Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4⁺ and CD8⁺ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10⁻³-10⁻¹²M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G₁ phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8⁺ than on CD4⁺ T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.     

CD122+CD8+ Treg suppress vaccine‐induced antitumor immune responses in lymphodepleted mice

Lymphodeleption prior to adoptive transfer of tumor‐specific T cells greatly improves the clinical efficacy of adoptive T‐cell therapy for patients with advanced melanoma, and increases the therapeutic efficacy of cancer vaccines in animal models. Lymphodepletion reduces competition between lymphocytes, and thus creates “space” for enhanced expansion and survival of tumor‐specific T cells. Within the lymphodepleted host, Ag‐specific T cells still need to compete with other lymphocytes that undergo lymphopenia‐driven proliferation. Herein, we describe the relative capacity of naïve T cells, Treg, and NK cells to undergo lymphopenia‐driven proliferation. We found that the major population that underwent lymphopenia‐driven proliferation was the CD122⁺ memory‐like T‐cell population (CD122⁺CD8⁺ Treg), and these cells competed with Ag‐driven proliferation of melanoma‐specific T cells. Removal of CD122⁺CD8⁺ Treg resulted in a greater expansion of tumor‐specific T cells and tumor infiltration of functional effector/memory T cells. Our results demonstrate the lymphopenia‐driven proliferation of CD122⁺CD8⁺ Treg in reconstituted lymphodepleted mice limited the antitumor efficacy of DC vaccination in conjunction with adoptive transfer of tumor‐specific T cells.     

Preferential elimination of CD28+ T cells in systemic lupus erythematosus (SLE) and the relation with activation-induced apoptosis

CD28 on T cells provides a potent costimulatory signal for T cell activation. Down-regulation of CD28 on peripheral T cells has been reported in certain clinical conditions, but full studies on the mechanism and biological significance have not been performed. Our extensive phenotype analysis of peripheral blood lymphocytes (PBL) from SLE patients revealed that the absolute number of CD28⁺ T cells of both CD4 and CD8 phenotypes was selectively decreased, while that of CD28⁻ T cells was maintained. CD28⁺ T cells from SLE patients exhibited mostly normal proliferative responses to both CD28-dependent and -independent stimulations. In contrast, CD28⁻ T cells were hyporesponsive to anti-CD3 stimulation in both SLE and normal controls. These results implied that the selective decrease of CD28⁺ T cells in SLE does not result from a hyporesponsiveness of CD28⁺ T cells. To investigate the reason for the selective loss of CD28⁺ T cells, we determined the appearance of apoptotic cells in culture with or without anti-CD3 stimulation. Apoptotic cells defined by merocyanine (MC)540 were gradually increased from 12 h to 24 h. Anti-CD3-induced apoptosis of CD28⁺ T cells was significantly accelerated in SLE, whereas apoptosis of CD28⁻ T cells was hardly detected in both SLE and normal controls. Comparative analysis between CD28⁺ and CD28⁻ T cells on CD95 (Fas) and Bcl-2 expression, which are related to activation-induced cell death (AICD), did not show a major difference, although CTLA4, which has been demonstrated to transmit an apoptosis-inducing signal, was expressed only on CD28⁺ T cells. Our results suggest that CD28-mediated costimulation influences T cell susceptibility to AICD and may be involved in T cell lymphopenia in SLE.     

CD4+ CD25+ Treg regulate the contribution of CD8+ T-cell subsets in repopulation of the lymphopenic environment

