Получение и иммунологические характеристики неинфекционного ВИЧ-1 клона mzNL4-3 с большой делецией вpol-последовательности

Inactivation ofintegrase and reverse transcriptase can revoke the replication of HIV virions, and non-infectious HIV particles are desirable virus-like particle (VLP) vaccine candidates. Here, we produced inactive in replication HIV-1 particles fit for vaccine and virological purposes by introducing a mutation into the pol sequence. Proviral DNA (pNLA-3) was cut at two points in the pol region using the Bal I restriction enzyme and then religated. HEK 293T cells were transfected with the resultant plasmid (pmzNL4-3) to produce mutated virions. To confirm a production of VLPs and evaluate their biological activity the p24 load and syncytium formation (MT2 cells) were analyzed. The assay indicated that mzNL4-3 virions were assembled and contained functional envelope glycoproteins (ENV). In addition, mzNL4-3 virions were not able to infect MT2 and HEK 293T cells. Furthermore, the immunogenicity of VLPs was investigated in a mouse model. According to the data on vaccinated mice, the titer of ENV-specific antibodies rose rapidly after a boosting injection. Moreover, lymphoid cells extracted from these mice proliferated after exposure to the antigen. The mzNL4-3 virus particles possessed immunogenic antigens of HIV and can effectively trigger humoral and CD4 immune responses. Non-infectious mzNL4-3 virions may also be used in biomedical experiments to improve the biological safety conditions. Moreover, the mzNL4-3 seems to be a promising candidate for further HIV-1 vaccine investigations.

PD-1/PD-L1 Blockade Can Enhance HIV-1 Gag-specific T Cell Immunity Elicited by Dendritic Cell-Directed Lentiviral Vaccines

Exhaustion of CD8(+) T cells and upregulation of programmed death 1 (PD-1), a negative regulator of T cell activation, are characteristic features of individuals chronically infected with human immunodeficiency virus type 1. In a previous study, we showed in mice that a dendritic cell-directed lentiviral vector (DCLV) system encoding the human immunodeficiency virus (HIV)-1 Gag protein was an efficient vaccine modality to induce a durable Gag-specific T cell immune response. In this study, we demonstrate that blocking of the PD-1/PD-1 ligand (PD-L) inhibitory signal via an anti-PD-L1 antibody generated an enhanced HIV-1 Gag-specific CD8(+) immune response following both a single round of DCLV immunization and a homologous prime/boost regimen. The prime/boost regimen combined with PD-L1 blockade generated very high levels of Gag-specific CD8(+) T cells comprising several valuable features: improved ability to produce multiple cytokines, responding to a broader range of Gag-derived epitopes, and long-lasting memory. This enhanced cellular immune response generated by DCLV immunization combined with anti-PD-L1 blockade correlated with improved viral control following challenge with Gag-expressing vaccinia virus. Taken together, our studies offer evidence to support the use of PD-1/PD-L1 blockade as an adjuvant modality to enhance antigen-specific immune responses elicited by T cell-based immunizations such as DCLV.

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4(+) and CD8(+) immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207(+), CD11c(+), CD4(+), CD8(+), iNOS(+) cellular densities. Cytokine (IFN-γ, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207(+) and CD11c(+) were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4(+) and CD8(+) were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS(+) was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-γ was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

Immunoglobulin Fc Fragment Tagging Allows Strong Activation of Endogenous CD4 T Cells To Reshape the Tumor Milieu and Enhance the Antitumor Effect of Lentivector Immunization

A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor benefits, mainly owing to multiple immune suppressions in established tumor lesions. In this study, we investigated if and how activation of endogenous CD4 T cells could be achieved to influence the suppressive tumor milieu and antitumor effect. We engineered a lentivector (lv) to express a nominal fusion Ag composed of hepatitis B surface protein and IgG2a Fc fragment (HBS-Fc-lv) to increase the magnitude of CD8 response but, more importantly, to induce effective coactivation of CD4 T cells. We found that, remarkably, immunization with HBS-Fc-lv caused significant regression of established tumors. Immunologic analysis revealed that, compared with HBS-lv without Fc fragment, immunization with HBS-Fc-lv markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor while substantially decreasing the regulatory T cell ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were dependent on the CD4 activation, which was Fc receptor mediated. Adoptive transfer of CD4 T cells from the HBS-Fc-lv-immunized mice could activate endogenous CD8 T cells in an IFN-γ-dependent manner. We conclude that endogenous CD4 T cells can be activated by lv expressing Fc-tagged Ag to provide another layer of help--that is, creating a Th1/Tc1-like proinflammatory milieu within the tumor lesion to boost the effector phase of immune responses in enhancing the antitumor effect.

