Impact of partial sleep deprivation on immune markers.

BACKGROUND: Sleep quality is considered to be an important predictor of immunity. Lack of sleep therefore may reduce immunity, thereby increasing the susceptibility to respiratory pathogens. A previous study showed that reduced sleep duration was associated with an increased likelihood of the common cold. It is important to understand the role of sleep in altering immune responses to understand how sleep deprivation leads to an increased susceptibility to the common cold or other respiratory infections. OBJECTIVE: We sought to examine the impact of partial sleep deprivation on various immune markers. PATIENTS AND METHODS: Fifty-two healthy volunteers were partially sleep deprived for one night. We took blood samples before the sleep deprivation, immediately after, and 4 and 7 days after sleep deprivation. We measured various immune markers and used a generalized estimating equation (GEE) to examine the differences in the repeated measures. RESULTS: CD4, CD8, CD14, and CD16 all showed significant time-dependent changes, but CD3 did not. The most striking time-dependent change was observed for the mitogen proliferation assay and for HLA-DR. There was a significant decrease in the mitogen proliferation values and HLA-DR immediately after the sleep deprivation experiment, which started to rise again on day 4 and normalized by day 7. CONCLUSIONS: The transiently impaired mitogen proliferation, the decreased HLA-DR, the upregulated CD14, and the variations in CD4 and CD8 that we observed in temporal relationship with partial sleep deprivation could be one possible explanation for the increased susceptibility to respiratory infections reported after reduced sleep duration.

Characterization of cytotoxic T-lymphocyte epitopes and immune responses to SARS coronavirus spike DNA vaccine expressing the RGD-integrin-binding motif.

Integrins are critical for initiating T-cell activation events. The integrin-binding motif Arg-Gly-Asp (RGD) was incorporated into the pcDNA 3.1 mammalian expression vector expressing the codon-optimized extracellular domain of SARS coronavirus (SARS-CoV) spike protein, and tested by immunizing C57BL/6 mice. Significant cell-mediated immune responses were characterized by cytotoxic T-lymphocyte (51)Cr release assay and interferon-gamma secretion ELISPOT assay against RMA-S target cells presenting predicted MHC class I H2-Kb epitopes, including those spanning residues 884-891 and 1116-1123 within the S2 subunit of SARS-CoV spike protein. DNA vaccines incorporating the Spike-RGD/His motif or the Spike-His construct generated robust cell-mediated immune responses. Moreover, the Spike-His DNA vaccine construct generated a significant antibody response. Immunization with these DNA vaccine constructs elicited significant cellular and humoral immune responses. Additional T-cell epitopes within the SARS-CoV spike protein that may contribute to cell-mediated immunity in vivo were also identified.

Targeting tumours by adoptive transfer of immune cells.

1. Surgery, radiotherapy and chemotherapy are the most widely used and well-established modalities for treating malignant diseases. Surgery is used to excise solid tumours and radiotherapy/chemotherapy are used for the treatment of liquid tumours and for solid tumours where there is a risk of micrometastases. A major drawback for both radiotherapy and chemotherapy is their lack of specificity for tumour cells. Both these treatments can destroy normal bone marrow cells and result in severe side-effects. 2. The impairment of haemapoiesis due to bone marrow destruction combined with the use of toxins in chemotherapy that inhibit the proliferation of immune cells leaves many patients immunocompromised. This complicates the development of prophylactic (vaccine) strategies for tumours where patients are undergoing conventional therapy. 3. An alternative approach is to expand and activate tumour-specific immune cells in vitro that can then be adoptively transferred back in large numbers. This is defined as adoptive immunotherapy and has the advantage of potentially bypassing the immuno-inhibitory effects of conventional therapies. 4. Transferred immune cells have been shown to mediate tumour regression in patients by both direct and indirect mechanisms. The immune cells used include tumour reactive T lymphocytes and dendritic cells, which elicit tumour specific responses. 5. Many novel cell-based immunotherapeutic strategies developed in murine tumour models are now being applied in human clinical trials. The malignancies targeted include melanoma, chronic myelogenous leukaemia and breast, ovarian, colon and kidney cancers. In the present review, we discuss these novel cell-based strategies and the implications they have for the future treatment of human malignancies.

Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers.

Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.

Peptides complexed with the protein HSP70 generate efficient human cytolytic T-lymphocyte responses.

