Lack of in vivo compartmentalization among HIV-1 infected naïve and memory CD4 T cell subsets

Viral compartmentalization between naïve and memory CD4⁺ T cell subsets has been described, but only for individuals who were receiving antiretroviral therapy (ART). We present here an extensive analysis of the viral quasispecies residing in the naïve, central and effector memory CD4⁺ T cell subsets in a number of therapy naïve individuals and representing an array of HIV-1 subtypes. We longitudinally analyzed subset-specific infection and evolution in a subtype B infected individual who switches from CCR5 to dual CCR5/CXCR4 coreceptor usage. We show that the central memory subset, the predominantly infected subset, harbors a more diverse viral population compared to the others. Through sequence analysis of the env C2V3 region we demonstrate a lack of viral compartmentalization among all subsets. Upon coreceptor switch we observe a pronounced increase in the infection level of the naïve population. Our findings emphasize the importance of all CD4⁺ T cell subsets to viral evolution.     

Maintenance of naïve CD8 T cells in nonagenarians by leptin, IGFBP3 and T3

Research into the age-associated decline in the immune system has focused on the factors that contribute to the accumulation of senescent CD8 T cells. Less attention has been paid to the non-immune factors that may maintain the pool of naïve CD8 T cells. Here, we analyzed the status of the naïve CD8 T-cell population in healthy nonagenarians (≥90-year-old), old (60-79-year-old), and young (20-34-year-old) subjects. Naïve CD8 T cells were defined as CD28⁺CD95⁻ as this phenotype showed a strong co-expression of the CD45RA⁺, CD45RO⁻, and CD127⁺ phenotypes. Although there was an age-associated decline in the percentage of CD28⁺CD95⁻ CD8 T cells, the healthy nonagenarians maintained a pool of naïve CD28⁺CD95⁻ cells that contained T-cell receptor excision circles (TREC)⁺ cells. The percentages of naïve CD28⁺CD95⁻ CD8 T cells in the nonagenarians correlated with the sera levels of insulin-like growth factor binding protein 3 (IGFBP3) and leptin. Higher levels of triiodothyronine (T3) negatively correlated with the accumulation of TREC⁻CD28⁻CD95⁺ CD8 T cells from nonagenarians. These results suggest a model in which IGFBP3, leptin and T3 act as non-immune factors to maintain a larger pool of naïve CD8 T cells in healthy nonagenarians.     

Low pre-infection levels and loss of central memory CD4+ T cells may predict rapid progression in SIV-infected pigtail macaques

CD4⁺ T lymphocyte subsets are targeted to different degrees by SIV infection. We studied central memory, effector memory, naïve, and regulatory T cell levels longitudinally in 11 SIV_mac251-infected pigtail macaques. Depletion of CD28⁺CD95⁺ central memory CD4⁺ T cells, but not other populations, correlated with both SIV viral load and disease progression. A low pre-infection level of central memory CD4⁺ T cells was also predictive of rapid disease progression. If confirmed in larger studies, our results suggest stratifying macaques for baseline central memory CD4⁺ T cells would be useful in defining both the pathogenesis of SIV disease and SIV vaccine efficacy.     

