Development of an Automatic Measurement Method for CD8 and PD-1 Positive T Cells Using Image Analysis Software.

BACKGROUND/AIM: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods. RESULTS: In the CD8+ T cell number measurement, there was a good correlation (r=0.738 by Pearson test) between the manual and automated counting methods. However, in the PD-1+ T cell measurement, there was a large difference in TIL numbers in both groups. After adjustment of the parameter settings, the correlation between the manual and automated methods in the PD-1+ T cell measurements improved (r=0.668 by Pearson test). CONCLUSION: An imaging software-based automatic measurement could be a simple and useful tool for evaluating the therapeutic effect of cancer immunotherapies in terms of TIL status.

Positron emission tomography imaging with 89Zr-labeled anti-CD8 cys-diabody reveals CD8+ cell infiltration during oncolytic virus therapy in a glioma murine model.

Determination of treatment response to immunotherapy in glioblastoma multiforme (GBM) is a process which can take months. Detection of CD8+ T cell recruitment to the tumor with a noninvasive imaging modality such as positron emission tomography (PET) may allow for tumor characterization and early evaluation of therapeutic response to immunotherapy. In this study, we utilized 89Zr-labeled anti-CD8 cys-diabody-PET to provide proof-of-concept to detect CD8+ T cell immune response to oncolytic herpes simplex virus (oHSV) M002 immunotherapy in a syngeneic GBM model. Immunocompetent mice (n = 16) were implanted intracranially with GSC005 GBM tumors, and treated with intratumoral injection of oHSV M002 or saline control. An additional non-tumor bearing cohort (n = 4) receiving oHSV M002 treatment was also evaluated. Mice were injected with 89Zr-labeled anti-CD8 cys-diabody seven days post oHSV administration and imaged with a preclinical PET scanner. Standardized uptake value (SUV) was quantified. Ex vivo tissue analyses included autoradiography and immunohistochemistry. PET imaging showed significantly higher SUV in tumors which had been treated with M002 compared to those without M002 treatment (p = 0.0207) and the non-tumor bearing M002 treated group (p = 0.0021). Accumulation in target areas, especially the spleen, was significantly reduced by blocking with the non-labeled diabody (p < 0.001). Radioactive probe accumulation in brains was consistent with CD8+ cell trafficking patterns after oHSV treatment. This PET imaging strategy could aid in distinguishing responders from non-responders during immunotherapy of GBM.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of rhesus macaque CD8alphaalpha homodimer.

As a T-cell co-receptor, CD8 binds to MHC class I molecules and plays a pivotal role in the activation of cytotoxic T lymphocytes. To date, structures of CD8 have been solved for two different mammals: human and mouse. The infection of rhesus macaques (Macaca mulatta) by simian immunodeficiency virus (SIV) is the best animal model for studying HIV. In this study, the rhesus macaque CD8 (rCD8) alphaalpha homodimer was obtained and rCD8alpha exodomain protein crystals were successfully obtained for further structural analysis. Diffraction data were collected to a resolution of 2.4 A. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.52, b = 56.28, c = 82.40 A. These data will facilitate further studies on the structural differences between these CD8 structures and the cellular immune responses of rhesus macaque.

Rapid immunosorting of transmembrane proteins of Lymphocytes from a cDNA expression library of COS-1 cells.

The proteins on lymphocyte surface play important roles in a wide range of immunological processes, but the profile and characterization of surface proteins remain to be further investigated, among which the method for fast screening of surface proteins needs to be established. In this study, a conventional cDNA clone library of hepatic lymphocytes from C57BL/6 mouse was constructed, and then the cDNA was inserted into a recombinant expression vector pSecTag-attR with a signal peptide and tag protein for fluorescence screening. The recombinant cDNA expression library was transfected into COS-1 cells, and the transfected cells with the expressed membrane proteins were labeled by fluorescence antibodies and isolated by fluorescence activated cell sorting. After two cycles of sorting, the purity of fluorescence positive cells with membrane proteins was up to 98%, and the representative membrane molecules on lymphocytes such as CD3, CD4, CD8, NK1.1 and NKG2D were detected in the library. These results demonstrated that the cDNA expression library containing transmembrane proteins provided an efficient and fast tool for the study of transmembrane proteins on hepatic lymphocytes.

CD8 alpha is expressed by human monocytes and enhances Fc gamma R-dependent responses.