Peripheral T‐cell expansion is of major relevance for immune function after lymphopenia. In order to promote regeneration, the process should result in a peripheral T‐cell pool with a similar subpopulation structure as before lymphopenia. We investigated the repopulation of the CD8⁺ central‐memory T cells (T_CM) and effector‐memory T cells (T_EM) pools after adoptive transfer of sorted CD8⁺ T cells from naïve, T_CM and T_EM subsets into T‐cell‐deficient hosts. We show that the initial kinetics of expansion are distinct for each subset and that the contribution to the repopulation of the CD8⁺ T‐cell pool by the progeny of each subset is not a mere function of its initial expansion. We demonstrate that CD4⁺CD25⁺ Treg play a major role in the repopulation of the CD8⁺ T‐cell pool and that CD8⁺ T‐cell subsets impact on each other. In the absence of CD4⁺CD25⁺ Treg, a small fraction of naïve CD8⁺ T cells strongly proliferates, correlating with further expansion and differentiation of co‐expanding CD8⁺ T cells. CD4⁺CD25⁺ Treg suppress these responses and lead to controlled repopulation, contributing decisively to the maintenance of recovered T_CM and T_EM fractions, and leading to repopulation of each pool with progeny of its own kind.     

Analysis of T cell receptor Vβ diversity in peripheral CD4+ and CD8+ T lymphocytes in patients with autoimmune thyroid diseases

Autoimmune thyroid diseases are characterized by intrathyroidal infiltration of CD4⁺ and CD8⁺ T lymphocytes reactive to self‐thyroid antigens. Early studies analysing T cell receptor (TCR) Vα gene usage have shown oligoclonal expansion of intrathyroidal T lymphocytes but not peripheral blood T cells. However, TCR Vβ diversity of the isolated CD4⁺ and CD8⁺ T cell compartments in the peripheral blood has not been characterized fully in these patients. We performed complementarity‐determining region 3 (CDR3) spectratyping as well as flow cytometric analysis for the TCR Vβ repertoire in peripheral CD4⁺ and CD8⁺ T cells from 13 patients with Graves' disease and 17 patients with Hashimoto's thyroiditis. Polyclonal TCR Vβ repertoire was demonstrated by flow cytometry in both diseases. In contrast, CDR3 spectratyping showed significantly higher skewing of TCR Vβ in peripheral CD8⁺ T cells but not CD4⁺ T cells among patients with Hashimoto's thyroiditis compared with healthy adults. We found trends towards a more skewed CDR3 size distribution in those patients having disease longer than 5 years and requiring thyroid hormone replacement. Patients with Graves' disease exhibited no skewing both in CD4⁺ and CD8⁺ T cells. These findings indicate that clonal expansion of CD8⁺ T cells in Hashimoto's thyroiditis can be detected in peripheral blood and may support the role of CD8⁺ T cells in cell‐mediated autoimmune attacks on the thyroid gland in Hashimoto's thyroiditis.     

Circulating CD4+CD8+ T lymphocytes in patients with Kawasaki disease

We serially monitored cell surface antigen expression on mononuclear cells in peripheral blood isolated from patients with Kawasaki disease (KD), and found, for the first time, that a markedly increased number of CD4⁺CD8⁺ T lymphocytes was present in some of the patients (11 of the 24 cases). The cases of five of these 11 patients were complicated with coronary artery lesion (CAL); the 13 patients with normal numbers of CD4⁺CD8⁺ T lymphocytes did not have CAL. The patients' age, sex and grade of systemic inflammation evaluated by peripheral leucocyte count and serum C-reactive protein levels were not correlated to the number of CD4⁺CD8⁺ T lymphocytes. Other cell surface antigen characteristics of the CD4⁺CD8⁺ T lymphocytes included CD3⁺, CD45RA⁺, CD45RO⁺, CD16^?, and HLA-DR⁺. These results indicate that the surface antigen characteristics of the KD peripheral blood examined were the same as those of Epstein–Barr virus infection without CD45RA⁺. These findings provide useful information for the analysis of the pathogenesis of KD.     

