Secretion of leukaemia inhibitory factor after allogeneic bone marrow transplantation: a study of CD4+ and CD8+ TCRαβ+ T-cell clones derived from four leukaemia patients.

Abstract: CD4+ and CD8+ TCRαβ+ T-cell clones were derived from 4 leukaemia patients early (4–6 weeks) after allogeneic bone marrow transplantation. Leukemia inhibitory factor (LIF) secretion in response to the activation signal accessory cells (AC) + phytohaemagglutinin (PHA) was investigated for each individual clone. Only a minority of CD4 + TCRαβ + T-cell clones secreted LIF in response to AC + PHA, whereas most T-cell clones were capable of LIF secretion when exogenous interleukin 2 was added together with AC + PHA. LIF secretion could also be demonstrated for CD8+ TCRαβ+ posttransplant T-cell clones. Thus, posttransplant CD4+ and CD8+ TCRαβ + clonogenic T-cells are capable of LIF secretion, and LIF secretion may be a T-cell effector mechanism in graft versus host disease, graft versus leukaemia effects or posttransplant haematopoietic reconstitution.     

The effect of α and γ-interferon on proliferation and production of IgE and β2-microglobulin in the human myeloma cell line U-266 and in an α-interferon resistant U-266 subline

An IFN-resistant subline (U-266^r_α) was established from the IFN-α-sensitive myeloma cell line U-266 by subculturing U-266 cells with increasing doses of INF-α. The U-266^r_α secreted IgE at a higher rate than the U-266 (7.2 × 10^?13 g/c/8 h as compared to 3.3 × 10^?13 g/c/8 h). The 2 cell lines were found to be equally high producers of β₂m (9.2 and 9.6 × 10^?13 g/c/8 h). The U-266 produced 2.9 times less IgE and 5 times more β₂m compared to the initial production rates at establishment. INF-α and recombinant IFN-αM₂ (rIFN-α₂) inhibited proliferation and concomitantly decreased the rate of IgE and β₂m secretion in U-266 but not in U-266 IFN^r_α, which in contrast was slightly stimulated by IFN-α with respect to growth, IgE and β₂m secretion. In addition, IFN-α at a concentration of 100 U/ml was shown to decrease the IgE and β₂m production without exerting more than minimal cytotoxicity on U-266 cells. No antiproliferative effect was found for IFN-γ or recombinant IFN-γ (rIFN-γ) on either of the 2 cell lines. IFN-γ and rIFN-γ were, however, found to stimulate the production of β₂m. Our results show that the U-266 and the derived IFN-α-resistant subline can be used as models for studying some of the biological effects of IFN-α and -γ in vitro. The clinical implications of these in vitro results, in particular the usefulness of serum determinations of immunoglobulin and β₂m concentrations for monitoring the tumor cell mass, are discussed.     

Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22− and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity

We analysed the cytokine profile of a T cell subset (CD4⁺ CD45 RC⁻) that confers protection against Trichinella spiralis infection in rats. These CD4⁺ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-γ and functional cytokine secretion for IL-4, IL-5, IFN-γ, TNF-α and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4⁺ CD45 RC⁺ or CD8⁺), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-γ in the protective CD4⁺ CD45 RC⁻ cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-γ or TNF-α was secreted by the protective CD4⁺ CD45 RC⁻ cells. Whereas the non-protective CD4⁺ CD45 RC⁺ cells secreted significant levels of IL-4, IFN-γ, mast cell differentiating activity and TNF-α but little IL-5 activity. Nonprotective CD8⁺ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-γ secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.     

Detection of tumor necrosis factor-α in bone marrow plasma and peripheral blood plasma from patients with aplastic anemia

