Perforin-dependent CD4+ T-cell cytotoxicity contributes to control a murine poxvirus infection

CD4(+) T cells are generally regarded as helpers and regulators of the immune response. Although cytolytic CD4(+) T cells have been described, whether those generated during the course of a viral infection play a role in virus control remains unknown. Here we show that during acute infection with ectromelia virus, the mouse homolog of the human virus of smallpox, large numbers of CD4(+) T cells in the draining lymph node and liver of resistant mice have a cytotoxic phenotype. We also show that these cells kill targets in vivo in a perforin-dependent manner and that mice with specific deficiency of perforin in CD4(+) T cells have impaired virus control. Thus, perforin-dependent CD4(+) T-cell killing of infected cells is an important mechanism of antiviral defense.     

CD8 T-cell recognition of acquired alloantigen promotes acute allograft rejection

Adaptive CD8 T-cell immunity is the principal arm of the cellular alloimmune response, but its development requires help. This can be provided by CD4 T cells that recognize alloantigen “indirectly,” as self-restricted allopeptide, but this process remains unexplained, because the target epitopes for CD4 and CD8 T-cell recognition are “unlinked” on different cells (recipient and donor antigen presenting cells (APCs), respectively). Here, we test the hypothesis that the presentation of intact and processed MHC class I alloantigen by recipient dendritic cells (DCs) (the “semidirect” pathway) allows linked help to be delivered by indirect-pathway CD4 T cells for generating destructive cytotoxic CD8 T-cell alloresponses. We show that CD8 T-cell–mediated rejection of murine heart allografts that lack hematopoietic APCs requires host secondary lymphoid tissue (SLT). SLT is necessary because within it, recipient dendritic cells can acquire MHC from graft parenchymal cells and simultaneously present it as intact protein to alloreactive CD8 T cells and as processed peptide alloantigen for recognition by indirect-pathway CD4 T cells. This enables delivery of essential help for generating cytotoxic CD8 T-cell responses that cause rapid allograft rejection. In demonstrating the functional relevance of the semidirect pathway to transplant rejection, our findings provide a solution to a long-standing conundrum as to why SLT is required for CD8 T-cell allorecognition of graft parenchymal cells and suggest a mechanism by which indirect-pathway CD4 T cells provide help for generating effector cytotoxic CD8 T-cell alloresponses at late time points after transplantation.Cytotoxic CD8 T-cell responses directed against MHC class I alloantigens are one of the principal mediators of acute allograft rejection (1, 2). Exceptionally, at very high precursor frequency, cytotoxic CD8 T cells can effect graft rejection autonomously (3), but generally, differentiation of naïve CD8 T cells to fully functional cytotoxic effector cells capable of mediating acute allograft rejection requires help from activated CD4 T cells. How such help is delivered remains unclear. Studies of conventional CD8 T-cell responses against nontransplant antigens have demonstrated that CD4 T-cell help does not involve cognate cell-surface interaction between the helper CD4 T cell and cytotoxic CD8 T cell, but that instead, help is delivered to an intermediary antigen presenting cell (APC), which is then licensed to prime CD8 T cells (4–6). A critical requirement for such help is the expression of both the CD4 and CD8 T-cell epitopes on the same APC, and an analogous three-cell cluster model (Fig. 1A) in transplantation is only possible if help for CD8 T cells is provided by CD4 T cells that recognize intact MHC class II alloantigen on the surface of donor APCs, via the so-called “direct pathway” (7). Murine studies have confirmed that restricting CD4 T-cell help to the direct pathway generates strong cytotoxic CD8 T-cell alloresponses that effect rapid allograft rejection (8).Open in a separate windowFig. 