DOCK8 is essential for T-cell survival and the maintenance of CD8+ T-cell memory

Deficiency in the guanine nucleotide exchange factor dedicator of cytokinesis 8 (DOCK8) causes a human immunodeficiency syndrome associated with recurrent sinopulmonary and viral infections. We have recently identified a DOCK8‐deficient mouse strain, carrying an ethylnitrosourea‐induced splice‐site mutation that shows a failure to mature a humoral immune response due to the loss of germinal centre B cells. In this study, we turned to T‐cell immunity to investigate further the human immunodeficiency syndrome and its association with decreased peripheral CD4⁺ and CD8⁺ T cells. Characterisation of the DOCK8‐deficient mouse revealed T‐cell lymphopenia, with increased T‐cell turnover and decreased survival. Egress of mature CD4⁺ thymocytes was reduced with increased migration of these cells to the chemokine CXCL12. However, despite the two‐fold reduction in peripheral naïve T cells, the DOCK8‐deficient mice generated a normal primary CD8⁺ immune response and were able to survive acute influenza virus infection. The limiting effect of DOCK8 was in the normal survival of CD8⁺ memory T cells after infection. These findings help to explain why DOCK8‐deficient patients are susceptible to recurrent infections and provide new insights into how T‐cell memory is sustained.     

Thymic and peripheral apoptosis of antigen-specific T cells might cooperate in establishing self tolerance

Aside from CD4⁺CD8⁺ double-positive (DP) thymocytes, the subpopulations of T lineage cells affected by negative selection are unknown. To address whether this process occurs in more mature cell types, we have compared the responses of purified single-positive (SP) murine thymocytes and peripheral T cells to the superantigen staphylococcal enterotoxin B (SEB) utilizing as antigen-presenting cells (APC) a fibroblast cell line expressing transfected I-E^k class II molecules. Whereas ∽ 70% of SEB-reactive SP thymocytes, either CD4⁺ or CD8⁺, undergo programmed cell death (apoptosis) and, therefore, negative selection, CD4⁺ and CD8⁺ antigen-specific peripheral T cells are predominantly activated and proliferate to APC+SEB. Thus, mature thymocytes and peripheral T cells, with identical patterns and levels of expression of CD4, CD8 and T cell receptor (TCR), are programmed to elicit different responses followingTCR stimulation. Unexpectedly, however activation of peripheral T cells was preceded by deletion of a large fraction of Vβ8⁺ T lymphocytes (SEB specific). This surprising phenomenon was also observed in in vivo studies: in fact, administration of SEB to adult mice resulted in depletion of the majority of antigen-specific T cells from the peripheral lymphoid tissues analyzed (lymph nodes and spleen). This depletion is the consequence of deletion as indicated by program cell death of Vβ8⁺ T cells and is followed by proliferation of the remaining SEB-reactive T cells. Clonal elimination of peripheral T cells may represent a mechanism by which tolerance to self antigens never expressed in and/or exported to the thymus is achieved.     

Normal Human Immune Peritoneal Cells: Subpopulations and Functional Characteristics

Normal human peritoneal cells collected during elective laparatomy from patients with gallbladder stones without clinically detectable inflammatory changes were characterized phenotypically with immunocytochemical method and flow cytometry, with special attention paid to the presence of memory cells. The responsiveness of normal PCs to mitogen and, specifically, the role of peritoneal macrophages in this process was studied. The peritoneal cells consisted of 45% of monocytes/macrophages (CD68⁺), as many as CD2⁺ T lymphocytes, 8% CD57⁺ NK and K 2% CD22⁺ B, cells. The CD4/CD8 ratio was 0.4. The peritoneal cells did not express interleukin-2 (CD25⁺) and transferrin receptors (CD71⁺) on their surface. Approximately 49% of the peritoneal cells were class II MHC antigen positive cells. Two per cent of S100⁺ dendritic cells were found. Flow cytometric two-colour analysis revealed that the majority of peritoneal CD4⁺ (92.4%) and CD8⁺ (73.1%) lymphocytes, while only 50.2% of CD4⁺ and 30.1% CD8⁺ peripheral blood cells expressed simultaneously the CD45RO (UCHL1) molecule, which is characteristic to the memory/effector T-cell subpopulation. Peritoneal T lymphocytes responded to the mitogens less than peripheral blood lymphocytes of the same individual. Supplementation of cell culture with anti-macrophage (anti-CD68) and anti-HLA-DR MoAb brought about a dose-dependent decrease of proliferative peritoneal cell response to ConA. The authors conclude that human peritoneal cell population comprises a high proportion of T lymphocytes and macrophages capable of presenting antigens to peritoneal lymphocytes. High prevalence of memory lymphocytes points to the preparedness of these cells to react with invading antigens most likely of gut bacterial origin.     

Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphoe from HIV-infected individuals

Individuals infected with HIV have elevated numbers of total and activated CD8⁺ lymphocytes in peripheral blood. CD8⁺ lymphocytes from HIV-infected individuals have been shown to mediate non-human histocompatibility-linked antigen (HLA)-restricted suppression of viral replication, HLA-restricted killing of cells expressing HIV antigens, and killing of uninfected lymphocytes. We studied CD8⁽ T lymphocytes that lysed autologous CD4+ lymphocytes, hetcrologous CD4¹ lymphocytes from HIV-infected individuals and uninfected CD4⁺ lymphocytes. Killing in all cases required T cell receptor (TCR)-mediated recognition or triggering. However, these CD8 cytotoxic T lymphocytes (CTL) killed HLA class I mismatched CD4* lymphocytes and CD4⁴ lymphocytes treated with a MoAb against HLA-A, B and C antigens (PA2.6) which blocks HLA class I-restricted killing. HLA class H-negativc CD4* T lymphoma cells (CEM.NK^R) were also killed by anti-CD3 inhibited CTL. Stimulation of peripheral blood lymphocytes (PBL) from HIV-infected individuals, but not uninfected controls, with concanavalin A (Con A) and IL-2, induced non-HLA-restricted TCR aft¹, CD8^f CTL which lysed CD4⁺ lymphocytes. Activation ofCD4’lymphocytes increased their susceptibility to CD8^f CTL-mediated lysis. In HIV infection, a population of non-HLA-restricted CTL which lyse activated CD4⁺ lymphocytes is expanded. The expansion of CTL with unusual characteristics is interesting, because the stimulus for this expansion is unknown. CTL which recognize activated CD4⁺ cells could play a role in immune regulation and the pathogenesis of A IDS.     

T Cells from BB-DP Rats Show a Unique Cytokine mRNA Profile Associated with the IDDMI Susceptibility Gene,LYP

Diabetes prone biobreeding rats display several abnormalites in T cell numbers, T cell function and T cell surface phenotype which are associated with the onset of spontaneous disease. One of the most pronounced abnormalities in these animals is a marked T cell lymphopenia which is evident in both CD4⁺ and CD8⁺ peripheral T cell subsets. To gain a better understanding as to the nature of T cell responses in these animals, we have utilized RT-PCR to analyze the cytokine mRNA profiles of mitogen activated peripheral T cells derived from lymphopenic and non-lymphopenic animals. Our results suggest that inheritance of the lymphopenia gene, Lyp, is associated with a unique cytokine profile most similar to that previously described for mouse medullary thymocytes. In addition, cell surface staining of peripheral T cells from diabetes prone animals revealed a high frequency of Thyl⁺ cells, which is characteristic of both thymocytes and recent thymic emigrants. Following thymectomy, T cell responsiveness to a number of different stimuli is greatly reduced on a cell for cell basis as is the absolute number of surviving T cells. Taken collectively, our results suggest that the majority of the peripheral T cell pool in these diabetic prone rats consists of short lived, recent thymic emigrants which most likely also contain the effector cells required for initiation of diabetes.     

B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas

Objective

To investigate B7-H4 expression and its correlation with the number of infiltrating T lymphocytes and cytokine production by those lymphocytes in human cervical cancer and to determine the effect of recombinant B7-H4 on the active peripheral blood T cells of the patients in vitro.

