Mikael Jondal

MHC Class I–Restricted CTL Responses to Exogenous Antigens

A number of different modified exogenous protein antigens can efficiently prime CTL responses. The biological significance of this for priming of normal CTL responses is unknown. Processing of exogenous antigen for MHC-I presentation seems an attractive possibility to bypass the necessity that all foreign antigens have to be translated within the cytosol of professional APC in order to prime CTL responses. Many microbes have a select host cell specificity that does not include APC. They kill their host cells or severely disturb the sophisticated cellular functions that are required in APC (84xNorkin, L.C. Clin. Microbiol. Rev. 1995; 8: 293–315PubMedSee all References, 45xHaywood, A.M. J. Virol. 1994; 68: 1–5PubMedSee all References). Another scenario for a virus-specific CTL response is thus that infected cells disintegrate (possibly as apoptotic bodies) and exogenous antigen from them is transferred to APC for further processing in different cellular compartments.There are many unresolved issues regarding processing of exogenous antigens for MHC-I presentation and the priming of CTL responses. First, which are the most important uptake mechanisms? Second, is there a special (MHC-II-like) compartment for “endosomal” processing, or do all APC “leak” antigen into the cytosol and thereby into the classical MHC-I processing pathway? Third, in a proposed endosomal-like compartment, which are the proteolytic enzymes involved in processing? Do they generate a spectrum of MHC-I-bound peptides distinct from those generated in the cytosolic pathway? Finally, how do MHC-I molecules gain access to the endosomal-like compartment, and how are trimeric peptide MHC-I–β2-microglobulin complexes transported to the cell surface and their membrane expression regulated?If exogenous antigens are instrumental in priming normal CTL responses, processing in noncytosolic, vesicular compartments, distinct from the endogenous classical class I processing pathway, might be important. If so, it may be relevant to reevaluate the endogenous/MHC-I and exogenous/MHC-II processing dogma and instead classify pathways in functional terms. In CTL activation, induction (by exogenous antigen) and tar- get cell recognition (by endogenous antigen) by class I–restricted peptide presentation would result from different processing pathways. This is an important issue in terms of vaccine development, as these should primarily be expressed in the most optimal processing compartment. If exogenous antigens are of major importance in generating CTL responses, vaccines based on live, replicating vectors (and nucleic acids) induce their protective effect in a nonviable form. In this transfer process, from the infected host cell to the professional APC cell, there might be a lesson to be learned in terms of formulations for direct uptake into APC cells. The potent clinical effect of the first human recombinant vaccine licensed for clinical use, which was based on self-assembly of the hepatitis B virus small surface antigen into 22 nm subviral particles, might be a “lesson of nature” in this context (Schirmbeck et al. 1996xSchirmbeck, R, Bohm, W, and Reimann, J. Intervirology, in press. 1996; See all ReferencesSchirmbeck et al. 1996).

Self-replicating Semliki Forest virus RNA as recombinant vaccine

Recombinant RNA based on the Semliki Forest virus (SFV) replicon was used to express the nucleoprotein of influenza virus in mice. Two strategies were employed to deliver the RNA. In the first, recombinant RNA was packaged into infectious suicide SFV particles which were used directly for immunization. The second approach involved injection of in vitro-synthesized RNA directly into the quadriceps muscle. Both approaches resulted in the generation of humoral responses with high antibody titres. Immunization with suicide particles showed that a strong, class I-restricted cytotoxic T-cell response can be obtained using only 100 infectious units. We conclude that the self-replicative recombinant SFV RNA may be quite useful as a nucleic acid vaccine.

Bcl-2, Bax and p53 expression in B-CLL in relation to in vitro survival and clinical progression.