Plasma levels of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were determined in healthy individuals and patients with aplastic anemia (ApAn). IFN-γ was not detected in normal peripheral blood plasma (PBP) or bone marrow plasma (BMP) and was present in PBP from only 2 of 22 patients and in BMP from 1 of 14 patients and the levels were low (< 1.5 U/ml). Elevated levels of TNF-α were present in BMP and PBP from patients but not in control (healthy donor) PBP and BMP. Eleven of twenty-four patients had elevated levels of TNF-α in their PBP and 6 of 13 patients had detectable levels of TNF-α in their BMP. Only one of the 14 healthy control donors had detectable TNF-α and the level was very low (7 pg/ml), while 13 of the 27 ApAn patients had detectable TNF-α (P = .009, chi-square test). Not surprisingly, the centers of the distributions of TNF-α concentrations of the controls and ApAn patients differed significantly (P < .017 for control and patient PBP and P < .056 for control and patient BMP, Wilcoxon rank-sum test). Spontaneous production of IFN-γ and TNF-α by cultured bone marrow mononuclear cells was observed in four of seven patients but not in the six healthy controls (P = 0.026). Spontaneous production of IFN-γ and TNF-α by cultured peripheral blood mononuclear cells from patients and controls was however similar. Phytohemagglutinin (PHA)-induced production of IFN-γ and TNF-α by cultured mononuclear cells did not differ significantly between ApAn patients and normal controls. The significance of overproduction of TNF-α in the pathophysiology of ApAn is discussed. © 1994 Wiley-Liss, Inc.     

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-α (TNF-α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34⁺ compartment from MDS patients (P = 0.039), which are absent in both CD34^? MDS cells (P = 0.53) and also CD34⁺ cells from normal subjects (P = 0.55). MDS CD34⁺ blood cells also showed oxidized pyrimidine nucleotides compared with CD34^? cells (P = 0.029). Within normal subjects no differences were seen between CD34⁺ and CD34^? bone marrow cell compartments. CD34⁺ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.     

Tumor necrosis factor-α overproduction in Fanconi's anemia

Various in vitro studies and clinical observations suggest that Fanconi's anemia (FA) patients are unable to detoxify adequately superoxide anions (O) released by activated phagocytes. Recent studies have shown that certain lymphokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) can significantly enhance O production by phagocytic cells. To ascertain lymphokine production in FA patients, we measured TNF-α and IFN-γ production in vivo and in vitro. TNF-α was detected in the plasma of 16 of 18 FA patients with concentrations ranging from 6 to 131 pg/ml (mean 31 pg/ml). TNF-α was detected in only one of 25 control (healthy donor) plasma, and the level was very low (7 pg/ml). IFN-γ levels in normal and patient plasma were negligible. Spontaneous and phytohemagglutinin (PHA)-induced production of IFN-γ and TNF-α by cultured peripheral blood mononuclear cells did not differ significantly between FA patients and normal controls. The significance of overproduction of TNF-α in vivo in the pathophysiology of FA is discussed. © 1993 Wiley-Liss, Inc.     

Inhibition of Plasmodium falciparum growth in vitro by CD4+ and CD8+ T cells from non-exposed donors

T cells from most adult non-exposed donors, which express a memory phenotype (CD45RO+), can respond by proliferation to P. falciparum asexual stages in vitro. Such cells may have arisen from exposure to environmental organisms. To address the efficacy of such cells in eliminating parasites and investigate the mechanisms involved, we have used an in vitro assay where parasite growth can be precisely monitored in the presence of different cell preparations. Unfractionated peripheral blood mononuclear cells (PBMC) from both malaria-exposed and non-exposed donors inhibited parasite growth by up to 62% in a two day assay. Purified T cells in the presence of adherent cells had a similar effect, but purified T cells alone or adherent cells alone had minimal effect. Antigens released at the time of schizont rupture were maximally effective in stimulating interferon-γ (IFNγ) production. Neutralizing antibodies to IFNγ showed a partial reduction of growth inhibition in some individuals tested suggesting that different mechanisms may be operative. Neutralizing antibody to TNFα had a partial effect in combination with anti-IFNγ. Antibodies to IL-1 and IL-4 had no effect. T cell fractionation experiments showed that while purified CD4⁺ T cells from some donors produced IFNγ and inhibited parasite growth, purified CD8⁺ T cells could inhibit parasite growth to a greater extent without production of detectable IFNγ. Four parasitised red blood cell clones (CD4⁺, TCRαβ⁺, IFNγ producing) derived from non-exposed donors inhibited parasite growth to comparable levels, but one clone showed low production of IFNγ whilst the other three produced high levels. Our data show that T cells from non-exposed donors have the potential to eliminate malaria parasites via non-IFNγ dependent mechanisms. Such mechanisms may contribute to a degree of innate resistance to malaria in vivo, and may be able to be targeted by malaria vaccine programs.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Lineage determination of CD7+ CD5− CD2− and CD7+ CD5+ CD2− lymphoblasts: Studies on phenotype,genotype, and gene expression of myeloperoxidase,CD3ϵ, and CD3δ