Methods

B7-H4 expression was detected in 67 cases of cervical cancer using immunohistochemical staining. Tumor-infiltrating CD8⁺T, CD4⁺T, and FOXP3⁺ (Forkhead Box P3) T lymphocytes and their levels of IFN-γ and TGF-β₁ production were determined by immunofluorescent double-staining. After the peripheral blood T lymphocytes of patients were co-cultured with B7-H4, proliferation, apoptosis, and cell subtypes were analyzed using flow cytometry. Cytokines in the supernatant were detected by ELISA.

Results

B7-H4 was expressed in 46% (31/67) of the cases of cervical cancer. The number of infiltrating CD8⁺T lymphocytes and their IFN-γ production in positive B7-H4 expression cervical cancers was significantly lower than in negative B7-H4 cases (P < 0.01, P < 0.05), but there was no significant difference between cases positive and negative for B7-H4 with respect to infiltrating FOXP3⁺T and CD4⁺T cells or TGF-β1 production. After co-culture with B7-H4 for 48 h, the patients’ activated T lymphocytes were arrested at G1/G2 phase. The Ki67 positive rates of CD4⁺T and CD8⁺T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, and they were lower than in the blank group. The proportion of CD4⁺T and CD8⁺T cells decreased, but CD4⁺T/CD8⁺T and the proportion of CD4⁺CD25⁺Foxp3⁺T cells increased. In addition, concentrations of IL-10 and TGF-β1 in the supernatant of co-cultured T cells increased significantly (P < 0.05, P < 0.05), but that of IFN-γ decreased. B7-H4 had no significant effect on apoptosis of the T cells.

Conclusion

B7-H4 is overexpressed in human cervical cancers, and it is associated with lower numbers of tumor-infiltrating CD8⁺T lymphocytes and therefore less IFN-γ production. In vitro, B7-H4 inhibits the proliferation of CD4⁺T and CD8⁺T but promotes the proliferation of Tregs and the secretion of IL-10 and TGF-β1. B7-H4 plays an important role in depressing the anti-tumor immunity of CD8⁺T cell in microenvironments of cervical cancer.     

The lack of CD8α cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56^lck. A T cell-specific transgene encoding CD8α truncated at the cytoplasmic domain (“tailless CD8α”), was introduced into CD8α-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. “Tailless CD8α” was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4^? T cells (6% of T lymphocytes) was found to have cell surface expression of “tailless CD8α” and endogenous CD8β indicating that these cells may belong to the CD8⁺ T cell lineage. A consistent result was obtained from CD8α-deficient mice bearing the “tailless CD8α” and the MHC class I-restricted 2C TcR transgenes. A small population of CD4 T cells expressing CD8β the “tailless CD8α” and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When “tailless CD8α” mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8⁺ CD4^? T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8α is not absolutely required but dramatically enhances the eficiency of thymic maturation of CD8⁺ T cells. The lack of CD8α cytoplasmic domain in peripheral CD8⁺ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.     

Antihypoxic and Antioxidant Effects of Exogenous Succinic Acid and Aminothiol Succinate-Containing Antihypoxants

We studied the mechanisms of induction of recombinase activity in peripheral T cells of patients with autoimmune type 1 diabetes mellitus. It was shown that the presence of CD40 on T cell membrane (not typical of these cells) is crucial for this process: expression of recombinase RAG-1 in diabetic patients was detected primarily in αβTCR⁺CD40⁺ lymphocytes; targeted CD40-dependent activation of intact T cells in vitro increases, while blockade of CD40 signal in the culture of stimulated T cells abolishes recombinase expression.     

