Javier Avila-Cariño

Autologous T lymphocytes may specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia

This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real‐time reverse transcription polymerase chain reaction (RT‐PCR) revealed that granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were γ‐interferon (IFN), interleukin (IL)‐2 and tumour necrosis factor (TNF)‐α, but not IL‐4. This profile suggests a type 1 anti‐B‐CLL T‐cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T‐cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T‐cell DNA synthesis proliferation. The proliferative T‐cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T‐cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T‐cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti‐CLL T‐cell immunity is warranted that may facilitate the development of effective anti‐tumour vaccines in CLL.

Truncated thioredoxin (Trx80) exerts unique mitogenic cytokine effects via a mechanism independent of thiol oxido-reductase activity.

Recently we discovered that a naturally occurring C‐terminally truncated thioredoxin (Trx80) is a potent mitogenic cytokine stimulating IL‐12 production from CD40+ monocytes. To further characterise Trx80 we have engineered cysteine to serine mutants of Trx80 corresponding to the active site cysteines of Trx (Trx80SGPS) and to the structural cysteine at position 72 (Trx80C72S). Trx80SGPS and Trx80C72S retained the cell stimulatory activity of Trx80 and increased peripheral blood mononuclear cell (PBMC) proliferation three‐ to five‐fold in vitro (P<0.01, n=18). Both Trx80SGPS and Trx80C72S significantly stimulated IL‐12 and IFN‐γ secretion from PBMCs in the same manner as Trx80 (P<0.01, n=9 and 10). The previously described Trx80 dimer is caused by non‐covalent interactions, and not by any intermolecular disulphide bonds.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

The thiol antioxidant N‐acetyl‐ l‐cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL‐2 and up‐regulation of the IL‐2 receptor. The 120‐kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen‐specific T cell clones (TCC). TCC were stimulated with anti‐CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down‐regulation of IL‐4, IL‐5 and IFN‐γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL‐4. Furthermore, they down‐regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 m m NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down‐regulation of IL‐4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL‐4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases.

B-CLL cells with unusual properties

In studies concerning the interaction of B‐CLL cells and Epstein‐Barr virus (EBV), we encountered one patient whose cells had several unusual properties. In addition to the B‐cell markers, the CLL cells expressed the exclusive T‐cell markers CD3 and CD8 and carried a translocation t(18,22)(q21;q11), involving the bcl‐2 and Igλ loci. The patient represents the 4th reported CLL case with this translocation. The CLL cells could be infected and immortalized by the indigenous and by the prototype B958 virus in vitro. The T‐cell markers were not detectable on the established lines. In all experiments the immortalized lines originated from the CLL cells. Their preferential emergence over virus‐infected normal B cells may be coupled to the high expression of the bcl‐2 gene due to the translocation. In spite of the sensitivity of CLL cells to EBV infection in vitro, no EBNA‐positive cells were detected in the ex vivo population. In vitro, we could generate cytotoxic function in T‐lymphocyte cultures which acted on autologous EBV‐infected CLL cells. Therefore we assume that if such cells emerged in vivo they were eliminated by the T‐cell response.

EBV Infection of B-CLL Cells in vitro Potentiates Their Allostimulatory Capacity if Accompanied by Acquisition of the Activated Phenotype

Epstein‐Barr virus (EBV)‐carrying immortalized lymphoblastoid cell lines (LCLs) stimulate autologous T lymphocytes in vitro. This T‐cell response is independent of the EBV‐specific cellular memory because it also occurs in experiments with cells of seronegative individuals. The question can be posed whether the T‐cell‐stimulatory potential of the LCL is coupled to its immortalized state. B‐CLL cells were exploited to study this question because the majority of clones, represented by different patients, can be infected with EBV but they rarely become immortalized. We have investigated the phenotypic changes and the T‐cell‐stimulatory capacity of EBV‐infected B‐CLL cells. One aliquot of CLL cells was infected with EBV, another was activated with a mixture of Staphylococcus aureus (SAC), IL‐2 and the supernatant from the T‐cell hybridoma MP6 (activation mixture, AcMx) and the third aliquot received both treatments. In accordance with the individual features of the clonal populations represented by each patient, the immunophenotypic changes imposed by these treatments differed. With the samples of 3 patients the allo‐stimulatory potential showed the following ranking order: EBV and AcMx‐treated cells > AcMx‐treated > EBV‐infected. An analysis of several activation‐related surface markers and adhesion molecules on the cells did not reveal any association between their expression and the EBV‐imposed potentiation of allostimulatory capacity. These results may be extrapolated to EBV‐genome‐carrying normal B cells, suggesting that they can persist in vivo only as long as they have the resting phenotype. Once they are activated, these cells may be recognized and eliminated by T lymphocytes.

