Tracking phenotypically and functionally distinct T cell subsets via T cell repertoire diversity

Antigen-specific T cell receptors (TCRs) recognise complexes of immunogenic peptides (p) and major histocompatibility complex (MHC) glycoproteins. Responding T cell populations show profiles of preferred usage (or bias) toward one or few TCRbeta chains. Such skewing is also observed, though less commonly, in TCRalpha chain usage. The extent and character of clonal diversity within individual, antigen-specific T cell sets can be established by sequence analysis of the TCRVbeta and/or TCRValpha CDR3 loops. The present review provides examples of such TCR repertoires in prominent responses to acute and persistent viruses. The determining role of structural constraints and antigen dose is discussed, as is the way that functionally and phenotypically distinct populations can be defined at the clonal level. In addition, clonal dissection of "high" versus "low" avidity, or "central" versus "effector" memory sets provides insights into how these antigen specific T cell responses are generated and maintained. As TCR diversity potentially influences both the protective capacity of CD8+ T cells and the subversion of immune control that leads to viral escape, analysing the spectrum of TCR selection and maintenance has implications for improving the functional efficacy of T cell responsiveness and effector function.     

Epstein-Barr virus is integrated between REL and BCL-11A in American Burkitt lymphoma cell line (NAB-2)

Epstein-Barr virus (EBV) initially isolated from the cultured Burkitt lymphoma (BL) cells, is one of the well-known oncogenic virus. The NAB-2 line, which was established from a North American Burkitt's tumor, was indicated to contain one copy of EBV DNA as the integrated form into chromosome 2p13 of the host genome. To demonstrate the integration site of EBV directly, and to clarify the relation between the integration sites and the oncogenes, fragments containing the nucleotide sequence of NAB-2 integration sites were cloned. EBV was integrated via the terminal repeats (TR), and integration sites located in the clone RP11-440P5 on chromosome 2, between two oncogenes, REL and BCL11A, which is apart from approximately 350 kbp from each other. Expression level of REL in NAB-2 was increased. The flanking region of chromosome 2 at the bilateral junction sites showed no homology to the junction sites of EBV. The integration site 2p13 overlaps with common fragile site, FRA2E. NAB-2 cells expressed almost all latent genes but LMP-2A that flanks the TR, indicating the type III of latent infection of EBV. Integration event in NAB-2 might alter the regulation of the oncogenes and provide advantage for continuous cell proliferation.     

Mucosal type mast cells express complement receptor type 2 (CD21)

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.     

T cell repertoire and Epstein-Barr virus-specific T cell response in chronic active Epstein-Barr virus infection: a case study

In a patient with chronic active Epstein-Barr virus infection associated with vasculitis and fulminant CD4+ T cell lymphoproliferative disorder, we probed the peripheral blood mononuclear cells (PBMC) for the presence of an EBV-specific T cell repertoire and tested the possible relationship between the lymphocytic infiltrate and the EBV-specific T cell response. Our results give credence to the presence of an apparently normal EBV-specific memory T cell response after in vitro reactivation of the patient's PBMC with autologous infected B lymphoblastoid cell lines. In keeping with the characterization of the vasculitis, certain T cell subsets were detected after expansion of skin lesion-infiltrating lymphocytes and were found to be infected with EBV. These particular T cell expansions were neither the effectors nor the targets of the in vitro reactivated EBV-specific T cells, thus excluding a simple relationship between EBV, the skin lesions, and the T cell expansions frequently observed in these patients.     

Identification of Epstein-Barr virus integrated sites in lymphoblastoid cell line (IB4)

IB4 is a lymphoblastoid cell line frequently used for the functional analysis of the latent genes of EBV. Previous study indicated that EBV whole genome is integrated tandemly as the linear viral genome into host genome of IB4, although sites of integration have not been determined. Through cloning of the junctional regions between EBV and host genomes, one of the integration sites was identified on the BamHI-C fragments around oriP sequences and another on the EcoRI-I fragment. Both of the integration sites were located on the clone RP11-119H12 of chromosome 4q25 and separated approximately 6.5 kbp from each other. The integration sites identified were apart from the genes of the host genome, indicating that both host gene and EBV latent genes are not altered by the integration event.     

Isolation, cloning, and complete nucleotide sequence of a phenotypically distinct Brazilian isolate of human T-lymphotropic virus type II (HTLV-II)