BACKGROUND: CD8 alpha enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8 alpha is also found on dendritic cells and rat macrophages, but whether CD8 alpha enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8 alpha co-activates FcR responses. Interestingly, CD8 alpha+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8 alpha evidence for CD8 alpha expression by mouse or human monocytes or macrophages was incomplete. RESULTS: We detected CD8 alpha, but not CD8 beta on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8 alpha mAb with monocytes is at least partly independent of FcR as anti-CD8 alpha mAb detect CD8 alpha by western blot and inhibit binding of MHC class I tetramers. CD8 alpha mRNA is also found in monocytes and THP-1 suggesting CD8 alpha is synthesized by monocytes and not acquired from other CD8 alpha+ cell types. Interestingly, CD8 alpha from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8 alpha mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8 alpha mAb in immune-complexes. CONCLUSION: Human monocytes express CD8 alpha. Co-engagement of CD8 alpha and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8 alpha on innate immune cells.

Mononuclear cell infiltration and its relation to the expression of major histocompatibility complex antigens and adhesion molecules in pancreas biopsy specimens from newly diagnosed insulin-dependent diabetes mellitus patients.

We examined pancreas biopsy specimens from 18 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients to elucidate the mechanism underlying beta cell destruction. Pancreas islets were seen in all patients and insulitis in eight patients. Infiltrating mononuclear cells consisted of CD4+T, CD8+T, B lymphocytes, and macrophages. Among them, CD8+T lymphocytes were predominant and macrophages followed. The expression of MHC class I antigens was increased in islet and endothelial cells in nine patients. MHC class II expression was increased in endothelial cells of the same patients. The expression of intercellular adhesion molecule-1 was increased in endothelial cells in two of the nine patients with MHC hyperexpression; in one of them, lymphocyte function-associated antigen-3 expression was also increased. Out of the eight patients with insulitis, seven showed MHC class I hyper-expression, whereas 2 of the 10 patients without insulitis showed the phenomenon (P < 0.05). The relation between insulitis and the hyperexpression of adhesion molecules was not evident. In conclusion, we revealed the close relation between CD8+T lymphocyte-predominant insulitis and MHC class I hyperexpression in islet cells. This suggests that infiltrating CD8+T lymphocytes recognize islet autoantigens in association with increased MHC class I molecules and act as major effector cells in autoimmune response against islet cells in IDDM pancreases. The role of adhesion molecules in the pathogenesis of IDDM still remains to be elucidated.

Development of a Cell Marker ELISA for the Detection of Goose T Cell Surface CD8α Molecules.

CD8 molecule is a key marker on T cell surface and is connected with the antigen recognition and activation of T lymphocytes. In order to provide a detection method for quantifying goose CD8α expression, this study raised the protein and antibody for goose CD8α and developed a feasible cell marker enzyme-linked immunoabsorbent assay (ELISA) method. Recombinant protein of the extracellular region gene of goCD8α was expressed in prokaryotic expression system, and specific polyclonal antibodies for goCD8α were raised and purified, which was further confirmed by Western-blot, immunofluorescence assay (IFA), and immunohistochemistry (IHC). A cell marker ELISA was established and optimized to detect the change of goCD8α expression between goose parvovirus (GPV)-infected and mock-infected goose peripheral blood mononuclear cells (PBMCs), which is consistent with our previously results of real-time quantitative PCR (qPCR). Cell marker ELISA can provide a new method to detect goCD8α in protein level and in a sensitive, specific, and simple way. This may provide a convenient and novel method for the detection of goCD8α expression.

A soluble form of the human CD8 alpha chain expressed in the baculovirus system: biochemical characterization and binding to MHC class I.

We have generated a soluble form of the CD8 molecule consisting of the entire extracellular domains of the human alpha chain, by expressing a mutated CD8 alpha cDNA in SF9 cells infected with a recombinant baculovirus. The truncated molecule was secreted into the medium mostly as a disulfide-linked homodimer in which a single cysteine residue in the hinge-like region (Cys143) was sufficient to assure covalent bonding. Soluble CD8 purified to homogeneity appears to be monodisperse as assessed by gel filtration analysis and contains only O-linked carbohydrates. To determine whether recombinant CD8 can interact with MHC class I molecules, we developed an assay that measures binding of MHC class I-bearing cell lines to purified CD8 adsorbed to plastic plates. The level of binding of cells to immobilized CD8 depended on the amount of CD8 bound to the plate and correlated with the levels of cell surface MHC class I expression. The binding was specifically inhibited by monoclonal antibodies directed either against CD8 or MHC class I molecules. This assay therefore provides a way to measure CD8 binding to MHC class I independently of other cell-cell interactions and should allow direct structure-function studies.