A T cell activation antigen,Ly6C,induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8⁺ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8⁺ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C⁺ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G₀/G₁ to S+G₂/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4⁺ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4⁺ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

Functional double-negative T cells in the periphery express T cell receptor Vβ gene products that cause deletion of single-positive T cells

A proportion of peripheral T cells lack surface expression of the CD4 or CD8 coreceptor molecules and hence are designated as “double negative” (DN). Most DN T lymphocytes express the Γ/β T cell receptor (TcR), but a minor fraction of them, in both humans and mice, express the α/β TcR. Whereas α/β⁺ DN T lymphocytes are infrequent (< 1%) in conventional lymphoid organs (spleen, blood, lymph node), they account for two-thirds of the T cells residing in adult bone marrow. Analysis of the TcR Vβ repertoire expressed by peripheral DN T cells revealed a high frequency of cells bearing autoreactive TcR that cause deletion of “single-positive” (SP) (CD4⁺CD8^? or CD4^?CD8⁺) T cells. Peripheral DN cells thus represent a cell type that is relatively resistant to clonal deletion. Furthermore, such cells have not been inactivated (anergized) in vivo since they proliferate and secrete interleukins in response to cross-linking by monoclonal antibodies specific for these Vβ gene products that are deleted in SP T cells. These results might help to understand the association of peripheral expansion of DN cells and development of autoimmune diseases.     

ORIGINAL ARTICLE: Women with Multiple Implantation Failures and Recurrent Pregnancy Losses have Increased Peripheral Blood T Cell Activation

Citation Yang KM, Ntrivalas E, Cho HJ, Kim NY, Beaman K, Gilman‐Sachs A, Kwak‐Kim J. Women with multiple implantation failures and recurrent pregnancy losses have increased peripheral blood T cell activation. Am J Reprod Immunol 2010 Problem We aim to determine whether peripheral blood T cell activation is associated with repeated implantation failures or recurrent pregnancy losses (RPLs). Method of study Women with a history of repeated implantation failure (n = 18) or RPLs (n = 17) comprise the study group. Normal fertile women (n = 11) are included as controls. Proportion of activated peripheral blood T cells (CD69⁺, CD154⁺) and Th1/Th2 cell ratios are measured by flow cytometric analysis. Results Proportions (%) of CD4⁺/154⁺ of CD4⁺ and CD8⁺/154⁺ of CD8⁺ cells were significantly higher in study group than those of controls. Proportions (%) of CD3⁺/69⁺ of CD3⁺ cells and CD8⁺/69⁺ of CD8⁺ cells were significantly increased in study group compared to controls. Proportion (%) of CD4⁺/69⁺ cells significantly correlated with % CD4⁺/154⁺ cells (P = 0.003). Activated cytotoxic T cells (CD8⁺/154⁺, CD8⁺/69⁺) inversely correlated with INF‐γ/IL‐10 producing CD3⁺/4⁺ T cell ratios. Proportion of activated CD3⁺/8⁺/69 and CD3⁺/8⁺/154⁺ cells was inversely correlated with IFN‐γ/IL‐10 expressing CD3⁺/4⁺ T cell ratios. Conclusion Women with MIFs or RPLs have increased T cell activation in peripheral blood lymphocytes, and T cell suppressor activation seems to be associated with decreased Th1 immunity. Further studies on T cell activation may elucidate molecular mechanisms controlling Th1 effectors.     

Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8⁺ CD28⁻ T cells with a corresponding reduction in the percentage of CD8⁺ CD28⁺ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4⁺ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8⁺ CD28⁺ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8⁺ CD28⁻ T cells in peripheral blood of HLA-A3⁺ but not HLA-A3⁻ HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR⁺ but not CD45RO⁺ cells was also found within the peripheral CD8⁺ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8⁺ CD28⁺ T cells both in controls and HH were able to expand in vitro; (ii) that CD8⁺ CD28⁻ T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8⁺ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard ⁵¹Cr-release assays when compared with CD8⁺ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8⁺ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.     

CD8high(CD57+) T cells in normal,healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines

We have previously identified two subsets of CD8⁺, CD57⁺ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8^high(CD57⁺)] and ii) natural killer cells expressing low levels of surface CD8 [CD8^low(CD57⁺)]. We investigated the cytotoxic and suppressive function of CD8^high(CD57⁺) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8^high(CD57⁺) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8^high(CD57⁺) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8^high(CD57⁺) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8^high(CD57⁺) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8^high(CD57⁺) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8^high(CD57⁺) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8⁺ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.