Microbial HSPs (heat-shock proteins) are implicated in the induction of the innate and adaptive arms of the immune response. We set out to determine whether peptides complexed with HSP70 generate efficient CTL (cytolytic T-lymphocyte) responses. Human dendritic cells pulsed with peptide-loaded microbial HSP70 complexes generate potent antigen-specific CTL responses. Using fluorescence anisotropy, we have calculated the peptide-binding affinity of mycobacterial HSP70 (K(D)=14 microM) and show that 120 pM HSP70-bound peptide is sufficient to generate a peptide-specific CTL response that is four orders of magnitude more efficient than the peptide alone. Through the generation of mycobacterial HSP70 truncations, we find that the minimal 136 amino acid, mycobacterial HSP70 peptide-binding domain is sufficient to generate CTL responses. The design of an HSP70 mutant, in which the peptide-binding site of HSP70 is filled with a bulky hydrophobic residue, leads to a large decrease in the peptide-binding affinity. This mutant HSP70 retains stimulatory capacity but is unable to generate CTL and has separated antigen delivery from immunostimulation of dendritic cells.

T cytotoxic 1 and T cytotoxic 2 CD8 T cells both inhibit IgE responses.

BACKGROUND: It is well recognized that CD8 T cells inhibit IgE responses. In this study, we investigated the mechanism of CD8 T cell-mediated IgE suppression by comparing the capacity of T cytotoxic 1 (Tc1) and T cytotoxic 2 (Tc2) CD8 T cells to inhibit IgE responses to ovalbumin (OVA). METHODS: Tc1 and Tc2 CD8 T cells were generated from OVA(257-264)-specific Vbeta5.2 T cell receptor (TcR) transgenic mice by stimulation with anti-CD3 and anti-CD28 under Tc1 and Tc2 polarizing conditions. Tc1 and Tc2 Vbeta5.2 TcR CD8 T cells (10(6)) were adoptively transferred to syngeneic mice, and following immunization with 100 micro of OVA/alum, serum IgE antibodies were measured by passive cutaneous anaphylaxis and expressed as the highest dilution that gave a detectable skin response. RESULTS: Both Tc1 and Tc2 CD8 T cells from OT-I mice inhibited IgE. CONCLUSION: Both Tc1 and Tc2 CD8 T cells promote Th1 immunity and inhibit IgE responses. This process appears to be independent of CD8 T cell-derived IFN-gamma, as both Tc2 (IFN-gamma-) and Tc1 (IFN-gamma+) CD8 T cells inhibited IgE.

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells.

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as "sentinels" of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Allergen-specific Th1 cells counteract efferent Th2 cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via IFN-gamma.

Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.

Inhibitory effects of endogenous and exogenous interferon-gamma on bronchial hyperresponsiveness, allergic inflammation and T-helper 2 cytokines in Brown-Norway rats.

Interferon-gamma (IFN-gamma) is an important cytokine involved in the regulation of allergen-induced immune responses. We examined the role of IFN-gamma in a Brown-Norway rat model of bronchial hyperresponsiveness (BHR) and airway eosinophilia, and its effects on the mRNA expression of T helper type 1 (Th1)/Th2 cytokine. Ovalbumin (OA)-sensitized animals were given either exogenous IFN-gamma (105 U/rat over 3 days, intraperitoneally) or anti-IFN-gamma blocking antibody (DB-1 0.3 mg/rat, intravenously) prior to exposure to OA aerosol and were studied 18-24 hr later. In sensitized animals, OA induced significant BHR, accumulation of eosinophils, T lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also increased eosinophils and CD8+ T cells in the airways. Exogenous IFN-gamma attenuated allergen-induced BHR (P<0.02, compared with sham-treated animals) together with a significant reduction in eosinophil and neutrophil numbers in BAL fluid (P<0. 005), and eosinophils and CD8+ T cells in airways (P<0.05). By contrast, anti-IFN-gamma antibody increased airway CD4+ T cells and BHR. Using reverse transcriptase-polymerase chain reaction, significant increases in Th2 [interleukin-4 (IL-4), IL-5 and IL-10], and IFN-gamma cytokine mRNA were found in the lungs of sensitized and OA-exposed animals, while exogenous IFN-gamma significantly suppressed IL-4, IL-5 and IL-10 mRNA expression, and anti-IFN-gamma antibody increased IL-4 and IL-5 mRNA expression. These results indicate that Th1 effects, such as those mediated by IFN-gamma, play a down-regulatory role to suppress the Th2 responses associated with allergen-induced BHR and eosinophilic inflammation.

