A single-chain H-2Db molecule presenting an influenza virus nucleoprotein epitope shows enhanced ability at stimulating CD8+ T cell responses in vivo.

We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.

Characterization of the CD4+ T cell response to Epstein-Barr virus during primary and persistent infection.

The CD8+ T cell response to Epstein-Barr virus (EBV) is well characterized. Much less is known about the evolution of the CD4+ T cell response. Here we show that EBV stimulates a primary burst of effector CD4+ T cells and this is followed by a period of down-regulation. A small population of EBV-specific effector CD4+ T cells survives during the lifelong persistent phase of infection. The EBV-specific effector CD4+ T cells accumulate within a CD27+ CD28+ differentiation compartment during primary infection and remain enriched within this compartment throughout the persistent phase of infection. Analysis of CD4+ T cell responses to individual epitopes from EBV latent and lytic cycle proteins confirms the observation that the majority of the effector cells express both CD27 and CD28, although CD4+ T cells specific for lytic cycle antigens have a greater tendency to express CD45RA than those specific for the latent antigens. In clear contrast, effector CD4+ T cells specific for cytomegalovirus (CMV) accumulate within the CD27- CD28+ and CD27- CD28- compartments. There are striking parallels in terms of the differentiation of CD8+ T cells specific for EBV and CMV. The results challenge current ideas on the definition of memory subsets.

Phenotypic and functional analysis of synovial natural killer cells.

Natural Killer (NK) cells are cells of the innate immune system with characteristic effector functions, including recognition and lysis of virus-infected or tumor cells and production of immunoregulatory cytokines, particularly interferon-gamma (IFN-gamma). NK cells account for between 10 and 15% of peripheral blood lymphocytes and are also present in synovial fluid and tissue where they might potentially contribute to amplification of the inflammatory process through interactions with macrophages and dendritic cells. This chapter outlines methods of assessing the phenotype of NK cells through analysis of NK-cell markers and the function of NK cells through cytotoxicity assays and measurement of cytokine production.

Expression of leukocyte immunoglobulin-like receptors and natural killer receptors on virus-specific CD8+ T cells during the evolution of Epstein-Barr virus-specific immune responses in vivo.

Antigen-primed cytotoxic T lymphocytes (CTL) may express leukocyte immunoglobulin-like receptors (LILRs) and natural killer receptors (NKRs). Published work suggests that expression of some of these receptors confers survival advantage, leading to the idea that cells expressing such receptors may accumulate as an antigen-specific response evolves. Here we tested this hypothesis by analyzing expression of CD85j (also known as LILRB1 or ILT2), KIRs, CD94, and CD161 by Epstein- Barr virus (EBV)-specific CTL during the primary and persistent phases of EBV infection in humans. During primary infection, few EBV-specific CTL expressed these receptors and this proportion was equally low in early persistent infection. Thus, expression of these molecules does not influence capacity to survive downregulation of the primary response. However, in donors persistently infected with EBV for many years, a significantly higher proportion of EBV-specific CTL expressed CD85j and NKRs, suggesting that cells expressing these receptors can accumulate with time. Using FACS analysis, we confirmed, at a single cell level, that expression of CD85j, defined by staining with the antibody VMP55, was associated with reduced capacity of EBV-specific CD8+ T cells to respond to antigen. Thus, in the later stages of persistent infection, protective immunity to EBV may be reduced due to the preferential accumulation of hyporesponsive EBV-specific CD8+ T cells.

Lack of Epstein-Barr virus- and HIV-specific CD27- CD8+ T cells is associated with progression to viral disease in HIV-infection.

OBJECTIVE: Despite readily detectable virus-specific CD8+ T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein-Barr virus (EBV)-specific CD8+ T cells was performed. DESIGN: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV-associated non-Hodgkin lymphoma's (NHL), both number and phenotype of virus-specific CD8+ T cells was studied longitudinally. METHODS: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells. RESULTS: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27- T cells, in contrast to rising numbers of EBV-specific CD27- CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS-related NHL had very low numbers of EBV-specific CD27- CD8+ T cells. Higher numbers of HIV-specific CD27- CD8+ T cells were associated with delayed disease progression. Virus-specific CD27- T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function. CONCLUSIONS: Taken together, our data indicate that virus-specific CD27- T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27- effector T cells may lead to progression of viral disease.

The immune response to Epstein-Barr virus.

Epstein-Barr virus is a gammaherpes virus that establishes persistent infection in the majority of humans. Despite the potent oncogenic potential of the virus it is relatively rarely associated with malignant disease. This review focuses on the cellular immune responses that successfully control Epstein-Barr virus infection in most individuals.

The evolution of antigen-specific CD8+ T cell responses after natural primary infection of humans with Epstein-Barr virus.