1.Possible mechanisms for provision of CD4 T-cell help to alloreactive CD8 T cells. Analogous to provision of CD4 T-cell help for cytotoxic CD8 T-cell responses against nominal protein antigen, simultaneous linked direct-pathway allorecognition of MHC class I and class II alloantigen on donor antigen presenting cells (APCs) by cytotoxic CD8 and helper CD4 T cells, respectively, is considered the dominant helper mechanism (A). Nevertheless, indirect-pathway CD4 T cells that recognize self-restricted alloantigen following internalization and processing by recipient APCs can also provide help; an unlinked four-cell cluster model has been proposed (B). Provision of linked help by indirect pathway CD4 T cells may be achieved by a three-cell cluster model in which the recipient APC simultaneously presents intact and processed MHC class I alloantigen to, respectively, the cytotoxic CD8 T-cell and helper CD4 T-cell (C).The indirect pathway of allorecognition, whereby CD4 T cells recognize processed alloantigen as self-restricted allopeptide (7, 9), is now viewed as being at least as important as the direct pathway for initiating and mediating allograft rejection. This importance partly reflects the unique ability of indirect-pathway CD4 T cells to act as helper T cells for generating sophisticated alloantibody responses (10), but it is now also clear that effective cytotoxic CD8 T-cell responses can be generated when CD4 T-cell help is restricted exclusively to the indirect pathway (11). This observation is surprising, because the delivery of indirect-pathway T-cell help is only readily explained by postulating the unlikely formation of a cumbersome four-cell cluster, comprising CD4 and CD8 T lymphocytes and both recipient and donor APC (Fig. 1B), a cluster in which there are no apparent cell surface ligands to enable physical linkage between the donor APC/recipient CD8 T-cell couplet and the recipient APC/CD4 T-cell couplet. This scenario raises concerns of potentially uncontrolled CD8 T-cell alloimmunity, because such “unlinked” help could in principle be provided by concurrent exposure to any unrelated antigen.The appreciation that many types of cells, but particularly dendritic cells (DCs), can capture (“trogocytose”) membrane fragments from other cells (12, 13) has prompted the proposal that cytotoxic CD8 T-cell alloimmunity may be initiated by a “semidirect” pathway, whereby intact donor MHC class I alloantigen is recognized after its acquisition onto the surface of recipient DCs. In support, murine studies have detailed in vivo capture of membrane alloantigen by host cells (14, 15). This phenomenon provides a possible mechanism by which the same recipient APC as represents intact MHC class I alloantigen could simultaneously present processed allopeptide for recognition by indirect-pathway CD4 T cells. This scenario would enable formation of a linked three-cell cluster (Fig. 1C) that obviates many of the concerns associated with the above four-cell cluster model. However, although we have recently demonstrated simultaneous presentation of intact and processed alloantigen by recipient DCs following heart transplantation (16), the contribution of trogocytosis of alloantigen to graft rejection has yet to be clarified. To have functional relevance, presentation of intact donor alloantigen by recipient APCs must, at least in certain circumstances, be more effective for triggering cytotoxic alloimmunity than conventional encounter on donor APCs, and this has not been shown. Addressing this key concern is challenging, because it requires development of an experimental model in which CD8 T-cell allorecognition of target MHC class I alloantigen is limited to the surface of recipient, but not donor APCs. One way this scenario could perhaps be achieved is by resolving the paradox created by the seminal observations that CD8 T cells can still effect allograft rejection when target MHC class I alloantigen is expressed by graft parenchyma only (17), but that rejection of vascularized allografts does not occur in the absence of secondary lymphoid tissue (SLT) (18). Why SLT is necessary for CD8 T-cell allorecognition of graft parenchymal cells has not been explained, but one elegant and compelling solution is that alloreactive CD8 T-cell activation occurs in lymphoid tissue because shed, intact MHC class I alloantigen is only presented by recipient APCs at these sites.Here, we show that rejection of heart allografts in mice lacking hematopoietic APCs is mediated by alloreactive CD8 T cells, and that the participation of CD8 T-cells is dependent upon the acquisition and presentation of intact MHC class I alloantigen by recipient DCs within host SLT. We further demonstrate that representation of intact alloantigen enables the delivery of essential help from indirect-pathway CD4 T cells. These findings provide strong functional support for the provision of indirect-pathway CD4 T-cell help to alloreactive CD8 T cells via a “linked” three-cell cluster model.     

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, C?té 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.     