Effect of CD8+ T-cell depletion on bronchial hyper-responsiveness and inflammation in sensitized and allergen-exposed Brown-Norway rats.

We examined the role of CD8+ T cells in a Brown-Norway rat model of asthma, using a monoclonal antibody to deplete CD8+ T cells. Ovalbumin (OA)-sensitized animals were given anti-CD8 antibody (0.5 mg/rat) intravenously 1 week prior to exposure to 1% OA aerosol and were studied 18-24 hr after aerosol exposure. Following administration of anti-CD8 antibody, CD8+ cells were reduced to <1% of total lymphocytes in whole blood and in spleen. In sensitized animals, OA exposure induced bronchial hyper-responsiveness (BHR), accumulation of eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also an increase in tissue eosinophils and CD2+, CD4+ and CD8+ T cells in airways. Anti-CD8 antibody caused a further increase in allergen-induced BHR (P<0.03, compared with sham-treated animals), together with a significant increase in eosinophil number in BAL fluid (P<0.05). While CD2+ and CD4+ T cells in airways were not affected by anti-CD8 treatment, the level of CD8+ T cells was significantly reduced in sensitized, saline-exposed animals (P<0.04, compared with sham-treated rats), and sensitized and OA-challenged rats (P<0.002, compared with sham-treated rats). Using reverse transcription-polymerase chain reaction, an increase of T helper (Th)2 cytokine [interleukin (IL)-4 and IL-5], and also of Th1 cytokine [interferon-gamma (IFN-gamma) and IL-2], mRNA in the lung of sensitized and OA-exposed animals was found; after CD8+ T-cell depletion, Th1 cytokine expression was significantly reduced (P<0.02), while Th2 cytokine expression was unchanged. CD8+ T cells have a protective role in allergen-induced BHR and eosinophilic inflammation, probably through activation of the Th1 cytokine response.

Ovalbumin-specific, MHC class I-restricted, alpha beta-positive, Tc1 and Tc0 CD8+ T cell clones mediate the in vivo inhibition of rat IgE.

In the following study, we demonstrate that medium responder PVG rats immunized i.p. with OVA complexed to the adjuvant aluminum hydroxide exhibit a moderate IgE response (400-1000 ng/ml). In these rats, we demonstrate that underlying the MHC class II-restricted CD4+ T cell response, there is an MHC class I-restricted CD8+ T cell component that plays an important role in restricting the magnitude and duration of the IgE response. We show that in vivo depletion of CD8+ T cells effects a massive increase in IgE (20-fold), and that they are MHC class I-restricted, OVA-specific, cytolytic cells that universally produce IFN-gamma (25-69 ng/ml) and IL-2 (7.6-22 U/ml), and occasionally secrete IL-4 (68-81 U/ml IL-4), and when adoptively transferred into CD8-depleted recipients, can effect a significant reduction in IgE (3- to 50-fold). We also demonstrate that this in vivo inhibition of IgE is dependent on the Ag-specific activation of the CD8+ T cells, and that the activated CD8+ T cells will suppress total/bystander IgE in an Ag-nonspecific manner. These data are consistent with a growing literature demonstrating sensitization of MHC class I-restricted CD8+ T cells by exogenous protein Ags delivered to mucosal sites, and may represent a mechanism whereby a selective pressure can be applied on the functional outcome of an immunoglobulin response to environmental allergens.

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells.

There is a growing body of evidence which suggests that CD8+ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4+ T cell responses to ovalbumin (OVA) by CD8+ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8+ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8+ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8+ T cells on days 12-18 greatly enhanced the IgE response, which rose to 26 micrograms/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 microgram/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8+ T cells from animals immunized on day 12 completely inhibited the IgE response. This effect was antigen specific; CD8+ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8+ T cell depletion decreased interferon (IFN)-gamma production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4+ T cells. Furthermore, CD8+ T cell depletion and addition of anti-IFN-gamma antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8+ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8+ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-gamma production by CD4+ T cells.

Depletion of CD8+ T cells following primary immunization with ovalbumin results in a high and persistent IgE response.