Our previous data have shown that isolated leukemic cells from progressive chronic lymphocytic leukemia (B‐CLL) patients respond to growth stimulation in vitro and express high levels of p53, immunoreactive with the configuration‐specific antibody PAb 240. We have now analyzed the in vitro survival of B‐CLL cells in relation to Bcl‐2, Baxα and p53 expression and compared this with the clinical progression of the disease. Leukemic cells from patients with progressive disease demonstrated higher in vitro survival, compared with non‐progressive B‐CLL and normal B cells. All cells were sensitive to treatment with a combination of glucocorticoid and cAMP. Bcl‐2 protein levels were not related to clinical progression, as measured by flow cytometry. Competitive PCR showed that Bcl‐2 mRNA was over‐expressed in most of the B‐CLL samples and that p53 mRNA expression was similar between B‐CLL groups and normal values and thus not related to clinical progression. However, since Baxα expression was lower in progressive than in non‐progressive patients, the Bcl‐2/Baxα ratio at the mRNA level was significantly higher in the progressive group. Our data suggest that the Bcl‐2/Baxα ratio is important for the regulation of B‐CLL cell survival, that p53 over‐expression in progressive B‐CLL is the result of post‐transcriptional modifications and that a directed PKA activation may potentiate the cytolytic effect of glucocorticoids in vivo.

Surface markers on human B and T lymphocytes. VIII. Association between complement and Epstein-Barr virus receptors on human lymphoid cells.

An association has been found between the Epstein‐Barr virus (EBV) and complement (C3d) receptors on human lymphoid cells. The evidence was fourfold: there was a correlation between the expression of these two receptors; inhibition experiments showed that the binding sites probably are close to each other in the cell membrane, although not identical; EBV and complement receptors have been found to co‐cap in either order; and lymphoid cell lines lacking complement receptors could not be superinfected with EBV.

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3β‐hydroxysteroid dehydrogenase (3βHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with ‘reporter cells’ containing the glucocorticoid receptor (GR) and a GR‐dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3βHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.—Pazirandeh, A., Xue, Y., Rafter, I., Sjövall, J., Jondal, M., Okret, S. Paracrine glucocorticoid activity produced by mouse thymic epithelial cells. FASEB J. 13, 893–901 (1999)

Crystal structure of an MHC class I presented glycopeptide that generates carbohydrate-specific CTL.

T cell receptor (TCR) recognition of nonpeptidic and modified peptide antigens has been recently uncovered but is still poorly understood. Immunization with an H-2Kb-restricted glycopeptide RGY8-6H-Gal2 generates a population of cytotoxic T cells that express both alpha/beta TCR, specific for glycopeptide, and gamma/delta TCR, specific for the disaccharide, even on glycolipids. The crystal structure of Kb/RGY8-6H-Gal2 now demonstrates that the peptide and H-2Kb structures are unaffected by the peptide glycosylation, but the central region of the putative TCR binding site is dominated by the extensive exposure of the tethered carbohydrate. These features of the Kb/RGY8-6H-Gal2 structure are consistent with the individual ligand binding preferences identified for the alpha/beta and gamma/delta TCRs and thus explain the generation of a carbohydrate-specific T cell response.

Human lymphocyte subpopulations: classification according to surface markers and-or functional characteristics.

Lymphocytes of birds and mammals can be subdivided into two major groups according to origin and function. If classified according to dependence or origin they are called thymus-dependent or T lymphocytes and bursa-derived or B lymphocytes (Raff 1973). Whereas in animal model systems the functional role of these cells have been assessed under varying experimental conditions, analogies have been provided for by the experiment of nature in human beings suffering from immunodeficiency disorders (Gatti 1972, Cooper e al. 1973). Such comparisons have made it possible to state quite emphatically that the human T and B lymphocyte populations exist with similar functional characteristics as in the other mammals. Whereas especially in the mouse, progress into the functional characteristics of the subpopulations of lymphocytes have been made largely on the basis of development of surface markers allowing actual physical separation of the various groups of cells, less is known about such ptossibilities in the human system. It is the purpose of the present article to present some of the surface markers we have used in our analysis of human lymphocytes under varying clinical and experimental conditions. The present article has no ambition to make a complete review of this field but will merely cover our own results and present views on human lymphocytes, their functional characteristics and distinguishing markers in healthy or diseased human beings. The reader is referred to the other articles in this issue to receive a more complete and maybe more objective picture.