Absence of CD4 T-Cell Help Provides a Robust CD8 T-Cell Response While Inducing Effective Memory in a Preclinical Model of Melanoma

Immunotherapy strategies for cancer are focused on inducing effective and specific cytotoxic responses mediated by CD8 T cells. On the other hand, immunosuppressive mechanisms induced by the tumor, such as the generation of tumor-specific CD4(+)CD25(+)FoxP3(+) Tregs, conspire against the efficacy of immunotherapies. It has been considered that, similar to what has been observed in the context of immunological responses towards microbes, CD4 help is indispensable for the development of a successful and long-lasting (memory) CD8 immune response. In the recent article, Côté et al. reported that, in a mouse model of melanoma, total ablation of CD4 help does not hamper the development of a specific antitumor memory CD8 response. In addition, ablation of CD4 was more successful than strategies to deplete CD25 Tregs in generating memory CD8 T cells. These data opens the door for therapies destined to induce effective antitumor immune responses by ablation of whole CD4 T-cell populations.

Immunoglobulin Fc fragment tagging allows strong activation of endogenous CD4 T cells to re-shape the tumor milieu and enhance the antitumor effect of lentivector immunization (162.4)

Abstract A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor benefits mainly due to the multiple immune suppressions in the established tumor lesions. In this study, we investigated if and how activation of endogenous CD4 T cells could be achieved to relieve the suppressive tumor milieu. We engineered a lentivector to express a nominal fusion Ag composed of HBsAg and IgG2a Fc fragment (HBS-Fc-lv) to increase the magnitude of CD8 response, but more importantly, to induce effective CD4 co-activation. We found that, remarkably, HBS-Fc-lv immunization caused significant regression of established tumors. Immunological analysis revealed that HBS-Fc-lv immunization markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor, while substantially decreased Treg ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were dependent on the Fc receptor mediated CD4 activation. Adoptive transfer of the CD4 T cells from the HBS-Fc-lv immunized mice could activate endogenous CD8 T cells via IFNgamma and CD40L dependent manner. We conclude that endogenous CD4 T cells can be activated by lentivector expressing Fc tagged Ag to provide another layer of help, i.e. creating a pro-inflammatory milieu within the tumor lesion to help the effector phase of immune responses to enhance the antitumor effect.

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.

CD40 ligand enhances immunogenicity of vector-based vaccines in immunocompetent and CD4+ T cell deficient individuals

Impairment of host immunity, particularly CD4+ T cell deficiency, presents significant complications for vaccine immunogenicity and efficacy. CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor superfamily (TNFSF), is an important co-stimulatory molecule and, through interactions with its cognate receptor CD40, plays a pivotal role in the generation of host immune responses. Exploitation of CD40L and its receptor CD40 could provide a means to enhance and potentially restore protective immune responses in CD4+ T cell deficiency. To investigate the potential adjuvanticity of CD40L, we constructed recombinant plasmid DNA and adenoviral (Ad) vaccine vectors expressing murine CD40L and the mycobacterial protein antigen 85B (Ag85B). Co-immunization of mice with CD40L and Ag85B by intranasal or intramuscular prime-boosting led to route-dependent enhancement of the magnitude of vaccine-induced circulating and lung mucosal CD4+ and CD8+ T cell responses in both normal (CD4-replete) and CD4+ T cell deficient animals, including polyfunctional T cell responses. The presence of CD40L alone was insufficient to enhance or restore CD4+ T cell responses in CD4-ablated animals; however, in partially depleted animals, co-immunization with Ag85B and CD40L was capable of eliciting enhanced T cell responses, similar to those observed in normal animals, when compared to those given vaccine antigen alone. In summary, these findings show that CD40L has the capacity to enhance the magnitude of vaccine-induced polyfunctional T cell responses in CD4+ T cell deficient mice, and warrants further study as an adjuvant for immunization against opportunistic pathogens in individuals with CD4+ T cell deficiency.

DOCK8 is essential for T‐cell survival and the maintenance of CD8+ T‐cell memory

Deficiency in the guanine nucleotide exchange factor dedicator of cytokinesis 8 (DOCK8) causes a human immunodeficiency syndrome associated with recurrent sinopulmonary and viral infections. We have recently identified a DOCK8-deficient mouse strain, carrying an ethylnitrosourea-induced splice-site mutation that shows a failure to mature a humoral immune response due to the loss of germinal centre B cells. In this study, we turned to T-cell immunity to investigate further the human immunodeficiency syndrome and its association with decreased peripheral CD4(+) and CD8(+) T cells. Characterisation of the DOCK8-deficient mouse revealed T-cell lymphopenia, with increased T-cell turnover and decreased survival. Egress of mature CD4(+) thymocytes was reduced with increased migration of these cells to the chemokine CXCL12. However, despite the two-fold reduction in peripheral naïve T cells, the DOCK8-deficient mice generated a normal primary CD8(+) immune response and were able to survive acute influenza virus infection. The limiting effect of DOCK8 was in the normal survival of CD8(+) memory T cells after infection. These findings help to explain why DOCK8-deficient patients are susceptible to recurrent infections and provide new insights into how T-cell memory is sustained.