CD8 lymphocyte counts and serum immunoglobulin A levels early in HIV infection as predictors of CD4 lymphocyte depletion during 8 years of follow-up.

OBJECTIVE: To assess the ability of the CD8 lymphocyte count and immunoglobulin (Ig) A level, measured at the early stage of HIV infection when the CD4 lymphocyte count remains relatively high, to predict the future rate of CD4 lymphocyte loss and hence the risk of AIDS. DESIGN: Cohort of recently infected haemophiliacs with relatively high CD4 lymphocyte counts followed for up to 8.5 years from baseline measurement of CD8 lymphocyte counts and IgA levels. SETTING: A regional haemophilia centre based in a major teaching hospital. PATIENTS: Eighty-four of 111 patients with haemophilia who seroconverted to HIV between 1979 and 1985 in whom CD8 lymphocyte counts and IgA levels were measured soon after seroconversion (mean, 2.7 years; maximum, 5 years) while CD4 lymphocyte counts remained relatively high (median, 600 x 10(6)/l; minimum, 300 x 10(6)/l). OUTCOME MEASURES: Development of severe immunodeficiency defined by a CD4 lymphocyte count falling below 50 x 10(6)/l, and AIDS. RESULTS: Individuals with high CD8 counts (P < 0.008) and high IgA levels (P < 0.003) at baseline experienced a more rapid rate of CD4 lymphocyte loss than those with low baseline levels. A score was derived to combine the predictive ability of CD8 count and IgA level. Estimated proportions with CD4 counts below 50 x 10(6)/l after 8 years of follow-up were 100, 30 and 14% for those with high, intermediate and low baseline scores, respectively. The CD8/IgA score showed similar ability to predict the future occurrence of AIDS (P < 0.0001; log-rank test). CONCLUSION: Immune activation seen in HIV infection, as reflected by raised CD8 counts and IgA levels, appears to be linked to the process of CD4 lymphocyte depletion. Measurement of these markers in the years following infection, when CD4 lymphocyte counts remain high, provides a first indication of a patient's long-term prognosis.

Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein.

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.

Preparation of CD8bright and CD8dim lymphocyte populations using two positive selection methods in tandem.

Two positive selection methods were compared for the ability to capture both the bright and dim subsets of CD8 lymphocytes in mononuclear cell (MC) preparations from ten healthy individuals. The first method utilized anti-CD8-coated magnetic beads; captured cells were then recovered using a polyclonal sheep anti-mouse Fab reagent. At all bead: CD8 cell ratios tested (4:1, 8:1, 16:1), the selected cells were > 94% CD8+, and these CD8 cells were enriched for CD8bright cells (77-85%) when compared to CD8 cells in the starting MC preparation (68%). The second method utilized anti-CD8-coated culture flasks; captured cells were recovered by physical dislodgement. The recovered cells were > 90% CD8+, and these CD8 cells were modestly enriched for CD8dim cells (52%) compared to starting CD8 cells (32%). To further enrich for CD8dim cells, we used these two methods in tandem (n = 10). MC were first incubated with anti-CD8-coated magnetic beads (4:1 ratio) to obtain a CD8bright-enriched population (97% of all cells CD8+, 83% of all cells CD8bright). Uncaptured cells were incubated with anti-CD4-coated magnetic beads, and the uncaptured cells from this step were then placed in an anti-CD8-coated flask. The recovered flask-selected cell population was highly enriched for CD8dim cells (87% of all cells CD8+, 85% of all cells CD8dim). CD8 cells in the CD8bright population were 94% CD3+ and 6% CD16+, whereas those in the CD8dim population were 29% CD3+ and 66% CD16+. In proliferative studies, CD8bright cells were preferentially activated by immobilized anti-CD3, whereas CD8dim cells were preferentially activated by exogenous IL-2. In assays of natural killer activity, CD8dim cells were markedly more active than CD8bright cells. This method provides an alternative to cell sorting for obtaining enriched populations of CD8bright and CD8dim lymphocytes.

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues.

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.

Endometrial T-lymphocyte subset infiltration during the ovine estrous cycle and early pregnancy.