Age-related accumulation of a novel CD44 + CD25lowgammadelta T-cell population in hematopoietic organs of the mouse

We discovered a novel population of gammadelta T cells in the mouse that accumulates with age in hematopoietic organs, but not in epithelia. These cells are CD25low (an unusual phenotype for gammadelta T cells in the mouse); express higher levels of TCRgammadelta and CD44 than do CD25- gammadelta T cells; mainly express Vgamma2, Vgamma3, and Vgamma4 chains; and are largely quiescent. A very similar cell population appears in the late stages of fetal thymus organ cultures, suggesting that the accumulation of CD44 + CD25lowTCRgammadelta + cells is a response to stress induced by aging in vivo or by culture in vitro. The precursors of CD44 + CD25lowTCRgammadelta + cells are generated during fetal or very young adult life, as this population was undetectable in aged recipients of bone marrow from old or young donors. CD44 + CD25lowTCRgammadelta + cells may be a biomarker of aging, but could also play a role in the inflammatory changes that accompany aging.     

Homeostatic cytokines orchestrate the segregation of CD4 and CD8 memory T-cell reservoirs in mice

Memory T cells (T(M)s) have been detected in many tissues but their quantitative distribution remains largely undefined. We show that in mice there is a remarkably biased accumulation of long-term CD4 T(M)s into mucosal sites (mainly gut, especially Peyer patches), and CD8 T(M)s into lymph nodes and spleen (in particular, peripheral lymph nodes [PLNs]). This distinction correlates with their differentiated expression of PLN- and gut-homing markers. CD8 and CD4 T(M)s selectively require the expression of PLN-homing marker CCR7 or gut-homing marker α4β7 for maintenance. PLNs and gut supply CD8 and CD4 T(M)s with their individually favored homeostatic cytokine, IL-15, or IL-7. Cytokine stimulation in turn regulates the different gut-homing marker expression on CD4 and CD8 T(M)s. IL-15 plays a major role in vivo regulating CD8 T(M)s homing to PLNs. Thus, the reservoir segregation of CD4 and CD8 T(M)s meets their individual needs for homeostatic cytokines and is under feedback control of cytokine stimulation.     

IL4 production and increased CD30 expression by a unique CD8+ T-cell subset in B-cell chronic lymphocytic leukaemia

Phenotypic and functional abnormalities within the residual non-B-cell compartment of B-cell chronic lymphocytic leukaemia (CLL) suggest an interaction between tumour cells and host immune effectors. To explore the possibility of a polarized Th1/Th2 response we have studied CD30 antigen expression and the pattern of cytokine production by purified CLL T cells. Activated T cells from CLL patients showed a significant increase in the expression of CD30 compared to normal controls. Accordingly, high levels of soluble CD30 were detected in supernatants from activated T-cell cultures, as well as in CLL serum samples. Messenger RNA for IL4 was found in both resting and, to a greater extent, in activated CLL T lymphocytes. The latter cells were also capable of releasing IL4. Three-colour immunofluorescence analyses revealed a strong CD30 expression in the CD3+/CD8+/CD28- large granular lymphocyte subset, which is considerably expanded in CLL. Production of IL4, as well as expression and release of CD30 by these T cells, was conclusively demonstrated at the clonal level. These findings document an expansion of a peculiar subset of 'Th2-like' cells in CLL, with an increased IL4 production and CD30 expression and release, that are likely to contribute to both the B-cell accumulation and immune-defects characteristic of this disease.     

Interleukin-10 promotes the maintenance of antitumor CD8(+) T-cell effector function in situ

Interleukin-10 (IL-10) is a multifunctional cytokine that can exert suppressive and stimulatory effects on T cells. It was investigated whether IL-10 could serve as an immunostimulant for specific CD8(+) cytotoxic T cell (CTL) in vivo after vaccination and, if so, under what conditions. In tumor prevention models, administration of IL-10 before, or soon after, peptide-pulsed primary dendritic cell immunization resulted in immune suppression and enhanced tumor progression. Injection of IL-10, however, just after a booster vaccine significantly enhanced antitumor immunity and vaccine efficacy. Analysis of spleen cells derived from these latter animals 3 weeks after IL-10 treatment revealed that the number of CD8(+) CD44(hi) CD122(+) T cells had increased and that antigen-specific proliferation in vitro was enhanced. Although cytotoxicity assays did not support differences between the various treatment groups, 2 more sensitive assays measuring antigen-specific interferon-gamma production at the single-cell level demonstrated increases in the number of antigen-specific responder T cells in animals in the vaccine/IL-10 treatment group. Thus, IL-10 may maintain the number of antitumor CD8(+) T cells. In adoptive transfer studies, the ability of IL-10 to maintain CTL function could be enhanced by the depletion of CD4(+) T cells. This suggests that IL-10 mediates contrasting effects on both CD4(+) and CD8(+) T cells that result in either immune dampening or immune potentiation in situ, respectively. Appreciation of this dichotomy in IL-10 immunobiology may allow for the design of more effective cancer vaccines designed to activate and maintain specific CD8(+) T-cell effector function in situ.     