Workshop F: Lymphocyte Activation, Abstract F1 bis F24

F. 1 T cell signaling pathways are differentially affected in oxidative stress-induced hyporesponsivenessF. 2 Mitogenic anti-CD28 antibody stimulation induces PLC-γ, PKC-θ and NF-κB activation leading to T cell proliferation without TCR ligationF. 3 Protein kinase C-β1, a cyclosporin A-sensitive regulator of T cell receptor-/CD28-activated signal transductionF. 4 Differential roles of O-acetylated ganglioside GD3 (CD60b,c) on human tonsillar B lymphocytesF. 5 Platelet factor 4 inhibits proliferation and cytokine release of activated human T cellsF. 6 The impact of B cells on the polarization of naïve Th cellsF. 7 Impaired upregulation of activation/maturation molecules on naïve B cells from patients with common variable immunodeficiencyF. 8 Regulation of Hsp90-associated cochaperone p23 during T cell activationF. 9 Direct correlation of B7 expression levels with T cell stimulationF. 10 Detection of CTLA-4 in the rat by monoclonal antibodiesF. 11 T cell proliferation measured by flow cytometric assessment of nuclear antigen Ki67F. 12 Minimal costimulatory requirements of T cell priming and Th1 differentiation: Investigations with targeted bifunctional antibody constructs on naïve human T lymphocytesF. 13 Involvement of the metastasis-associated protein CD44v6 in T lymphocyte activationF. 14 The expression of surface CTLA-4 is time-dependent and regulated by multiple factorsF. 15 CD4 cross-linking prior to TCR/CD3 stimulation enhances the expression of activation antigens on purified naïve helper T cellsF. 16 CTLA-4 lacking the cytoplasmic domain costimulates IL-2 production in the T cell hybridoma DO11.10F. 17 Binding of the extracellular domain of CD22 to sialic acid residues on the B cell surface is needed for inhibition of the BCR-induced Ca²⁺ flux as demonstrated by the use of a novel sialic acid analogueF. 18 Expression and function of surface CTLA-4 on polarized T helper lymphocytesF. 19 MART-1 peptide stimulates Jurkat T cells independent of TCR expressionF. 20 Flottilin-1 and 2 reside in lipid rafts of B cells, T cells, and macrophagesF. 21 Induction of Notch1 synthesis in murine splenic B cells by LPS and anti-CD40/IL-4F. 22 The effectiveness of antigen presentation by dendritic cells to CD4⁺ T helper cells but not CD8⁺ cytotoxic T cells is depending on the maturational stateF. 23 Similar morphodynamic interaction of macrophages or dendritic cells with antigen-specific T cells despite dramatic differences in their capacity to activate T cellsF. 24 Importance of tryptophan in position 2 for inhibitory interaction of short peptides with T cell-expressed DPP IV (CD26)     

Expression and co-stimulatory function of B7-2 on murine CD4+ T cells

Co-stimulatory signals mediated by the interaction of B7-1/B7-2 with CD28 are important for the activation of CD4⁺ T cells stimulated with antigen on antigen-presenting cells. There are controversies about the expression and function of B7-1/B7-2 on CD4⁺ T cells. The aim of this study was to analyze the expression of B7-1/B7-2 on naive and memory CD4⁺ T cells and the co-stimulatory function in the activation of naive CD4⁺ T cells stimulated by TCR ligation. Present results indicate that memory CD4⁺ T cells express B7-2 molecules on their surface, whereas naive CD4⁺ T cells do not. Neither memory nor naive CD4⁺ T cells expressed B7-1 molecule on their surface, although B7-1 mRNA was faintly expressed in memory T cells. B7-2 molecules expressed on memory T cells co-stimulated CD4⁺ naive T cells stimulated with plate-coated anti-CD3 to produce IL-2. Naive CD4⁺ T cells were shown to express B7-2 after co-stimulation with B7-2 and TCR ligation, because the naive T cells stimulated with anti-CD3 and B7-2CHO expressed B7-2 on their surface, although it remained to be studied whether the co-stimulation with B7-2 directly induced B7-2 expression on naive T cells. Our present results indicate that memory CD4⁺ T cells play some role in the activation of naive CD4⁺ T cells through the co-stimulation with B7-2 molecules.     

Antigen-nonspecific activation of CD8+ T lymphocytes by cytokines: relevance to immunity, autoimmunity, and cancer