Mathematical model of the primary CD8 T cell immune response: stability analysis of a nonlinear age-structured system

The primary CD8 T cell immune response, due to a first encounter with a pathogen, happens in two phases: an expansion phase, with a fast increase of T cell count, followed by a contraction phase. This contraction phase is followed by the generation of memory cells. These latter are specific of the antigen and will allow a faster and stronger response when encountering the antigen for the second time. We propose a nonlinear mathematical model describing the T CD8 immune response to a primary infection, based on three nonlinear ordinary differential equations and one nonlinear age-structured partial differential equation, describing the evolution of CD8 T cell count and pathogen amount. We discuss in particular the roles and relevance of feedback controls that regulate the response. First we reduce our system to a system with a nonlinear differential equation with a distributed delay. We study the existence of two steady states, and we analyze the asymptotic stability of these steady states. Second we study the system with a discrete delay, and analyze global asymptotic stability of steady states. Finally, we show some simulations that we can obtain from the model and confront them to experimental data.

Polyfunctional analysis of Gag and Nef specific CD8+ T-cell responses in HIV-1 infected Indian individuals

Polyfunctional CD8+ T-cells have been described as most competent in controlling viral replication. We studied the impact of antigen persistence on the polyfunctional immune responses of CD8+ T-lymphocytes to HIV Gag and Nef peptides and polyclonal stimuli in 40 ART naïve HIV infected individuals and analyzed the alterations in T-cell functionality in early and late stages of infection. Significantly elevated level of global response and polyfunctional profile of CD8+ T-cells were observed to polyclonal stimulation, than HIV specific antigens in chronically infected individuals. However no key differences were observed in CD8+ T-cell functional profile in any of the 15 unique subsets for Gag and Nef specific antigens. The subjects in early stage of infection (defined as a gap of 6 months or less between seroconversion and enrolment and with no apparent clinical symptoms) had a higher degree of response functionality (4+ or 3+ different functions simultaneously) than in the late stage infection (defined as time duration since seroconversion greater than 6 months). The data suggest that persistence of antigen during chronic infection leads to functional impairment of HIV specific responses.

TH2 heterogeneity: Does function follow form?

T(H)2 immune responses are required for the 2 fundamental pathological processes characteristic of allergic disease: IgE-mediated hypersensitivity and eosinophilic inflammation. The 3 established T(H)2 cytokines, IL-4, IL-5, and IL-13, each play a nonredundant role in allergic disease pathology. The recent explosion of T(H) subpopulations combined with the wide availability of polychromatic cytokine staining has facilitated the discovery of T(H)2 lineage heterogeneity. In this article we review T(H)2 heterogeneity and ask the following question: At what point do these subpopulations graduate from in vitro curiosities to immunologically robust therapeutic targets? We propose criteria to establish a T-cell subset as a biologically relevant entity and address the evidence to support these T(H)2 subpopulations having a unique function or specific contribution to allergic pathology or host defense.

2-Gy whole-body irradiation significantly alters the balance of CD4+ CD25- T effector cells and CD4+ CD25+ Foxp3+ T regulatory cells in mice.

CD4(+)CD25(+) T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4(+)CD25(+)Foxp3(+) Treg cells has not been determined. The proportion, phenotypes and function of CD4(+)CD25(+) Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4(+)CD25(+) Treg cells and CD4(+)CD25(+)Foxp3(+) Treg cells to CD4(+) T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4(+)CD25(+) Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4(+)CD25(-) T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4(+)CD25(+)Foxp3(+) Treg cells than in CD4(+)CD25(-) T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4(+)CD25(+)Foxp3(+) Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4(+)CD25(+)Foxp3(+) Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.

Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production

BackgroundInsect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system.ResultsTransgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs.ConclusionTransgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.