c-Myc acts downstream of IL-15 in the regulation of memory CD8 T-cell homeostasis

A subset of CD8 T cells in normal mice, expressing high levels of activation markers such as CD44, shares many properties with antigen-specific memory CD8 T cells. Homeostasis of CD44(high) CD8 T cells depends upon cytokines such as interleukin-15 (IL-15); however, the downstream signaling pathways regulating IL-15-dependent homeostatic proliferation are poorly defined. Surprisingly, we show here that haploinsufficiency of the protooncogene c-myc leads to a highly selective decrease in CD44(high) CD8 T cells in mice. Although steady-state proliferation and survival of CD44(high) CD8 T cells appeared not to be dependent on c-Myc, homeostatic proliferation of c-myc(+/-) CD44(high) CD8 T cells in lymphopenic hosts was strongly reduced, and the residual homeostatic proliferation of these cells appeared to occur independently of IL-15. Moreover, c-myc(+/-) CD44(high) CD8 T cells responded very poorly to purified IL-15 in vitro. Backcrossing of c-myc(+/-) mice to IL-15(-/-) mice revealed that the number of CD44(high) CD8 T cells decreased in an additive fashion in mice heterozygous for c-myc and IL-15. Finally homeostatic proliferation of antigen-specific memory CD44(high) CD8 T cells was also impaired in c-myc(+/-) mice. Collectively, our data identify c-Myc as a novel downstream component of the IL-15-dependent pathway controlling homeostatic proliferation of memory CD44(high) CD8 T cells.     

Serum soluble CD8 molecule is a marker of CD8 T-cell activation in HIV-1 disease

To characterize CD8 T-cell activation during HIV-1 infection we measured serum soluble CD8 (sCD8) levels longitudinally in seroconverters and in individuals with established HIV infection who were in different stages of illness. CD8 T-cell activation occurs very early in HIV infection. Serum sCD8 levels were elevated in 91.5% of the first seropositive samples in seroconverters. Furthermore, CD8 T-cell activation persists throughout HIV infection. sCD8 predicted the occurrence of AIDS in HIV-seropositive individuals and so the addition of serum sCD8 levels to CD4 T-cell measurements increased the power in predicting the onset of AIDS. The serum level of sCD8 was particularly relevant to the prediction of subsequent CD4 T-cell fall relatively early in infection, for example, in the 3 years after seroconversion. However, later in HIV infection, for example within 2 years prior to development of AIDS, sCD8 levels were less predictive. sCD8 correlated with levels of beta 2-microglobulin and neopterin, which reflect activation of cell types other than CD8. Thus, serum sCD8 level can be a useful marker of specific CD8 T-cell activation, and is an independent predictor of prognosis in HIV infection.     

Antigen-bearing dendritic cells regulate the diverse pattern of memory CD8 T-cell development in different tissues

Memory T cells of the effector type (T(EM)) account for the characteristic rapidity of memory T-cell responses, whereas memory T cells of the central type (T(CM)) account for long-lasting, vigorously proliferating memory T-cell responses. How antigen-stimulated (primed) T cells develop into different memory T-cell subsets with diverse tissue distributions is largely unknown. Here we show that after respiratory tract infection of mice with influenza virus, viral antigen associated with dendritic cells (DCs) was abundant in lung-draining lymph nodes (DLN) and the spleen for more than a week but was scant and transient in nondraining lymph nodes (NDLN). Correspondingly, activated CD8 T cells proliferated extensively in DLN and the spleen but minimally in NDLN. Strikingly, however, although most persisting CD8 T cells in DLN and spleen exhibited the T(EM) phenotype, those persisting in NDLN exhibited the T(CM) phenotype. Reducing antigen exposure by depleting DCs at the peak of primary T-cell responses enhanced the development of T(CM), whereas subjecting primed CD8 T cells from NDLN to additional antigen stimulation inhibited T(CM) development. These findings demonstrate that differences in persistence of antigen-bearing DCs in various tissues regulate the tissue-specific pattern of memory CD8 T-cell development. The findings have significant implications for design of vaccines and immunization strategies.     