Development of T lymphocytes and their survival in the periphery are dependent on signals emanating from cytokine receptors as well as the T cell antigen receptor (TCR). These two signaling pathways play distinct and complementary roles at various stages of T cell development, maturation, survival, activation and differentiation. During immune response to foreign antigens initiated by TCR signaling, cytokines play a key role in the expansion of activated T cells. Even though the initial activation of T cells occurs via the TCR, this requirement can be overcome under certain circumstances. During lymphopenia, cytokines trigger memory CD8⁺ T cells to undergo antigen non-specific homeostatic expansion, whereas naïve CD8⁺ T cells require both cytokines and TCR signaling. Recent reports show certain combinations of cytokines can induce proliferation and effector functions of naïve CD8⁺ T cells without concomitant stimulation via the TCR. While such antigen non-specific stimulation of naïve T cells might significantly boost the adaptive immune response, it could also have an undesirable effect of triggering potentially autoreactive cells. Understanding the mechanisms and the regulation of cytokine-driven stimulation of naïve CD8⁺ T cells may lead to novel strategies of intervention for autoimmune diseases. On the other hand, in vitro expansion of naïve CD8⁺ T cells by certain combinations of cytokines could be used to generate tumor-specific cells with ideal properties for cellular immunotherapy of cancer.     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.     

Inhibition of Notch signalling has ability to alter the proximal and distal TCR signalling events in human CD3+ αβ T-cells

The Notch signalling pathway is an important regulator of T cell function and is known to regulate the effector functions of T cells driven by T cell receptor (TCR). However, the mechanism integrating these pathways in human CD3⁺ αβ T cells is not well understood. The present study was carried out to investigate how Notch and TCR driven signalling are synchronized in human αβ T cells. Differential expression of Notch receptors, ligands, and target genes is observed on human αβ T cells which are upregulated on stimulation with α-CD3/CD28 mAb. Inhibition of Notch signalling by GSI-X inhibited the activation of T cells and affected proximal T cell signalling by regulating CD3-ζ chain expression. Inhibition of Notch signalling decreased the protein expression of CD3-ζ chain and induced expression of E3 ubiquitin ligase (GRAIL) in human αβ T cells. Apart from affecting proximal TCR signalling, Notch signalling also regulated the distal TCR signalling events. In the absence of Notch signalling, α-CD3/CD28 mAb induced activation and IFN-γ production by αβ T cells was down-modulated. The absence of Notch signalling in human αβ T cells inhibited proliferative responses despite strong signalling through TCR and IL-2 receptor. This study shows how Notch signalling cooperates with TCR signalling by regulating CD3-ζ chain expression to support proliferation and activation of human αβ T cells.     

Potentiation of TLR9 responses for human naïve B-cell growth through RP105 signaling

Toll-like receptor 9 (TLR9) signals induce important pathways in the early defense against microbial pathogens. Although TLR9 signaling can activate memory B cells directly, efficient naïve B cell responses seem to require additional, but as yet unidentified, signals. We explored the effects of RP105 (CD180) on CpG DNA-activated naïve and memory B cells from normal controls and patients with common variable immunodeficiency (CVID). RP105 dramatically enhanced CpG DNA-induced proliferation/survival by naïve B cells but not by memory B cells. This enhancement was mediated by TLR9 upregulation induced by RP105, leading to Akt activation and sustained NF-κB activation. CpG DNA-activated CVID B cells showed enhancement of proliferation/survival by RP105 and produced specific IgM antibody to Streptococcus pneumoniae polysaccharides in response to interleukin-21 stimulation. Thus, RP105 strongly affects expansion of the naïve B-cell pool, and suggests that the putative RP105 ligand (s) upon cytokine stimulation facilitates antibody-mediated acute pathogen clearance.     

A 150-kDa molecule of macrophage membrane stimulates interleukin-2 and interferon-γ production and proliferation of ovalbumin-specific CD4+ T cells

In the present study, we describe the potential co-stimulatory role of a macrophage membrane-associated protein of 150 kDa (M150). The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was found to be a single molecule on two-dimensional gel electrophoresis. The molecule was re-constituted in phosphatidyl choline vesicles and tested for its ability to promote the proliferation and the secretion of lymphokines from T helper (Th) cells. The reconstituted M150 induced a significant proliferation of anti-CD3 monoclonal antibody (mAb)-stimulated ovalbumin-specific CD4⁺ T cells. Further, Th cells activated with this molecule in the presence of anti-CD3 mAb mainly secreted interleukin (IL)-2 and interferon-γ but not IL-4. M150 could not promote the proliferation of Th cells, or lymphokine secretion in the absence of anti-CD3 mAb. These observations suggest that M150 acts by selectively activating a Th1-like immune response.     