The gene expression of myeloperoxldase (MPO), CD3? and CD3δ molecules, the gene rearrangement of T-cell receptor (TCR)δ, γ, and β and Immunoglobulin heavy (IgH) chain, and the expression of cell-surface antlgens were investigated In seven cases of CD7+ CD5? CD2? and four cases of CD7+ CD5+ CD2? acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5? CD2? blasts included four categories: Undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeioblasts. The CD7+ CD5+ CD2? blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCRβ and IgH. Four cases were G for TCRδ and TCRγ. The others were of the monoclonally rearranged gene (R) for TCRδ and G for TCRγ or R for both TCRδ and TCRγ. The expression or In vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed In all 11 CD7+ CD5? CD2?and CD7+ CD5+ CD2?, and some CD7+ CD5+ CD2+ (CD3? CD4? COB-) cases, but not in CD3± CD4+ CD8+ or CD3+ CD4+ CD8? cases. CD3? mRNA, but not CD3δ mRNA, was detected in two CD7+ CD5? CD2? cases, while mRNA of neither of the two CD3 moleculea was detected In the other tested CD7+ CD5?CD2? cases. In contrast, mRNA of both CD3e and CD36 were detected in ail CD7+ CD5+ CD2? cases, indicating that CD7+ CD5+ CD2? blasts at least belong to T-lineage. The blasts of two CD7+ CD5? CD2? cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3?, and CD3δ) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myelold-lineage. The co-expression of the genes of MPO and CD3e In a CD7+ CD5? CD2? case or MPO, CD3?, and CD3δ in a CD7+ CD5+ CD2? case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during Immature stages of differentiation. This heips understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML). The detection of CD3?, but not CD3δ mRNA, in two CD7+ CD5? CD2?cases indicated that the gene expression of CD3? occurs at a more immature stage of differentiation than that of the CD3δ chain. © 1994 Wiley-Liss, Inc.     

Differential nitric oxide and TNF-α production of murine Kupffer cell subfractions upon priming with IFN-γ and TNF-α

Abstract: Aims/Background: We have previously shown a striking heterogeneity of naive murine Kupffer cells (KC) that depends on cell size. Methods: In the present study, we demonstrate a shift in response of KC fractions separated on cell size by countercurrent elutriation upon priming with tumor necrosis factor-α (TNF-α) or interferon-γ (IFN-γ). Results: Whereas unprimed large KC are most active in the production of TNF-α and nitric oxide (NO), after priming of KC with TNF-α predominantly small and intermediate sized KC produce TNF-α in response to bacteria. Priming with IFN-γ enhanced NO production in all KC. A strong synergy, with respect to production of NO, was observed when KC subfractions were exposed to a combination of TNF-α and IFN-γ. Concerning TNF-α production, priming of KC subfractions seemed to induce a shift of activity from large KC to smaller KC. Conclusions: The present data demonstrate a clear heterogeneity among murine KC with respect to immunologic response to stimuli. These results demonstrate that KC have different functions in immunologic reactions that seem to be related to size.     

Phenotypical analysis and cytokine release of liver-infiltrating and peripheral blood T lymphocytes from patients with chronic hepatitis of different etiology