Tumor specificity of viral replication has no impact on antitumoral immunity, but dictates antiviral immune response and toxicity of oncolytic therapy

The immune response against cellular antigens due to viral infection is an important mechanism for autoimmune diseases, but can be also exploited for HCC immunotherapy. However, surprisingly little is known about the role of tissue specific viral replication on the induction of antiviral and anti-tissue immune responses. To investigate the impact of the specificity of viral replication on the immune responses we constructed a highly tumor-selective oncolytic adenovirus, whose replication is dependent on telomerase activity as well as aberrant transcriptional p53-status of the infected target cell. Viral DNA- and mRNA-levels in the liver due to unspecific replication of an unselective adenovirus correlated with strong stimulation of innate immune response genes and with liver toxicity, while telomerase/p53-specific replication prevented innate immune responses in the liver as well as hepatotoxicity. Compared to unspecific viral replication tumor restricted replication elicited a significantly lower antiviral CD8 immune response, whereas the specificity of viral replication had no impact on the induction of the antitumoral CD8-immune response after oncolytic virotherapy. In a mouse model with lung metastases, virotherapy of the primary tumor with the tumor selective virus significantly prolonged the survival by inhibition of virally uninfected lung metastases, demonstrating the efficacy of the antitumoral immune response. In contrast, unspecific viral replication resulted in early death of the animals due to liver failure, while untreated animals died early because of lung metastases. In summary our results demonstrate that tumor specificity of viral replication does not impair viral infection induced antitumoral immune response, but dictates antiviral immune responses and toxicity of virotherapy.

Tumor‐reactive CD8+ T‐cell responses after vaccination with NY‐ESO‐1 peptide, CpG 7909 and Montanide® ISA‐51: association with survival

Peptide-based vaccines have led to the induction of antigen-specific CD8(+) T-cell responses in patients with NY-ESO-1 positive cancers. However, vaccine-induced T-cell responses did not generally correlate with improved survival. Therefore, we tested whether a synthetic CpG 7909 ODN (deoxycytidyl-deoxyguanosin oligodeoxy-nucleotides) mixed with NY-ESO-1 peptide p157-165 and incomplete Freund's adjuvants (Montanide(R) ISA-51) led to enhanced NY-ESO-1 antigen-specific CD8(+) immune responses in patients with NY-ESO-1 or LAGE-1 expressing tumors. Of 14 HLA-A2+ patients enrolled in the study, 5 patients withdrew prematurely because of progressive disease and 9 patients completed 1 cycle of immunization. Nine of 14 patients developed measurable and sustained antigen-specific CD8(+) T-cell responses: Four had detectable CD8+ T-cells against NY-ESO-1 after only 2 vaccinations, whereas 5 patients showed a late-onset but durable induction of NY-ESO-1 p157-165 specific T-cell response during continued vaccination after 4 months. In 6 patients, vaccine-induced antigen-specific T-cells became detectable ex vivo and reached frequencies of up to 0.16 % of all circulating CD8(+) T-cells. Postvaccine T-cell clones were shown to recognize and lyse NY-ESO-1 expressing tumor cell lines in vitro. In 6 of 9 patients developing NY-ESO-1-specific immune responses, a favorable clinical outcome with overall survival times of 43+, 42+, 42+, 39+, 36+ and 27+ months, respectively, was observed.

Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+CD25Hi regulatory T cells

Regulatory CD4(+)CD25(Hi) T cells (Treg) and programmed death-1 (PD-1) molecule have emerged as pivotal players in immune regulation. However, the underlying mechanisms by which they impact antigen-specific CD8(+) immune responses in cancer patients and how they interact with each other under physiologic conditions remain unclear. Herein, we examined the relationship of PD-1 and its abrogation to the function of Treg in patients with melanoma using short-term in vitro assays to generate melanoma-specific T cells. We identified Treg in the circulation of vaccinated melanoma patients and detected PD-1 expression on vaccine-induced melanoma antigen-specific CTLs, as well as on and within Treg from patients' peripheral blood. Programmed death ligand (PD-L) 1 expression was also detected on patients' Treg. PD-1 blockade promoted the generation of melanoma antigen-specific CTLs and masked their inhibition by Treg. The mechanisms by which PD-1 blockade mediated immune enhancement included direct augmentation of melanoma antigen-specific CTL proliferation, heightening their resistance to inhibition by Treg and direct limitation of the inhibitory ability of Treg. PD-1 blockade reversed the increased expression of PD-1 and PD-L1 on melanoma antigen-specific CTL by Treg, rescued INF-gamma and IL-2 or INF-gamma and tumor necrosis factor-alpha co-expression and expression of IL-7 receptor by melanoma antigen-specific CTL which were diminished by Treg. PD-1 blockade also resulted in down-regulation of intracellular FoxP3 expression by Treg. These data suggest that PD-1 is importantly implicated in the regulation of Treg function in melanoma patients.