Epstein-Barr virus (EBV) is a persistent, gamma-herpes virus that infects 90% of the human population. Primary infection, particularly if it is delayed until adolescence or beyond, may cause acute infectious mononucleosis and persistent infection may be associated with the development of several malignancies. CD8(+) T cells play a critical role in controlling both the primary and persistent phases of infection. This review summarises work that has been done characterising the primary immune responses to EBV. It goes on to describe the down regulation of the primary immune response and to discuss some of the factors that may be involved in determining the death or survival of populations of antigen-specific CD8(+) T cells. Finally it describes features of the populations of memory cells that mediate the long-term control of EBV in healthy seropositive individuals. The studies show differences in the responses to epitopes from lytic cycle versus latent proteins and highlight the complexity of naturally occurring, in vivo, immune responses. A clear understanding of the means by which CD8(+) T cells control EBV is important if we are to successfully develop vaccines and other forms of immunotherapy for the virus and its related malignancies.

Early acquisition of cytolytic function and transcriptional changes in a primary CD8+ T-cell response in vivo.

Functional studies show that programming of CD8+ T cells occurs early after initial antigen encounter within as little as 2 hours. To define the molecular basis of these events, we transferred TCR transgenic T cells from F5 Rag-/- mice into naive recipients and stimulated them with recombinant vaccinia expressing the immunodominant influenza epitope NP366-374. Transcription in epitope-specific cytotoxic T lymphocytes (CTLs) was analyzed using Affymetrix 430 2.0 GeneChips and quantitative polymerase chain reaction (PCR). We demonstrated an early transcriptional burst with the greatest number of genes reaching peak expression 12 hours after stimulation. Using in vivo cytotoxicity assays we demonstrated that early up-regulation of cytolytic genes was accompanied by acquisition of killing capacity within 24 hours of stimulation. However, T-cell proliferation was not observed until 48 hours. We therefore conclude that clonal expansion rather than acquisition of effector function is the rate-limiting step in the development of a primary CTL response.

Human naive CD8 T cells down-regulate expression of the WNT pathway transcription factors lymphoid enhancer binding factor 1 and transcription factor 7 (T cell factor-1) following antigen encounter in vitro and in vivo.

The transcription factors lymphoid enhancer binding factor 1 (LEF1) and transcription factor 7 (TCF7) (T cell factor-1 (TCF-1)) are downstream effectors of the WNT signaling pathway, which is a critical regulator of T cell development in the thymus. In this study, we show that LEF1 and TCF7 (TCF-1) are not only expressed in thymocytes, but also in mature T cells. Our data demonstrate that Ag encounter in vivo and engagement of the TCR or IL-15 receptor in vitro leads to the down-regulation of LEF1 and TCF7 (TCF-1) expression in human naive CD8 T cells. We further show that resting T cells preferentially express inhibitory LEF1 and TCF7 (TCF-1) isoforms and that T cell activation changes the isoform balance in favor of stimulatory TCF7 (TCF-1) isoforms. Altogether, our study suggests that proteins involved in the WNT signaling pathway not only regulate T cell development, but also peripheral T cell differentiation.

Human CD4+ T cells are predominantly distributed among six phenotypically and functionally distinct subsets.

Human T cells are heterogeneous, varying in terms of their phenotype, functional capabilities, and history of Ag encounter. The derivation of a functionally relevant model for classifying CD4+ T cells has been hampered by limitations on the numbers of parameters that may be measured using classical four-color flow cytometry. In this study we have taken advantage of the introduction of reagents for five-color flow cytometry to develop a detailed, functionally meaningful scheme for classifying human CD4+ T cells. We show that CD4+ T cells are predominantly distributed among six of eight possible compartments, identified by the expression of CCR7, CD45RA, and CD28. We demonstrate novel phenotypic and functional correlates that justify the choice of these three molecules to define CD4+ T cell compartments. We note that CD4+ T cells with different Ag specificities are distributed differently among the six described subsets. On the basis of these results, we propose a cross-sectional model for classification of peripheral CD4+ T cells. Knowledge of where T cells lie on this model informs about their functional capacity and can reflect their history of Ag exposure.

Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets.

Memory T cells are heterogeneous in terms of their phenotype and functional properties. We investigated the molecular profiles of human CD8 naive central memory (T(CM)), effector memory (T(EM)), and effector memory RA (T(EMRA)) T cells using gene expression microarrays and phospho-protein-specific intracellular flow cytometry. We demonstrate that T(CM) have a gene expression and cytokine signaling signature that lies between that of naive and T(EM) or T(EMRA) cells, whereas T(EM) and T(EMRA) are closely related. Our data define the molecular basis for the different functional properties of central and effector memory subsets. We show that T(EM) and T(EMRA) cells strongly express genes with known importance in CD8 T cell effector function. In contrast, T(CM) are characterized by high basal and cytokine-induced STAT5 phosphorylation, reflecting their capacity for self-renewal. Altogether, our results distinguish T(CM) and T(EM)/T(EMRA) at the molecular level and are consistent with the concept that T(CM) represent memory stem cells.

Epstein-Barr virus, arthritis, and the development of lymphoma in arthritis patients.