The role of CD8+ T cells in the regulation of IgE was investigated by in vivo CD8+ T-cell depletion. Following intraperitoneal immunization with ovalbumin (OVA), OVA-specific T cell responses were first detected in the draining lymph nodes (LN) and subsequently in the spleen. In vitro depletion of LN CD8+ cells reduced the OVA-specific LN CD4+ T cell response; while depletion of splenic CD8+ cells enhanced the OVA-specific splenic CD4+ T cell response, suggesting an early helper and later suppressor function. CD8+ cell depletion in vivo up to 7 days after immunization failed to enhance IgE production, but depletion of CD8+ T cells between day 12 and day 18 increased IgE levels to over 10 microg/ml. Adoptive transfer of 1 x 10(7) purified CD8+ T cells (> 95% CD8+ > 98% CD3+ < 1% NK, < 1% CD4+) from parallel immunized rats or of 1 x 106 cells of an OVA-specific, MHC class I restricted CD8+ T cell line suppressed the IgE, but not IgG, response by > 95%. These results provide further evidence that CD8+ T cells inhibit IgE production, possibly by preventing the generation of adequate amounts of appropriate CD4+ T cell help.

Generation of rat MHC class-I-restricted ovalbumin-specific IgE inhibitory CD8+ T cell clones.

Allergic disease presents an increasing health problem in industrialised countries. Despite improved diagnosis and drug therapy, the number of fatalities from asthma and anaphylaxis continues to rise. A key element in the aetiology of the allergic response to inert protein antigens is an inherent imbalance in the atopic allergen specific CD4+ T cells, resulting in a dominance of the pathological, IL-4 producing, T helper 2 phenotype which favours B cell IgE production. Previous work from our laboratory, and elsewhere, has established that CD8+ T cells play an important role in regulating the magnitude and duration of IgE responses and may be vital for the restoration of IgE immunologic homoeostasis in 'normal' non-atopic humans and low/medium responder rodents. We have found that CD8+ T cell depletion during a critical period after immunization produces a high and persistent IgE response in otherwise IgE hyporesponsive animals. We and others have demonstrated that small, but important, quantities of soluble antigen can enter the MHC class I pathway and, using ovalbumin (OVA)-transfected antigen-presenting cells, have succeeded in cloning OVA-specific, MHC class I-restricted rat alpha/beta T cell receptor (TcR) positive CD8+ T cells. These cells universally secrete IFN-gamma (5-69 ng/ml) and IL-2 (7.6-37.0 U/ml) and occasionally IL-4 (16.0-8/1.0 IU). When adoptively transferred, 1 x 10(6) of these cloned cells inhibit IgE production in vivo by as much as 50-fold and this effect can be titrated down to 1 x 10(3) cells. The IgE regulatory activity of the clones appears unrelated to their cytolytic potential or to their capacity to make IFN-gamma.

IFN-gamma and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles.

In this study, we have investigated the effects of IL-4 and IFN-gamma on the growth and differentiation of CD8+ T cells in vitro. Rat splenic CD8+ T cells expressed higher levels of IL-4, IL-5, IL-10, and IFN-gamma mRNA, as measured by reverse-transcription PCR, than CD4+ T cells from the same source, whereas CD4+ T cells expressed more IL-2 and IL-6 mRNA. CD8+ T cells were cultured for 6 days with PMA, ionomycin, and IL-2, and their ability to proliferate, to express mRNA for IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-gamma, and to secrete IFN-gamma and IL-2 was determined. IL-4 could act as a growth factor for CD8+ T cells during primary stimulation, but inhibited proliferation of the restimulated 6-day-cultured CD8+ T cells. The highest levels of mRNA for IL-2 and IL-6 were detected in control cultures in which little mRNA for IL-4, IL-5, or IL-10 was detected. Addition of IL-4 to the primary cultures reduced the capacity of the restimulated cells to express mRNA for IL-2, and for IL-6, but enhanced expression of mRNA for IL-4 and IL-5. Addition of IFN-gamma to the cultures, alone or in conjunction with IL-4, or addition of IFN-gamma-specific neutralizing Ab, had little effect. However, in conjunction with IL-4, anti-IFN-gamma enhanced expression of mRNA for IFN-gamma, and for IL-10. These results indicate that IL-4 and IFN-gamma regulate the differentiation of CD8+ T cells and the growth of cytotoxic and other types of CD8+ T cells, and suggest pathways through which CD8+ T cells may interact with other immune cells.