T-lymphocytes were quantitated within luminal, stromal and glandular areas of ovine endometrium. In experiment 1, ovariectomized (OVX), estrus (E) and day 13 (D13) ewes (six/group) received 500 micrograms of phytohemagglutinin (PHA) or vehicle in ligated right and left uterine horns, respectively. At 48 h, uteri were removed for the immunohistochemical evaluation of T-lymphocyte subsets. In experiment 2, T-lymphocytes were quantitated within non-pregnant and pregnant uterine horns on day 19. For experiment 1, mean numbers of T4 and T8 lymphocytes within luminal and stromal areas of PHA-treated horns were greatest (P less than 0.05) for D13 ewes and least (P less than 0.05) for E ewes. Numbers of T6 lymphocytes for these same areas were greatest (P less than 0.05) for PHA-treated horns of OVX ewes. Overall, the T4/T8 ratio (P less than 0.004) and mean number of T19 cells (P less than 0.009) were increased by PHA. Numbers of CD45R lymphocytes were not affected by PHA but were greater (P less than 0.05) in glandular and luminal than stromal areas. For experiment 2, mean numbers of endometrial T4, T6, T8 and T19 lymphocytes were similar (P greater than 0.05) between non-pregnant and pregnant horns; however, the number of CD45R lymphocytes was greater (P less than 0.05) in endometrial tissue of pregnant than non-pregnant horns. The data indicate that the in vivo response of specific ovine T-lymphocytes to PHA was generally dependent upon reproductive stage and the presence of conceptus tissue influenced the infiltration of CD45R lymphocytes.

Identification of a bone marrow-derived CD8αα+ dendritic cell-like population in inflamed autoimmune target tissue with capability of inducing T cell apoptosis.

DCs play critical roles in promotion of autoimmunity or immune tolerance as potent APCs. In our anti-GBM GN model, WKY rats develop severe T cell-mediated glomerular inflammation followed by fibrosis. A DC-like cell population (CD8αα(+)CD11c(+)MHC-II(+)ED1(-)) was identified in the inflamed glomeruli. Chimera experiments demonstrated that the CD8αα(+) cells were derived from BM. The CD8αα(+) cells infiltrated glomeruli at a late stage (Days 28-35), coincident with a rapid decline in glomerular inflammation before fibrosis. The CD8αα(+) cells isolated from inflamed glomeruli were able to migrate rapidly from the bloodstream into inflamed glomeruli but not into normal glomeruli, suggesting that the migration was triggered by local inflammation. Despite high-level expression of surface and cellular MHC class II molecules, in vitro experiments showed that this CD8αα(+) DC-like cell induced apoptosis but not proliferation in antigen-specific CD4(+) T cells from T cell lines or freshly isolated from lymph nodes; they were not able to do so in the absence of antigens, suggesting induction of apoptosis was antigen-specific. Furthermore, apoptotic T cells were detected in a large number in the glomeruli at Day 32, coincident with the infiltration of the cells into glomeruli, suggesting that the cells may also induce T cell apoptosis in vivo. A potential role of this CD8αα(+) DC-like population in peripheral immune tolerance and/or termination of autoimmune inflammation was discussed.

[Pathogenetic and clinical significance of peripheral activated T-lymphocyte subsets in Basedow's disease]. / Pathogenetische und klinische Bedeutung von Subpopulationen aktivierter T-Lymphozyten im peripheren Blut bei Morbus Basedow.

The peripheral activated T-cell subsets which express concurrently DR and CD antigens were determined in 22 patients with Graves' disease using the fluorescence-rosette double labelling technique. In the active phase of the disease a decrease in the percentage of CD8+ cells, an increase in the CD4+/CD8+ ratio and an elevation of various DR+CD+T-lymphocytes was found. A concurrent marked increase in DR+CD4+ cells accounts for an abnormality in the equilibrium of activated T-lymphocyte subsets and an increase in DR+CD4+/DR+CD8+ ratios. In comparison with the active phase the DR+CD3+ and DR+CD4+ cells decreased in remission, but DR+CD8+ cells remained unchanged. The decrease in the ratio of CD4+/CD8+ cells was less marked than that of the DR+CD4+/DR+CD8+ cells, which fell into the normal range. Our results suggest an excessive activation of T-helper cells as well as a deficiency in T-suppressor cells which may play an important role in the pathogenesis of Graves' disease.

Crystal structure of a soluble form of the human T cell coreceptor CD8 at 2.6 A resolution.