Analysis of human T-lymphotropic virus type II (HTLV-II) isolates from North America and Europe have demonstrated the existence of two molecular subtypes of the virus, HTLV-IIa and HTLV-IIb. Recently, studies on HTLV-II infections in Brazil have revealed isolates that are related phylogenetically to the HTLV-IIa subtype but have a HTLV-IIb phenotype with respect to the transactivating protein, tax. To more clearly define this relationship, HTLV-II was isolated from peripheral blood of an IVDA from Sao Paulo, Brazil (SP-WV), and the complete provirus was cloned and sequenced. Comparison of HTLV-II(SP-WV) nucleotide sequences to other available complete HTLV-II proviral sequences revealed that HTLV-II(SP-WV) is most closely related to HTLV-II(Mo), the prototypic HTLV-IIa subtype sequence. Phylogenetic analysis of LTR, env, and tax regions unequivocally demonstrated that HTLV-II(SP-WV) and all other Brazilian sequences examined are members of the IIa subtype. The predicted amino acid sequences of the major coding regions of HTLV-II(SP-WV) are also most closely related to HTLV-II(Mo), with the important exception of tax. The tax protein encoded by HTLV-II(SP-WV) is 96-99% identical to the tax of IIb isolates and is similar in that it has an additional 25 amino acids at the carboxy-terminus compared to the HTLV-II(Mo) tax with which it shares 91% identity. Analysis of tax stop codon usage of a number of HTLV-IIa isolates from North American, Europe, and Brazil demonstrated that isolates from the last region appear to be unique in their extended tax phenotype. It could be demonstrated that the extended tax proteins in the HTLV-IIb and Brazilian isolates had equivalent ability to transactivate the viral LTR, and studies with deletion mutants indicated that the extended C-terminus is not essential for transactivation. In contrast, the HTLV-IIa tax was found to have a greatly diminished ability to transactivate the viral LTR, which appeared to be a consequence of reduced expression of the protein. The studies show that although the Brazilian strains do not represent an entirely new subtype based on nucleotide sequence analysis they are a phenotypically unique molecular variant within the HTLV-IIa subtype.     

Normal suppressive T cell function of Epstein-Barr virus induced B cell activation in type 1 (insulin dependent) diabetes mellitus

Several studies have demonstrated abnormalities of T cell regulation of Epstein-Barr virus-induced B cell activation in systemic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, and systemic sclerosis. However, a normal suppressive peripheral T cell function was observed in Graves' disease. To investigate whether this abnormality is a common feature to other autoimmune diseases, we studied T cell regulation of Epstein-Barr virus induced B cell activation in 15 newly diagnosed type 1 (insulin dependent) diabetes mellitus patients and 10 normal control subjects. Peripheral B lymphocytes infected with Epstein-Barr virus were cultured for 20 days in the presence or absence of autologous T cells at different ratios (1:1 and 1:4). IgM and IgG secretions into the supernatants were determined using an enzyme-linked immunosorbent assay. The extent of suppression when T cells were added, as measured by a suppression ratio, was not significantly different in type 1 (insulin dependent) diabetes mellitus patients and normal subjects. We conclude that in type 1 (insulin dependent) diabetes mellitus, the autoimmune reactivity is not dependent upon a generalized suppression defect. It can be hypothesized, therefore, that in type 1 diabetes mellitus as well as in Graves' disease, a local or organ specific suppressor deficit may induce the autoimmune phenomena.     

Interleukin 2 receptor traffic in a murine cytolytic T cell line

We have analyzed different parameters of the interleukin 2 receptor (IL2R) traffic in a murine IL2-dependent T cell line, B6.1. These cells express about 10(5) IL2R, of which approximately 10% are of high affinity (HAR). About 90% of all mature immunoprecipitable receptor molecules in the cell are on the cell surface. Measurements of the half life and of the rate of receptor synthesis indicate that these cells produce approximately 500 receptor molecules per cell and min. IL2 deprival for less than 6 h does not affect this rate. B6.1 cells internalize IL2 via their HAR only, with a maximal rate of about 500 molecules per cell and min. Among the receptors present at a given time on the cell surface, about 15% are internalized within 30 min. Receptor internalization is independent of IL2. The data obtained argue against rapid recycling of the HAR. The rate of receptor synthesis can be reconciled with the rate of IL2 internalization and the observation that internalization occurs only via HAR by assuming that cell surface low-affinity receptors can be transformed into HAR by associating with other molecules.     

Entry of human immunodeficiency virus (HIV) into MT-2, human T cell leukemia virus carrier cell line

Summary The ultrastructural features of early events in human immunodeficiency virus (HIV) infection of HTLV-I-carrying MT-2 lymphocytes were investigated by electron microscopy. Within 10 min after virus inoculation at 37°C, the virus entered the cell in two ways; (1) the virus attached to the lymphocyte membrane and the viral core entered the cell after fusion of the viral envelope with the cell membrane, and (2) part of the cell membrane to which the virus was attached became invaginated, the virus became trapped in a phagosome and the viral core entered after the fusion of viral membrane with the vacuolar membrane. Thereafter, some cells were observed to form syncytia with multiple nuclei. When the proportion of anti-HIV antibody-reactive cells present exceeded 90%, virus production was strongly activated, and budding on the cell membrane was frequently observed.     

Interaction of Epstein-Barr virus with leukaemic B cells in vitro. I. Abortive infection and rare cell line establishment from chronic lymphocytic leukaemic cells.