Evidence for in vivo clonal proliferation of unique population of blood CD4-/CD8- T cells bearing T-cell receptor alpha and beta chains in two normal men.

Rare T lymphocytes bearing CD3 surface antigen and T-cell receptor (TCR) alpha and beta chains, but lacking both CD4 and CD8 antigens, viz, TCR alpha beta+CD4-8- cells, appear at a frequency of 0.1% to 2% in peripheral blood TCR alpha beta+ cells of normal donors. Here we report two unusual cases, found among 100 healthy individuals studied, who showed an abnormally elevated frequency of these T cells, ie, 5% to 10% and 14% to 19%. Southern blot analyses of the TCR alpha beta+CD4-8- clones all showed the identical rearrangement patterns for each individual, demonstrating that these are derivatives of a single T cell. The same rearrangement patterns were also observed for the freshly isolated lymphocytes of TCR alpha beta+CD4-CD8- fraction, which excludes the possible bias in the processes of in vitro cloning. These TCR alpha beta+CD4-8- T cells were found to express other mature T-cell markers such as CD2, CD3, and CD5 antigens, as well as natural killer (NK) cell markers (CD11b, CD16, CD56, and CD57 antigens) for both individuals. Further, although lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities were observed for both freshly sorted lymphocytes of TCR alpha beta+CD4-8- fraction and in vitro established clones, NK-like activity was not detected.     

TLR3 ligand stimulates fully functional memory CD8+ T cells in the absence of CD4+ T-cell help

We investigated whether Toll-like receptor ligands (TLR-Ls) can bypass the requirement for CD4(+) T-cell help in the induction of fully efficient memory CD8(+) T cells (cytotoxic T lymphocytes [CTLs]). "Helpless" CTLs were induced by a synthetic CD8(+) T-cell epitope administered with TLR3-L and TLR9-L, but not with TLR2/6-L, TLR4-L, or TLR7-L. The up-regulation of MHC-I and costimulatory molecules by dendritic cells following TLR stimulation was not sufficient for the priming of "helpless" CTLs, which depended essentially on the induction of a strong IFN-alpha/beta response. The "helpless" CTLs induced by TLR-Ls differentiated into fully functional memory CTLs able to proliferate as well as their "helped" counterparts upon challenge, in the absence of CD4(+) T-cell help.     

Visual demonstration of hepatitis C virus-specific memory CD8(+) T-cell expansion in patients with acute hepatitis C

Hepatitis C virus (HCV)-specific CD8(+) T cells in peripheral blood mononuclear cells (PBMCs) from patients infected with HCV were quantitatively analyzed by flow cytometry using an HLA-B*3501-HCV epitope tetrameric complex. In chronic hepatitis C, tetramer(+)CD8(+) T cells were detected at frequencies ranging from 0.05% to 0.12% of total CD8(+) T cells. The number of tetramer(+)CD8(+) T cells in acute phase PBMCs from patients with acute hepatitis C was about 3 to 5 times higher than in recovery phase PBMCs from the same patients and in PBMCs from patients with chronic hepatitis C. Expanding tetramer(+)CD8(+) T cells in PBMCs from patients with acute hepatitis C express a CD28(+)CD45RA(-) memory T-cell phenotype. In contrast, tetramer(+)CD8(+) T cells in PBMCs from patients with chronic hepatitis C did not predominantly express this phenotype. These tetramer(+)CD8(+) T cells did not have perforin in their cytoplasma. The present study visually showed that a high number of circulating HCV-specific CD8(+) T cells in acute phase PBMCs from patients with acute hepatitis C are mostly memory T cells.     

Impact of viral infection on acute exacerbation of asthma in out-patient clinics: a prospective study

Background

The prevalence of viral infection triggering asthma exacerbation and its impact on the symptoms and duration of exacerbation are unclear.