Inhibition of TNF receptor signaling by anti-TNFα biologicals primes naïve CD4 T cells towards IL-10 T cells with a regulatory phenotype and function

TNFα is a potent pro-inflammatory cytokine playing a pivotal role in several autoimmune diseases. Little is known about the mechanism of TNFα blocking agents on naïve T cell differentiation. Here, we report that neutralizing TNFα during priming of naïve CD4⁺ T cells by dendritic cells favors development of IL-10⁺ T helper cells. TNFα counteracts IL-10⁺ T cell priming mainly via TNFRI receptor signaling. While initial T cell activation was not affected, neutralization of TNFα negatively affected sustained T cell differentiation in later stages of T cell priming. Whole genome gene expression analysis revealed an extended regulatory gene profile for anti-TNFα-treated T cells. Indeed, neutralizing TNFα during naïve T cell priming enhanced the suppressive function of anti-TNFα-treated T cells. Taken together, inhibition of TNFα–TNFR interaction shifts the balance of Th cell differentiation towards IL-10 expressing suppressive T cells, which may be one of the beneficial mechanisms in TNFα blocking therapies.     

Human memory T cell differentiation into Th2-like effector cells is dependent on IL-4 and CD28 stimulation and inhibited by TCR ligation

Freshly isolated memory T cells primarily produced IL-2 and small amounts of IL-4 and IFN-γ after stimulation in vitro. Priming for 5 days in vitro with anti-CD28 monoclonal antibodies (mAb) alone markedly increased production of IL-4. In comparison to fresh cells, the increase in the amount of IL-4 secreted reflected a marked increase in the number of IL-4-producing cells. Stimulation with immobilized anti-CD3 mAb during priming limited subsequent IL-4 production. By contrast, IFN-γ production from in vitro primed memory T cells was directly correlated to the concentration of priming anti-CD3 mAb. IL-2 production by all restimulated cells was decreased. The differentiation of IL-4-producing cells could be blocked by antibody to IL-4 and enhanced by the addition of recombinant IL-4 as well as antibody to IFN-γ. Of note, the IL-4-producing effector cells induced from in vitro priming derived from the early CD27^pos memory T cell subset, whereas the small CD27^neg differentiated memory subset produced IL-4 without in vitro priming. The results indicate that memory T cells can be directed to differentiate into IL-4-producing effector cells by stimulation via CD28 and IL-4, whereas increasing engagement of the TCR limits Th2 memory cell differentiation.     

Co-stimulatory pathways controlling activation and peripheral tolerance of human CD4+CD28− T cells

Co-stimulation mediated by the CD28 molecule is considered critical in the activation of CD4⁺ T cells. In patients with rheumatoid arthritis and infrequently in normal individuals, CD4⁺ T cells lacking CD28 expression are expanded and contain clonogenic populations. To analyze whether these cells are independent of co-stimulatory requirements or whether they use co-stimulatory signals distinct from the CD28 pathway, we have compared CD4⁺ CD28⁺ and CD4⁺ CD28^?T cell clones isolated from rheumatoid arthritis patients. Accessory cells supported the induction of CD25 expression as well as of proliferative responses after anti-CD3 cross-linking and prevented the induction of anergy in CD4⁺ CD28^? T cell clones. In contrast to CD4⁺CD28⁺ T cells, the presence of accessory cells did not enhance the secretion of interleukin (IL)-2, interferon-γ, or IL-4. The co-stimulatory signals did not involve CD28/CTLA-4–CD80/CD86 receptor-ligand interactions. The proliferative response of CD4⁺CD28^? T cells could not be blocked by anti-CD2, anti-CD18, and anti-CD58 antibodies, suggesting that these receptor-ligand interactions cannot provide CD28^? independent co-stimulation. Our data suggest that CD4⁺CD28^? T cells require co-stimulatory signals for optimal induction of cell growth and CD25 expression as well as for the prevention of anergy. The co-stimulatory receptor-ligand interaction is independent of the CD28 pathway and may be involved in the oligoclonal expansion of the CD4⁺ CD28^? T cell subset in rheumatoid arthritis.     