Thioredoxin-80 is a product of alpha-secretase cleavage that inhibits amyloid-beta aggregation and is decreased in Alzheimer's disease brain

Thioredoxin‐1 (Trx1) is an endogenous dithiol reductant and antioxidant that was shown to be decreased in Alzheimers disease (AD) neurons. A truncated form of Trx1, thioredoxin 80 (Trx80), was reported to be secreted from monocytes having cytokine activity. Here, we show that Trx80 is present in human brain in an aggregated form. Trx80 localizes mainly to neurons and is dramatically decreased in AD brains. Trx80 levels in cerebrospinal fluid (CSF) correlate with those of the classical AD biomarkers amyloid‐β (Aβ) 1–42 and total tau. Moreover, Trx80 measurements in CSF discriminate between patients with stable mild cognitive impairment, prodomal AD and mild AD. We report that ADAM10 and 17, two α‐secretases processing the Aβ precursor protein, are responsible for Trx80 generation. In contrast to the periphery, Trx80 has no pro‐inflammatory effects in glia, either by itself or in combination with Aβ or apolipoprotein E. Instead, Trx80 inhibits Aβ(1–42) aggregation and protects against its toxicity. Thus, a reduction in Trx80 production would result in increased Aβ polymerization and enhanced neuronal vulnerability. Our data suggest that a deficit in Trx80 could participate in AD pathogenesis.

Search for the critical characteristics of phenotypically different B cell lines, Burkitt lymphoma cells and lymphoblastoid cell lines, which determine differences in their functional interaction with allogeneic lymphocytes

SummaryBurkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity to lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.

Induction of the lytic viral cycle in Epstein Barr virus carrying Burkitt lymphoma lines is accompanied by increased expression of major histocompatibility complex molecules

Six Epstein Barr virus (EBV) genome-carrying Burkitt lymphoma (BL) cultures (P3HR-1, Raji, Akata, Daudi, Rael and Jijoye) were induced to enter the lytic cycle. Phorbol esther (TPA), n-butyrate, 5-azacytidine (5AzaC) or anti-IgG were used according to their known inducing capacity on these cell lines. Concomitantly with the appearance of the viral early antigens (EA) in a proportion of cells, the expression of major histocompatibility complex (MHC) class II antigens increased in the cultures. On P3HR-1 and Raji cells class I expression also increased. The enhancement of MHC expression correlated with the efficiency of induction and required only an early event of the viral lytic cycle. Treatment of 3 EBV-negative lymphoma lines (BJAB, Ramos and BL41) with TPA plus n-butyrate or 5AzaC did not influence MHC expression. Moreover, BL lines which carry the EBV genome after having been infected in vitro and which cannot be induced for the viral lytic cycle did not change MHC expression after treatment with the inducing agents. In mixed cultures the allo-stimulatory capacity of induced cells with elevated MHC expression was stronger compared to the untreated ones.

RECOGNITION OF B-CLL CELLS EXPERIMENTALLY INFECTED WITH EBV BY AUTOLOGOUS T LYMPHOCYTES

We compared 5-day-old cultures of two B-CLL clones experimentally infected with EBV for their interaction with autologous T lymphocytes. The clone which was strongly activated by the virus stimulated autologous T cells. It was also damaged by the cytotoxic T cells which were generated in mixed cultures with autologous lymphoblastoid cell lines (LCL). Cultured, non-infected CLL cells were not lysed by these effectors. The other B-CLL clone, which was activated to considerably lesser extent by the virus, did not stimulate the autologous T lymphocytes. While, also in this case cytotoxic function was generated in the mixed T cell-LCL culture, the effectors did not damage the EBV-infected CLL cells. The results with B-CLL cells can be regarded as a model for the EBV genome carrier normal B lymphocytes. They substantiate the current concept that such cells persist in seropositive healthy individuals undisturbed by the specific immune response as long as they maintain the phenotype of resting cells. However, after activation they can be recognized and eliminated by T cells.

Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

Background Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). Principal Findings In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. Significance Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.