A secreted fragment of the extracellular portion of human CD8 alpha has been expressed in CHO cells, and a deglycosylated and proteolyzed form of this fragment has been crystallized. We report here the crystal structure of this fragment as refined at 2.6 A resolution. The structure was solved by molecular replacement using a superposition of ten variable domains from immunoglobulin light chains as the search model. Only the N-terminal 114 amino acids of CD8 alpha are visible in the electron density maps. The domain formed by these residues possesses a fold typical of immunoglobulin variable domains and associates to form Fv-like homodimers.

CD8 lymphocytosis and pseudotumoral splenomegaly in HIV infection.

Three patients infected with human immunodeficiency virus (HIV) presented with pseudotumoral splenomegaly, CD8 lymphocytosis (3.5-5.1 x 10(9)/l), and hypergammaglobulinaemia. Spleen and bone marrow showed diffuse CD8 lymphocyte and plasma-cell infiltration. Amplification of the T-cell-receptor gamma chain gene did not reveal any clonal T-cell population. Phenotypic analysis showed a predominance of CD8/CD57 suppressor T cells with expression of activation markers (DR and CD38). No cytotoxic T lymphocytes specific for HIV could be detected. The three patients shared the HLA haplotype A1, B8, DR3. The association with this haplotype suggests a genetically determined host immune response to HIV.

Circulating TCR gammadelta cells in the patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a disorder with a wide range of immunological abnormalities. The results of the studies undertaken in the last decade indicated that SLE pathogenesis was mainly connected with the breakdown of the activation control of B and T cells, generating humoral or cell-mediated responses against several self-antigens of affected cells. The last studies demonstrate that the role of gammadelta T lymphocytes in autoimmune diseases can be especially important. Flow cytometry techniques were used to investigate the number and percentage of TCR gammadelta T cells and their most frequent subtypes in peripheral blood of 32 patients with SLE and 16 healthy volunteers. We also correlated TCR gammadelta cells number with the level of T CD3+, T CD4+, T CD8+, and NK (CD16) cells (cytometric measurements) and SLE activity (on the basis of clinical investigations). Our studies were preliminary attempts to evaluate the role of that minor T cell subpopulation in SLE. Absolute numbers of cells expressing gammadelta TCR in most SLE blood specimens were significantly lower than in the control group (P<0.006). However, since the level of total T cell population was also decreased in the case of SLE, the mean values of the percentage gammadelta T cells of pan T lymphocytes were almost the same in both analysed populations (7.1% vs 6.3%, respectively). In contrast to Vdelta2+ and Vgamma9+ subtypes of pan gammadelta T cells, Vdelta3+ T cells number was higher in SLE patients (20 x 10 cells/microl) than in healthy control group (2 x 2 cells/microl) (P=0.001). However, we found no differences between the numbers of pan gammadelta T lymphocytes and studied their subtypes in the patients with active and inactive disease. These cell subpopulations were doubled in the treated patients with immunosuppressive agents in comparison with untreated ones; however, data were not statistically significant. Our study indicated that Vdelta3+ subtype of gammadelta T cells seems to be involved in SLE pathogenesis; however, we accept the idea that the autoimmunity does not develop from a single abnormality, but rather from a number of different events.

HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus.

A major problem in the management of SLE patients is to predict a flare or to distinguish between active and quiescent disease. Serological markers are widely used to assess disease activity, but many patients have close to or normal values for these parameters while exhibiting obvious disease-related signs and symptoms. This study aimed to determine which serological parameters, among ESR, ANA and anti-dsDNA antibody titres, CH50 and the HLA-DR expression on circulating T-lymphocyte subsets, best reflected the development of SLE flares. Sixty SLE patients were included, 34 with quiescent disease throughout the entire follow-up period and 26 who experienced an SLE flare defined as having active disease. According to univariate analysis, all parameters were significantly higher for patients with active disease, with the percentage of CD8+DR+ cells being the most significant parameter (P = 10-7). Multivariate logistic regression analysis identified three independent variables enabling the identification of a lupus flare: CH50, the CD8+DR+ and CD4+DR+ cell percentages among total lymphocytes. The CD8+DR+ cell percentage is the biological parameter most significantly associated with a flare (P < 0.001), even more powerful than CH50 (P < 0.01). HLA-DR expression on CD8+ lymphocytes clearly coincided with disease evolution in seven patients enrolled as having quiescent disease, but who experienced one flare during follow-up that subsequently resolved. The percentage of circulating CD8+DR+ lymphocytes appears to be a biological marker which accurately reflects disease activity. A larger prospective study is needed to demonstrate the real efficacy of this marker in predicting an exacerbation in SLE patients.