PURPOSE OF REVIEW: Rheumatoid arthritis is a complex multisystem disorder. The manifestations of joint disease are usually clinically apparent, but the effects of the concomitant abnormalities of immune function are more subtle. It has been suggested that patients with rheumatoid arthritis have an impaired capacity to control infection with Epstein-Barr virus. Epstein-Barr virus has oncogenic potential and is implicated in the development of some lymphomas. This review analyses the relation between Epstein-Barr virus, rheumatoid arthritis, and the risk of lymphoma and considers the effect of immunosuppression on this triad. RECENT FINDINGS: Recent publications provide evidence for an altered Epstein-Barr virus-host balance in patients with rheumatoid arthritis, who have a relatively high Epstein-Barr virus load. Large epidemiologic studies confirm that lymphoma is more likely to develop in patients with rheumatoid arthritis than in the general population. The overall risk of development of lymphoma has not risen with the increased use of methotrexate or biologic agents. Histologic analysis reveals that most lymphomas in rheumatoid arthritis patients are diffuse large B cell lymphomas, a form of non-Hodgkin lymphoma. Epstein-Barr virus is detected in a proportion of these. SUMMARY: Overall, patients with rheumatoid arthritis have approximately a twofold increased risk of experiencing lymphoma. Some, but not all, of this increased risk reflects an increase in Epstein-virus-associated lymphomas. This in turn may be influenced by the elevated Epstein-Barr virus load found in rheumatoid arthritis patients and may reflect subtle impairment of antiviral immunity in this group of patients.

A subset of natural killer cells is greatly expanded within inflamed joints.

OBJECTIVE: To determine whether natural killer (NK) cells are present within inflamed joints and whether they might play a role in amplifying the inflammatory process. METHODS: Paired samples of peripheral blood and synovial fluid were obtained from 22 patients with inflammatory arthritis. The frequency and phenotype of the peripheral and synovial NK cells were analyzed using a panel of monoclonal antibodies. Further experiments were performed to investigate the functional capacity of the synovial NK cells. RESULTS: The study showed that the CD3-, CD56(bright) subset of NK cells was greatly expanded within inflamed joints. Our experiments suggested that this subset of cells was preferentially recruited from the periphery and that NK cells may be further activated by cytokines present within the joint. Furthermore, synovial NK cells responded to a combination of interleukin-12 (IL-12) and IL-15, cytokines that are secreted by cells of the monocyte/macrophage lineage, by rapidly secreting interferon-gamma, a cytokine that can, in turn, activate macrophages. CONCLUSION: A subset of NK cells was expanded within inflamed joints. The functional properties of these NK cells rendered them good candidates for a role in interacting with the macrophage/monocyte population within the joint, thus amplifying the production of proinflammatory cytokines.

Clonal selection, clonal senescence, and clonal succession: the evolution of the T cell response to infection with a persistent virus.

We have analyzed the CD8(+) T cell response to EBV and find that a larger primary burst size is associated with proportionally greater decay during the development of memory. Consequently, immunodominance and clonal dominance are less marked in memory than primary responses. An intuitive interpretation of this finding is that there is a limit to the number of cell divisions a T cell clone can undergo, and that the progeny of clones that have expanded massively during a primary immune response are more prone to die as a result of senescence. To test this hypothesis, we have derived a mathematical model of the response of different T cell clones of varying avidity for Ag in the primary and persistent phases of viral infection. When cellular survival and replication are linked to T cell avidity for Ag and Ag dose, then high-avidity T cells dominate both the primary and secondary responses. We then incorporated a limit in the number of cell divisions of individual T cell clones to test whether such a constraint could reproduce the observed association between cell division number and alterations in the contribution of clones to the response to persistent infection. Comparison of the model output with the experimental results obtained from primary and persistent EBV infection suggests that there is indeed a role for cellular senescence in shaping the immune response to persistent infection.

CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation.

Human NK cells may be divided into a CD56(dim) subset and a CD56(bright) subset. In peripheral blood, CD56(dim) NK cells dominate, whereas in lymph nodes, CD56(bright) NK cells are more common. In this study we show that CD56(bright) NK cells accumulate within inflammatory lesions in a wide variety of clinical diseases affecting several different anatomical sites. We demonstrate that when activated by the monokines IL-12, IL-15, and IL-18, these NK cells promote TNF-alpha production by CD14(+) monocytes in a manner that is dependent on cell:cell contact. Conversely, CD14(+) monocytes synergize with monokines to promote IFN-gamma production by these NK cells. Again, this interaction is dependent on cell:cell contact. The experiments show that CD56(bright) NK cells accumulate in inflammatory lesions and, in the appropriate cytokine environment, can engage with CD14(+) monocytes in a reciprocal activatory fashion, thereby amplifying the inflammatory response. Such a positive feedback loop is likely to be important in the pathogenesis of chronic inflammatory conditions such as rheumatoid arthritis.