Methods

Asthma and healthy control subjects were recruited from the First Affiliated Hospital of Guangzhou Medical University between February 2012 and February 2013. Nasal swabs were collected, and respiratory viruses were detected by polymerase chain reaction (PCR). All patients completed questionnaires and a lung function test. Some were followed up for 4 weeks, and symptom changes were evaluated via asthma diaries.

Results

In total, 70 patients with acute asthma exacerbations were recruited. Among them, 34 patients (48.6%) completed the 4-week follow-up study. Another 65 patients with stable asthma and 134 healthy volunteers were also included in this study. The rate of positive viral detection via PCR in acute asthma exacerbation patients was 34.2% (24/70), which is significantly higher than that of stable asthma (12/65; 18.5%; P=0.038) and normal control patients (18/134; 13.4%; P<0.001). Among the viral-positive subjects, the number of viral copies was significantly higher in acute asthma exacerbation patients [(5.00±4.63) ×10⁷ copies/L] (mean ± SD) than those in stable asthma patients [(1.24±1.44) ×10⁶ copies/L; P<0.001] or in healthy controls [(1.44±0.44) ×10⁶ copies/L; P<0.001], whose viral loads were not significantly different from one another (P=0.774). During the 4-week follow-up period, the cough scores on days 1 and 3 were significantly higher in the viral-positive group than in the viral-negative group (day 1: P=0.016; day 3: P=0.004). However, there were no significant differences between these two groups for other tested symptoms, such as dyspnea and total recovery time (P>0.05).

Conclusions

Respiratory viruses may be involved in acute asthma exacerbations, inducing more prominent and persistent cough symptoms.     

Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines

Four human CD8+ T-cell subsets, naive (CCR7+CD45RA+), central memory (TCM, CCR7+CD45RA-), effector memory (TEM, CCR7-CD45RA-), and CD45RA+ effector memory cells (TEMRA, CCR7-CD45RA+) were compared for their capacity to proliferate and differentiate in response to antigen or homeostatic cytokines. Cytokine responsiveness and interleukin-15 receptor expression were low in naive T cells and progressively increased from TCM to TEM and TEMRA. In contrast, the capacity to accumulate in response to T-cell receptor (TCR) or cytokine stimulation showed a reciprocal pattern and was associated with resistance to cell death and Bcl-2 expression. Whereas all TCR-stimulated cells acquired a CD45RA-CCR7- phenotype, cytokine-stimulated cells maintained their phenotype with the exception of TCM cells, which expressed CCR7, CD45RA, and perforin in various combinations. Single CD8+ TCM cells, but not TEM cells, could be expanded with cytokines, and the obtained clones displayed several distinct phenotypes, suggesting that TCM cells are heterogeneous. Consistently, CCR4 expression in the CD8+ TCM pool discriminated CCR4+ type 2 polarized cells (Tc2) and CCR4-CTL precursors. Finally, ex vivo bromodeoxyuridine (BrdU) incorporation experiments revealed that memory subsets have different in vivo proliferation rates, with CCR4-TCM having the highest turnover and TEMRA the lowest. These results show that human CD8+ memory T-cell subsets have different proliferation and differentiation potentials in vitro and in vivo. Furthermore, they suggest that TEMRA cells are generated from a TCM subset upon homeostatic proliferation in the absence of antigen.     

Large granular lymphocyte leukaemia is characterized by a clonal T-cell receptor rearrangement in both memory and effector CD8(+) lymphocyte populations

Large granular lymphocyte (LGL) leukaemia is a disease with increased numbers of circulating granular lymphocytes and an increased percentage of clonally rearranged CD8(+)CD57(+) cells. To determine whether LGL cells are also found in other lymphocyte subsets, CD8(+) cells from 10 LGL patients were sorted into CD57(+) and CD57(-) fractions and analysed for clonality using a T-cell receptor gamma (TCR gamma) polymerase chain reaction (PCR). In nine patients, a clonal TCR rearrangement was identified in the CD8(+)CD57(+) cells, and in one patient, the TCR rearrangement was oligoclonal in the CD8(+)CD57(+) fraction. In eight out of nine of the clonally rearranged patients, the same band was also present in the CD8(+)CD57(-) fraction. To define the relationship between CD57(-) and CD57(+) LGL populations, CD8(+)CD57(-) and CD8(+)CD57(+) cells were sorted from five patients and cultured in the presence of anti-CD3 plus CD28 antibodies. The CD57(+) cells died of apoptosis before d 7, while the CD57(-) cells proliferated and differentiated into CD57(+) cells. Clonal analysis identified the same band in both cultured subpopulations and in the uncultured CD8(+) cells. Immunophenotypical analysis showed that CD8(+)CD57(-) cells expressed memory cell markers, while the CD8(+)CD57(+) cells exhibited effector characteristics. These results suggest that LGL disease originates in a CD57(-) memory T-cell compartment that continually generates CD57(+) (effector cell) progeny.     