Leukaemia B cell populations, each with an individual pattern of monoclonal surface immunoglobulin expression, were obtained from 23 patients with chronic lymphocytic leukaemia (CLL) and, following exposure to a potent dose of Epstein-Barr (EB) virus in vitro, were monitored for expression of the virus associated nuclear antigen EBNA, for activation of immunoglobulin synthesis and for virus-induced transformation to an established cell line. Although possessing the EB virus receptor, CLL cells were generally refractory (vis-à-vis normal adult B cells) to the full effects of the viral infection. All the leukaemic populations tested developed a small proportion of EBNA positive cells within a few days post-infection, but in most instances this disappeared with no subsequent evidence of viral activity. In certain cases, however, the EBNA staining became more intense, involving a larger fraction of the population and persisting for some weeks, but again this was not accompanied by virus-induced immunoglobulin synthesis or transformation. In contrast, the leukaemic cells from a single patient, tested on three separate occasions, regularly responded to EB virus infection with the rapid establishment of an EBNA positive B cell line in which the restricted pattern of surface and cytoplasmic immunoglobulin expression (gamma lambda) exactly matched that present on the original leukaemic cells.     

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

Complement receptor type II (CR2/CD21) is the major receptor for C3d fragments on immune complexes. CD21 also serves as the receptor for Epstein-Barr virus in humans. On mature B cells, CD21 reduces the threshold of BCR signaling together with CD81, Leu13 and CD19, but it also occurs on other cells of the immune system where it performs unknown functions. A soluble form of CD21 (sCD21) is shed from the cell surface and is found in human blood plasma. An as-yet-unknown protease is thought to be responsible for this shedding. Altered levels of sCD21 occur in plasma in certain clinical conditions. We show here by mass spectrometry that sCD21 in human plasma of healthy donors is predominantly a short form of CD21 without the exon-11-encoded sequences. Whereas the N terminus of sCD21 was found unmodified, the C terminus is truncated, implying that only the extracellular portion of CD21 is shed. Peripheral blood B cells, but not T cells, contribute to the plasma CD21-pool. CD21 shedding is induced by stimulation with PMA plus Ca(2+) ionophore, or by stimulation of the BCR with anti-IgM+anti-CD40.     

Activated guinea-pig C3 and the immune adherence receptor (a complement receptor) on cell membranes.

By treating C3 with purified C1, C4 and C2 in the fluid phase, haemolytically inactive C3 was prepared. This was shown to bind to human erythrocytes by use of radio-labelled (Fab')2 antibody to guinea-pig C3. The activated C3 preparation inhibited immune adherence between EAC43 and human erythrocytes. These findings indicate that the activated C3 attaches to the immune adherence receptor on human erythrocytes. In addition the fluid-phase activated C3 adhered to thymus cells and sheep erythrocytes, whereas EAC43 did not. Thus the immune adherence receptor may be present on so called immune adherence-negative cells, but in insufficient concentration to form rosettes with EAC43.     

Characterization of T cell responses to herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) using a TNF-β ELISpot cytokine assay

Summary. We examined the suitability of a TNF-β cytokine ELISpot assay for assessing various aspects of the T cell response to herpes simplex viruses. The number of T cells responding to HSV-1 or HSV-2 was measured by TNF-β ELISpot assay. The number of T cells producing TNF-β in response to HSV-1 was high, ranging from 76 to 222 per 10⁵. HSV-1-specific TNF-β-secreting responder cell frequencies fluctuated over time in individual donors. Comparable fluctuations were not observed in the T cell frequencies to phytohemaglutinin (PHA). Responder cell frequencies to glycoproteins gB and gD of HSV-2 accounted for a large number of the HSV-2-specific T cells as measured using the TNF-β ELISpot assay. Type-specific and type-common components of the T cell response to HSV-1 and HSV-2 could be estimated with this assay. Type-common responder cells typically accounted for 25–30%. Finally, CD4 and CD8 TNF-β-producing T cells were stimulated by HSV-1 at a CD4:CD8 ratio of 2:1, indicating that both major subsets of T lymphocytes are activated by HSV. Revised September 27, 1996 Accepted February 18, 1997     

Characterization of a chemokine receptor CCR5-negative T cell line and its use in determining human immunodeficiency virus type 1 phenotype

A human CD4-positive T cell line from a donor homozygous negative for the chemokine receptor CCR5 was established, characterized, and used for determining the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) isolates. Clones of this IL-2 dependent human T-cell lymphotropic virus type 1 (HTLV-I) immortalized cell line, named IsnoR5 clones 1 and 2, are susceptible to infection by HIV-1 isolates that use CXCR4 as a coreceptor but resistant to infection by CCR5 tropic HIV-1 viruses. HIV-1 isolates whose replication is inhibited in IsnoR5 cells in the presence of the bicyclam AMD 3100, a CXCR4 specific inhibitor, utilize a coreceptor distinct from CCR5 and CXCR4. Using a panel of primary HIV-1 isolates we have shown that a single T cell line is sufficient to discriminate between use of CCR5, CXCR4 or an alternative coreceptor. As IsnoR5 clone 1 cells revealed the existence of even minor populations of CXCR4-using virus variants, they could be useful for the early identification of changes in coreceptor usage in HIV infected individuals facilitating the timely introduction of appropriate clinical treatments.