Low-dose antigen-experienced CD4+ T cells display reduced clonal expansion but facilitate an effective memory pool in response to secondary exposure

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T-cell receptor (TCR)-transgenic CD4⁺ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4⁺ T cells generated in response to low-dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high-dose antigen stimulation and by naïve CD4⁺ T cells, resulting in higher numbers of antigen-specific CD4⁺ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low-dose antigen-experienced CD4⁺ T cells, we also observed accelerated recall responses upon injection of antigen-bearing antigen-presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long-lasting memory CD4⁺ T cells in response to secondary exposure.     

Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells

We have previously shown that CD4⁺ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4⁺ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4⁺ T cells only when CD4⁺ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4⁺ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4⁺ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4⁺ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

Similar co-stimulation requirements of CD4+ and CD8+ primary T helper cells: role of IL-1 and IL-6 in inducing IL-2 secretion and subsequent proliferation.

Primary murine CD4+ and CD8+ T helper (Th) cells provide help for various immune responses by secreting lymphokines which activate effector cells. The purpose of the present study was to investigate the co-stimulatory signals that, together with T cell receptor (TCR) cross-linking, induce phenotypically distinct primary Th cells to secrete IL-2 and proliferate. We isolated highly purified populations of primary CD4+ or CD8+ T cells and stimulated them in vitro with platebound anti-CD3 mAb. TCR cross-linking by anti-CD3 mAb induced both IL-2 receptor expression and responsiveness to exogenous IL-2, but was not sufficient to induce either IL-2 secretion or T cell proliferation. Rather, for both CD4+ and CD8+ primary Th cells, IL-2 secretion and proliferation required both TCR cross-linking and antigen presenting cell (APC)-derived co-stimulatory signals. Based on G-10 adherence and sensitivity to gamma-irradiation, the APC populations able to induce primary CD4+ Th cells and primary CD8+ Th cells to secrete IL-2 were indistinguishable. In addition, we found that either IL-1 or IL-6 could replace the requirement for APC-derived co-stimulatory signals for IL-2 secretion and proliferation by both primary CD4+ Th cells and primary CD8+ Th cells. Thus, the present study has examined and compared the co-stimulatory requirements of rigorously purified subsets of IL-2-secreting primary CD4+ and primary CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Memory and naïve B-cell subsets in patients with multiple sclerosis

Memory and naïve B cells are considered to play distinct roles in immune regulation. However, the roles of memory and naïve B-cell subsets in multiple sclerosis (MS) have not yet been elucidated. In this study, we examined whether memory and naïve B-cell subsets differ between patients with MS and healthy subjects and whether interferon beta (IFNβ)-1b can affect these subsets in patients with MS. We also studied these subsets in relapsing and remitting stages of MS. Subjects included 31 patients with relapsing–remitting MS in the remitting stage, of which 15 were treated with IFNβ-1b and 16 were not treated, and 22 healthy control subjects. For 11 of the 16 untreated patients, blood samples were also obtained in the relapsing stage. Expression of CD5, CD80, CD86, CCR5, CXCR3, CD11a, and CD49d in memory and naïve B cells in blood samples was examined by flow cytometry. The percentages of CD86⁺ cells and CCR5⁺ cells in the naïve B-cell subset were significantly higher in untreated patients than in control subjects or IFNβ-treated patients. In patients with MS, the percentages of CD86⁺ cells and CCR5⁺ cells in the naïve B-cell subset and the percentage of CD5⁺ cells in the memory B-cell subset were significantly greater in the remitting stage than in the relapsing stage. These results indicate that memory and naïve B-cell subsets, especially CD86⁺ naïve B cells, CCR5⁺ naïve B cells, and CD5⁺ memory B cells, might be useful in the study of the pathogenesis of and therapy for MS.