Effects of altered glucocorticoid sensitivity in the T-cell lineage on thymocyte and T-cell homeostasis

The homeostatic regulation that controls total thymocyte and peripheral T‐cell numbers is not clearly understood. We describe here a direct hormonal influence of endogenous levels of glucocorticoids (GCs) on thymocyte and peripheral T‐cell homeostasis independent of indirect systemic effects of GCs. The results were obtained by generating transgenic mice with an altered GC sensitivity targeted to thymocytes and peripheral T cells by increasing or decreasing glucocorticoid receptor (GR) expression specifically in thymocytes and peripheral T cells. A twofold increase in GC sensitivity resulted in a major decrease in thymocyte number, affecting all subpopulations, although single‐positive CD8+ cells were less influenced. In the thymus, this was due to increased apoptosis in the organ, whereas proliferation of thymocyte populations was unaffected. In the periphery, a pronounced reduction in T‐cell number was seen, demonstrating an effect of endogenous GCs also on T‐cell homeostasis. The effects were confirmed in transgenic mice with reduced GR expression, which showed increased thymocyte and T‐cell numbers. Thus, our data demonstrate that physiological GC levels are directly involved in controlling the size of both thymocyte and T‐cell pools.

NK cell-induced cytotoxicity is dependent on a Ca2+ increase in the target.

In previous work we showed that programed cell death (PCD) in thymocytes is mediated by a sustained increase in cytosolic Ca2+ concentration, resulting in the activation of an endogenous endonuclease, DNA fragmentation, and cell death. In this study we investigated the roles of Ca2+ and DNA fragmentation in target cell killing by natural killer (NK) cells. The effector cells induced a rapid, sustained increase in cytosolic Ca2+ concentration in Jurkat target cells. Buffering the target cell cytosolic Ca2+ with the Ca2+‐selective dye, quin‐2, prevented target cell killing. Extensive DNA fragmentation was associated with killing in every target tested, and this response was also blocked by quin‐2. The endonuclease inhibitor, aurintricarboxylic acid, inhibited both DNA fragmentation and killing without influencing the Ca2+ increase in target cells. Thus, it is concluded that NK cell killing depends on a Ca2+ increase and appears to involve endogenous endonuclease activation in target cells.— McConkev. D. J.; Chow, S. C.; Orrenius, S.; Jondal, M. NK cell‐induced cytotoxicity is dependent on a Ca2+ increase in the target. FASEB J. 4: 2661‐2664; 1990.

Sensitization to TRAIL-induced apoptosis and modulation of FLICE-inhibitory protein in B chronic lymphocytic leukemia by actinomycin D

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of the cell death pathway and exerts tumoricidal activity in vivo with minimal toxicity. In order to investigate the therapeutic potential of TRAIL in B chronic lymphocytic leukemia (B-CLL) we have analyzed the expression of TRAIL receptors (TRAIL-Rs) in leukemic cells from B-CLL patients and their in vitro sensitivity to apoptosis induced by recombinant human TRAIL. We have found TRAIL-R1 and -R2 death receptor, and TRAIL-R3 and -R4 decoy receptor mRNA expression in most of the 57 B-CLL patients studied (R1 82%, R2 100%, R3 96% and R4 82%). TRAIL-R1 and R2 proteins were expressed on the surface and within the cells, whereas R3 and R4 decoy receptors were almost exclusively expressed in the cytoplasm. Despite TRAIL death receptor expression, B-CLL cells were relatively resistant to induction of apoptosis by recombinant human TRAIL (300 ng/ml). However, the susceptibility to TRAIL-induced apoptosis was increased by treatment of B-CLL cells with actinomycin D (Act D). Western blot analysis showed higher constitutive expression of the long form of FLICE-inhibitory protein (FLIPL) in B-CLL as compared to normal tonsillar B cells. Act D treatment down-regulated both long and short FLIP expression, which was correlated with the increase in B-CLL sensitivity to TRAIL. Although the surface TRAIL death receptor expression was up-regulated both by cell culture and by Act D treatment, the changes were not correlated with a gain in susceptibility to TRAIL. In addition, neither decoy receptors nor Bcl-2 expression were affected by Act D. Our findings suggest the possible involvement of FLIP in regulating TRAIL-mediated apoptosis in B-CLL.