Au cours de la réponse immunitaire, comme au cours de la sélection thymique, les interactions cellulaires sont dépendantes de la formation d'un contact étroit entre cellules caractérisé par une synapse. De nombreuses molécules associées aux synapses neurologiques ou neuro-musculaires se sont également exprimées au niveau des cellules immunitaires et impliquées dans la régulation des contacts. Nous avons donc recherché la présence d'autres récepteurs neurologiques au niveau des cellules immunitaires. La neuropilin-1 (Np-1), responsable de la régulation de l'orientation et des contacts des neurones, a été mise en évidence à la fois sur les cellules dendritiques (DC) et les lymphocytes T (LT). De même, la sémaphorin-3A (Sema-3A), un de ces principaux ligands, est exprimée et sécrétée au niveau de ces cellules. La Np-1 est impliquée dans la formation des contacts entre les LT-DC et Thymocytes-cellules épithéliales thymiques. Sa régulation est dépendante de l'activation T médiée par le TcR et/ou par l'interleukine-7 concernant les thymocytes. La production et la sécrétion de la Sema-3A, est exclusivement dépendante de la stimulation du TcR. La Sema-3A interfère avec la prolifération des LT via l'inhibition de la polymérisation de l'actine ce qui empêche la polarisation du TcR au site du contact cellulaire. Le blocage de la Sema-3A, par des anticorps neutralisants provoque une augmentation de la prolifération T. La Sema-3A joue également un rôle au niveau de l'adhésion des thymocytes en bloquant la capacité d'adhésion de ces derniers aux molécules de la matrice extracellulaire (laminine et fibronectine). De plus, la Sema-3A induit la chemorépulsion des thymocytes Np-1 positifs.

Le rôle des lymphocytes T CD4+ dans la réponse immune anti-tumorale s'est révélé être de plus en plus important au cours de ces dix dernières années. Nous nous sommes attachés à l'étude de leur fonction dans les lymphomes malins non hodgkiniens de type B. <br />Dans la première partie de ce travail de thèse, nous avons étudié un modèle à partir des cellules fraîches splénique d'un patient porteur d'un lymphome B splénique de la zone marginale. Nous avons pu déterminer un effet fonctionnel des lymphocytes T CD4+ réactifs par rapport aux cellules malignes B autologues. Ces cellules T CD4+ sont capables d'induire une différenciation des lymphocytes B tumoraux en plasmocytes, et cette induction a été dévoilée pour la première fois dans un système cellulaire autologue. <br />Nous avons ensuite étudié la capacité fonctionnelle des lymphocytes T CD4+ réactifs sur une lignée de lymphome B folliculaire obtenue dans notre laboratoire. Nous avons obtenu un effet cytotoxique par les cellules T CD4+ totales autologues venant des lymphocytes du sang périphérique. Cependant, cette cytotoxicité s'est montrée à la fois sur les cellules B malignes et les cellules B normales lymphoblastoïdes. Un clonage a été ensuite réalisé dans le but d'écarter les clones non spécifiques et de trouver des clones T cytotoxiques spécifiques des cellules malignes. Parmi les six clones obtenus, trois sont spécifiques et ils possèdent un TCR identique Vb17-Db1-Jb1.2. Ces clones exercent une cytotoxicité contre les cellules tumorales en reconnaissant l'antigène tumoral présenté par la molécule HLA-II DP et leur mécanisme de lyse correspond à la voie perforine/granzymes. <br />Ces deux résultats nous ont permis de conclure que les cellules T CD4+ peuvent induire un effet direct anti-tumoral avec des mécanismes variés. Ce travail donne de nouveaux arguments concernant le rôle pivot des lymphocytes T CD4+ dans l'immunité anti-tumorale et permet d'envisager l'identification de l'antigène tumoral.

Le lymphocyte T est une cellule clé de l'immunité spécifique. Dans le but de créer un outil de compréhension et de prédiction de certains mécanismes immunitaires, une modélisation de la réponse immunitaire du lymphocyte T est proposée. L'activation du lymphocyte par la reconnaissance d'un peptide apprêté par une cellule présentatrice d'antigène est une étape essentielle de cette réponse immunitaire. Cette activation a été modélisée par un système d'équations différentielles ordinaires, de type cinétique chimique, représentant l'évolution temporelle des concentrations de différentes protéines lymphocytaires (TCR/CD3, CD28, CD69, CD25, IL-2). Afin de considérer une quantité d'antigène variable dans l'organisme, un modèle de prolifération virale a aussi été écrit, basé sur des exemples de modèles proies/prédateurs, obtenant ainsi un système d'équations différentielles ordinaires mettant en jeu un virus donné, les cellules cibles du virus, saines ou infectées, et l'action des lymphocytes T cytotoxiques. Un couplage de ces deux modèles (activation lymphocytaire T et prolifération virale) permet une approche de simulation de la réponse lymphocytaire T spécifique à une infection virale.

<p>Tularemia is a zoonosis caused by the small, fastidious, gram-negative rod Francisella tularensis that appears over almost the entire Northern Hemisphere. In Sweden, tularemia has appeared mainly in restricted areas in northern parts of central Sweden.</p><p> The disease can be transmitted through several routes: direct contact with infected animals, by vectors, through contaminated food or water or through inhalation of aerosolized bacteria. Distinct clinical forms of the disease are seen, depending on the route of transmission. During the last years, tularemia has emerged in new areas in central Sweden, south of the endemic area. The emergence of tularemia in the County of Örebro prompted the investigations presented in this thesis.</p><p> We performed a case-control study, using a mailed questionnaire, to identify risk factors for acquiring tularemia in Sweden (Paper I). After multivariate analysis, mosquito bites and cat ownership could be associated with tularemia in all studied areas while farming appeared as a risk factor only in endemic areas.</p><p> In Paper II, we evaluated a PCR analysis, targeting the tul4 gene, used on samples from primary lesions in patients with ulceroglandular tularemia. The method performed well, with a sensitivity of 78% and a specifi city of 96%. The clinical characteristics of tularemia in an emergent area in Sweden were studied Paper III), using case fi les and a questionnaire. Of 278 cases of tularemia reported during the years 2000 to 2004, 234 had been in contact with a doctor from the Department of Infectious Diseases at Örebro University Hospital, and were thus included. The ulceroglandular form of the disease was seen in 89% of the cases, with the primary lesion, in most cases, on the lower leg. An overwhelming majority of cases occurred during late summer and early autumn, further supporting transmission by mosquitoes. Erythemas overlying the affected lymph node areas were seen in 19% of patients with forms of tularemia affecting peripheral lymph nodes. Late skin manifestations, of various appearances, were seen in 30% of the cases, predominantly in women. A raised awareness of tularemia among physicians in the county during the course of the outbreak was found, as documented by the development of shorter doctor’s delay and less prescription of antibiotics inappropriate in tularemia.</p><p> Finally, we developed a simplifi ed whole-blood lymphocyte stimulation test, as a diagnostic tool in tularemia (Paper IV). The level of IFN-γ, as a proxy for lymphocyte proliferation, was measured after 24-h stimulation. Additionally, a tularemia ELISA with ultra-purifi ed LPS as the antigen was evaluated, showing a high sensitivity. The lymphocyte stimulation test, when performed on consecutive samples from subjects with ongoing tularemia was able to detect the disease earlier in the course of the disease than both the new ELISA and the tube agglutination test. Furthermore, all tularemia cases became positive in the lymphocyte stimulation test within 12 days of disease. In conclusion, this thesis describes risk factors for acquiring tularemia as well as the clinical characteristics of the disease in Sweden. Additionally, a Francisella PCR analysis and a tularemia ELISA based on highly purifi ed LPS is evaluated, and a simplified lymphocyte stimulation test, for early confirmation of the disease, is developed.</p><p>Henrik Eliasson, Department of Infectious Diseases,</p><p>Örebro University Hospital, SE-701 85 Örebro, Sweden,</p><p>henrik.eliasson@orebroll.se</p>

<p>The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment. </p><p>We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002. </p><p>In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes. </p><p>The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.</p>

<p>Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined.</p><p>In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to > 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells.</p><p>Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease.</p><p>Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response.</p><p>Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins.</p><p>In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.</p>

Graduation date: 1994

Le polymorphisme et le polygénisme des complexes majeurs d’histocompatibilité (CMH) limitent les succès de la transplantation. En effet, les disparités, tant d’antigènes mineurs que majeurs, exposent le patient transplanté au risque de rejet et imposent l’administration d’un traitement immunosuppresseur. Ce dernier affecte de façon non spécifique l’ensemble des réponses immunitaires et augmente le risque d’infections mortelles et de cancers. En outre, ce traitement ne semble pas prévenir le rejet chronique. Des découvertes récentes ont confirmé l’existence de lymphocytes appelés régulateurs (Tregs) dont le rôle est de garantir l’homéostasie des réponses immunes afin qu’elles ne deviennent incontrôlées et pathologiques. Les Tregs classiquement décrits expriment de manière constitutive l’antigène CD4+, la chaîne alpha du récepteur de l’interleukine (IL)-2 (CD25) et le facteur de transcription Foxp3. Ils représentent 5 à 10% des lymphocytes CD4+ totaux. Les Tregs sont capables de réguler des lymphocytes alloréactifs et ont été décrits comme responsables du maintien de la tolérance d’allogreffe chez la souris. Mais jusqu'alors, les modèles employés pour démontrer l'importance des Tregs en transplantation utilisaient soit un traitement immunosuppresseur transitoire, soit des transferts de cellules T dans des souris lymphopéniques. Toutefois, ces derniers ne permettent pas de distinguer l'effet des Tregs sur la prolifération homéostatique des lymphocytes effecteurs de leur effet sur la réponse allogénique. Dans notre travail, nous montrons que les Tregs jouent un rôle prépondérant dans l’acceptation spontanée d’allogreffes en l’absence d’immunosuppresseur et en dehors d’un contexte lymphopénique chez la souris. En effet, la déplétion des Tregs du receveur par l’administration d’anticorps anti CD25 amplifie les réponses allogéniques de type Th1 et Th2 et, par conséquent, déclenche le rejet d’allogreffe. Les propriétés régulatrices des Tregs ne sont cependant pas illimitées. En effet, dans un second travail, nous décrivons, d’une part, leur incapacité à contrôler la production d’IL-17 par des lymphocytes CD4+CD25pos mémoires et, d’autre, part leur implication directe dans la différenciation de cellules Th17 au départ de lymphocytes CD4+CD25neg alloréactifs. Nous concluons donc que si les Tregs naturellement présents chez le receveur jouent un rôle primordial dans la protection du greffon contre des réponses de type Th1 ou Th2, ils pourraient néanmoins favoriser une voie alterne du rejet d’allogreffe dépendante de l’IL 17. / Major histocompatibility complex (MHC) polymorphism is a major hindrance to transplantation success. Both minor and major antigen disparities between donor and recipient increase the risk of transplant rejection. This is thwarted by the administration of an immunosuppressive therapy that unspecifically affects all immune responses therefore increasing the risk of infections and cancers. Besides, this treatment does not seem to prevent chronic rejection. Recent studies have confirmed the existence of lymphocytes called regulatory T cells (Tregs), whose role is to maintain the general immune homeostasis and to protect the individual from autoimmune diseases. The classically described Tregs express constitutively the CD4 antigen, the alpha chain of the interleukin (IL)-2 receptor (CD25) and the transcription factor Foxp3. They represent 5 to 10% of total CD4+ T cells. Tregs are able to control alloreactive responses and were described to be responsible for the maintenance of allograft tolerance in mice. So far, the tolerogenic capacities of Tregs have been demonstrated either in mice treated with immunomodulatory antibodies (induced Tregs) or by adoptive co-transfer of Tregs and effector cells into lymphopenic mice. However, the latter has the disadvantage of not being able to distinguish the effect of Treg on lymphopenia-induced homeostatic proliferation from their effect on alloreactive responses. Herein, we show that Tregs play a crucial role in spontaneously accepted allografts in the absence of immunosuppressive therapy and in non-lymphopenic condition. Indeed, the depletion of the recipient’s Tregs through the administration of an anti-CD25 antibody enhances type Th1 and type-Th2 allogeneic responses, consequently triggering allograft rejection. However, the regulatory properties of Tregs are not unlimited. Indeed, we found that Tregs are unable to control allogeneic IL-17 production by memory CD4+ T cells and are even necessary for de novo Th17 differentiation. We conclude, therefore, that Tregs naturally present in the recipient play a critical role in protecting the allograft. Nevertheless, despite this context of regulation, IL-17-producing alloreactive T cells, beyond the control of Tregs, could mediate an alternative pathway of allograft rejection.

Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined. In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to &gt; 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells. Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease. Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response. Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins. In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.

The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment. We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002. In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes. The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.