Pituitary epithelial cell implants reverse the accumulation of CD4-CD8- lymphocytes in thymus glands of aged rats.

Although implantation of GH3 pituitary epithelial cells has been shown to reverse thymic aging in rats, the differentiation pattern of T-lymphocytes within the reconstituted thymus glands has not been investigated. Syngeneic GH3 cells were implanted into 22-month-old female (old) Wistar-Furth rats. Eight weeks later, thymus glands and thymocyte subpopulations were compared to those in aged (24-month-old) and young (3-month-old) female Wistar Furth rats. We confirmed that implantation of GH3 cells increased (P less than 0.05) not only thymus size but also the number of thymocytes isolated from aged rats. Flow cytometric analysis using dual color direct immunofluorescence with fluorescein isothiocyanate-labeled anti-CD4 (W3/25) and phycoerythrin-labeled anti-CD8 (OX 8) monoclonal antibodies revealed that thymus glands from young rats had approximately 20% CD4-CD8- and 30% CD4+CD8+ cells. Thymus glands from old rats contained greater than 50% more CD4-CD8- cells and a reduced percentage of CD4+CD8+ lymphocytes compared to those of young rats (P less than 0.05). Moreover, both of these age-associated changes were reversed (P less than 0.05) by implanting GH3 cells. GH3-treated aged rats also had a significantly (P less than 0.05) greater proportion of CD4+CD8- thymocytes compared to aged control rats. There were no differences among the three groups of rats in the percentage of CD4-CD8+ thymocytes or in the percentage or intensity of cells expressing the T-cell markers CD3, T-cell receptor, or OX19. These results show that in aged rats, intrathymic maturation is inhibited at the key transitional stage where double negative cells differentiate into double positive cells, which may limit the diversity of the T-cell repertoire. The data also extend earlier results by demonstrating that GH3 epithelial cells promote not only growth, but also the differentiation of T-lymphocytes in the aging rat thymus.     

Epidemiology of respiratory viral infections in children enrolled in a study of influenza vaccine effectiveness

Background

Influenza-like illness (ILI) confers a high annual morbidity in young children. We report the epidemiology of ILIs in children who participated in an influenza vaccine effectiveness study during the 2010 Southern Hemisphere influenza season in Sydney, Australia.

Methods

Children aged 0·5–3 years were prospectively recruited from child care centres (CCCs). We classified them as fully vaccinated, partially vaccinated and unvaccinated according to their receipt of unadjuvanted vaccines containing influenza A (H1N1)pdm09. For 13 weeks commencing 30 July 2010, parents reported when their children developed an ILI (fever ≥37·8°C/feverishness plus ≥1 respiratory symptom) and collected nose and/or throat swabs for multiplex respiratory virus polymerase chain reaction (PCR) testing. Health impacts were assessed by telephone interview at enrolment and two weeks after each ILI.

Results

There were 124 ILIs reported in 105 of 381 enrolled children. Swabs were taken in 117 ILIs: 175 viruses were identified from 103 swabs. Adeno- and rhinoviruses were most frequently identified; 44% of swabs yielded multiple viruses. No virus was associated with more severe symptoms, although rhinovirus-related ILIs lasted longer. Nose swabs had a higher virus detection rate than throat swabs. Influenza-vaccinated children were 1·6 times (P = 0·001) more likely than unvaccinated children to have a non-influenza ILI.

Conclusion

Adeno- and rhinoviruses were the most common viruses causing ILI. Swabs taken by parents are an effective method for sample collection. Influenza-like illness was more common in children vaccinated against influenza in this observational study, but prior health-seeking behaviour may have contributed to this difference.