Cytokines released by infiltrating T cells may contribute to the hepatic injury in chronic hepatitis. Therefore, we characterized peripheral blood- and liver-infiltrating T cells from patients with chronic hepatitis of different etiology and determined the T cell phenotypes and the cytokine release. Liver tissue and peripheral blood-derived T cells from patients with autoimmune hepatitis and primary biliary cirrhosis predominantly expressed CD4-molecules and the α- and β-chains of the T cell receptor (TCR). In chronic viral hepatitis B and C, liver- and blood-derived T cells were preferentially CD8+ T cells expressing the αβ TCR. Mitogenic stimulation with irradiated Daudi lymphoma cells and phytohemagglutinin led to a strong release of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) by T cells in patients with chronic hepatitis and in healthy controls. T cells from patients with primary biliary cirrhosis and some patients with autoimmune hepatitis showed a significantly higher secretion of interleukin-4 (IL-4) and interleukin-10 (IL-10) than T cells from patients with chronic viral hepatitis or healthy controls. Histologic inflammatory activity did not correlate with the amount of cytokines released after mitogenic activation. In conclusion, liver tissue and peripheral blood T cells of patients with autoimmune hepatitis and primary biliary cirrhosis were dominated by CD4+ TCR αβ+ T helper/inducer cells, whereas in chronic viral hepatitis an enrichment of CD8+ TCR αβ+ cytotoxic/suppressor T cells was observed. In addition, analysis of the cytokine release showed that T cells in autoimmune and chronic viral liver diseases secreted high amounts of IFN-γ and TNF-α, cytokines predominantly secreted by T_hl-like cells. The secretion of the T_h2 cytokines. IL-4 and IL-10, however, was increased in autoimmune hepatitis and primary biliary cirrhosis. These data show that in autoimmune and chronic viral liver diseases different functional T cell subsets are activated.     

Microfilarial clearance in loiasis involves elevation of Th1 and Th2 products and emergence of a specific pattern of T-cell populations

Diethylcarbamazine (DEC) induced clearance of microfilaraemia in loiasis is associated with severe posttreatment reactions. To define the switch from hypo- to hyper-responsiveness associated with DEC treatment, phenotypic alterations of T-lymphocytes, characterized by flow cytometry, and cytokines, determined by enzyme linked immunosorbent assay, were monitored in a microfilaraemic patient. In contrast to reports on onchocerciasis and lymphatic filariases, no elevation of interleukin (IL)-6 and tumour necrosis factor (TNF)-α was observed. The most severe side effects coincided with an elevation of interferon (IFN)-γ on day 3, followed by IL-10, transforming growth factor (TGF)-β2 and macrophage inflammatory protein-1α (MIP-1α) peaking on day 5. Phenotypieally, T-cell activation markers CD38, CD54 and CD25 were significantly expressed before treatment, with high CD38 expression still existing one year after clearance of microfilaraemia. Treatment-related increases were observed with anti-CD 122, anti-HLA-DR and anti-CD69. CD28 was expressed before treatment on almost 100% qf'CD4⁺ and CD8⁺ T cells and dropped to 20% by day 5, reaching again baseline levels on day 21. Furthermore, there emerged 20% TCRαβ^-/CD3⁺ T cells and 10%) anti-βV5(e) ⁺ T cells, altogether indicating a specific pattern of T-helper (Th)1 and Th2 cytokines as well as expansion of certain pauciclonal T-cell populations in response to microfilarial clearance.     

Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4⁺ and CD8⁺ T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4⁺ and CD8⁺ TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8⁺ T-cell expansion, delaying differentiation into IFN-γ⁺ TNF⁺ multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4⁺ CD25^hi Foxp3⁺ T-cell population, which did not arise from endogenous natural T_reg but rather were peripherally generated from naïve Foxp3⁻ precursors.     

CD8+ T Cells of Tc2 Phenotype Mediate a GVL Effect and Prevent Marrow Rejection

Donor T cells mediate both beneficial and detrimental immune reactions in the setting of allogeneic BMT (alloBMT). T cells mediate the GVL effect and prevent marrow rejection, but also induce GVHD. In an attempt to favorably influence the balance of these allogeneic responses, we have evaluated the effect of donor CD4⁺, Thl/Th2 and CD8⁺, Tcl/Tc2 functional T cell subsets in murine marrow transplantation models. Our studies have identified the CD8⁺Tc2 population (which is a cytolytic effector secreting the type II cytokines IL-4, IL-5, and IL-10) as a subset capable of mediating the GVL effect and preventing marrow rejection with reduced GVHD. We have also shown that the Tc2 subset can be generated in humans. These studies indicate that administration of donor CD8⁺ T cells of Tc2 phenotype represents a strategy for improving alloBMT outcome.     

Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites

Increasing evidence indicates that hepatic CD8α⁺ dendritic cells (DCs) are important antigen cross‐presenting cells (APC) involved in the priming of protective CD8⁺ T‐cell responses induced by live‐attenuated Plasmodium sporozoites. Experimental proof for a critical role of CD8α⁺ DCs in protective pre‐erythrocytic malaria immunizations has pivotal implications for vaccine development, including improved vectored subunit vaccines. Employing Batf3^?/? mice, which lack functional CD8α⁺ DCs, we demonstrate that deficiency of these particular APCs completely abolishes protection and corresponding signatures of vaccine‐induced immunity. We show that in wild‐type, but not in Batf3^?/?, mice CD8α⁺ DCs accumulate in the liver after immunization with live irradiation‐attenuated P. berghei sporozoites. IFN‐γ production by Plasmodium antigen‐specific CD8⁺ T cells is dependent on functional Batf3. In addition, our results demonstrate that the dysfunctional cDC‐CD8+ T‐cell axis correlates with MHC class II upregulation on splenic CD8α^? DCs. Collectively, these findings underscore the essential role of CD8α⁺ DCs in robust protection induced by experimental live‐attenuated malaria vaccines.     

Expansion of CD8+CD57+ T cells after allogeneic BMT is related with a low incidence of relapse and with cytomegalovirus infection

Summary. Peripheral blood lymphocytes of 46 recipients of lymphocyte-depleted bone marrow allografts were pheno-typically analysed over a period of 1 year. We investigated the repopulation of lymphocyte subpopulations and their relation with clinical parameters such as graft-versus-host disease (GVHD), graft-versus-leukaemia and cytomegalovirus (CMV) infection. The number of repopulated T cells varied strongly between the blood samples of the recipients. In 45% of the recipients the number of T cells recovered to or above normal levels within 3 months after bone marrow transplantation (BMT), whereas the other recipients remained below normal up to 1 year after BMT. In recipients with a high repopulation, the CD8⁺ T-cell subset contributed more to this high repopulation than the CD4⁺ T-cell subset. We showed that the majority of T cells of these recipients expressed the a/3 T-cell receptor, CD8, CD57 and CDllb. HLA-DR was also highly expressed reflecting the activation stage of T cells in these recipients. BMT recipients with a high repopulation of CD8⁺ T cells showed a lower incidence of leukaemic relapse than recipients with a low repopulation. The 3-year probability of relapse was 19% versus 64% (P=O03), respectively. The relative high number of CD8⁺ T cells at 3 months after BMT was not associated with the incidence of GVHD. In contrast, occurrence of CMV infection after BMT was significantly higher in these recipients. Our results indicate that CD8⁺ T cells, predominantly CD57⁺, of BMT recipients with an expansion of these cells represent an in vivo activated cell population. This CD8⁺ T-cell population may consist partially of cytotoxic cells with anti-leukaemic activity as suggested by a low relapse rate. The signal for the strong expansion of these CD8+CD57+ T cells after BMT is still unclear, but association with CMV infection suggests that viral antigens are involved.     

Oligoclonal expansion of CD8+ CD57+ T cells with restricted T-cell receptor beta chain variability after bone marrow transplantation

A major expansion of CD8+57+ lymphocytes expressing an alpha-beta T- cell receptor (TCR) is frequent after bone marrow transplantation (BMT). We examined the clonality of the TCR beta gene repertoire in these expanded CD8+57+ cells after allogeneic or autologous BMT. We performed a polymerase chain reaction (PCR) analysis of the V beta chain usage in CD8+57+ cells purified from nine BMT recipients with a series of oligonucleotides specific for 20 V beta gene families. PCR products from selected TCR beta gene rearrangements were sequenced. The CD8+57+ cells from eight of nine patients used a restricted set of V beta families, with a marked predominance of two to three V beta gene families per patient, whereas the control autologous CD57- subset expressed the whole 20 V beta families. A direct sequencing analysis confirmed the V beta 16 and V beta 17 clonality in six patients, showing a striking homology in the CDR3 sequences of the V beta 16 products. The CD8+57+ cells, but not the CD57- cells, displayed an oligoclonal pattern of TCR rearrangements as shown by PCR analysis of TCR gamma gene rearrangements. Such an oligoclonal expansion of CD8+57+ cells, using a restricted set of the V beta gene families, may result from a specific TCR stimulation of a limited number of T-cell clones in BMT recipients.     

Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+ T cells

Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8(+) T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8(+) T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8(+) T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation.