Interleukin-6 receptor blockade enhances CD39+ regulatory T cell development in rheumatoid arthritis and in experimental arthritis.

OBJECTIVE: The rationale for blocking interleukin-6 (IL-6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti-IL-6 receptor (IL-6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti-IL-6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA. METHODS: Mice with collagen-induced arthritis (CIA) were treated with a mouse anti-IL-6R antibody (MR16-1), and changes in Treg, Th1, and Th17 cells were assessed at key time points during the course of the disease. Peripheral blood from 15 RA patients was collected on day 0 and after 3 months of tocilizumab treatment for flow cytometry analysis of Th17 and Treg cells. RESULTS: In MR16-1-treated mice, Th17 cell frequencies were unchanged, whereas Treg cell frequencies were increased. The Treg cell phenotype showed marked changes, with an increase in the frequency of CD39+ Treg cells in the lymph nodes and spleen. Interestingly, similar CD39+ Treg cell expansion was observed in RA patients who were tocilizumab responders at 3 months, with no change in Th17 cell frequency. Moreover, fluorescence-activated cell-sorted CD39+ Treg cells from responder RA patients were functionally able to suppress the proliferation of conventional T cells. CONCLUSION: In both CIA and RA, the frequency of functionally suppressive CD39+ Treg cells is increased as a result of anti-IL-6R treatment, whereas Th17 cells are unaffected. The modification of Treg cell frequency and phenotype may be one of the mechanisms involved in the therapeutic effect of IL-6 blockade in RA.

Protection from articular damage by passive or active anti-tumour necrosis factor (TNF)-α immunotherapy in human TNF-α transgenic mice depends on anti-TNF-α antibody levels.

Active anti-tumour necrosis factor (TNF)-α immunization with the kinoid of TNF-α (TNF-K) induces polyclonal anti-TNF-α antibodies and ameliorates arthritis in human TNF-α (hTNF-α) transgenic mice (TTg). We compared the efficacy of TNF-K to that of infliximab (IFX) and of TNF-K and IFX co-administration, and evaluated whether the titres of anti-hTNF-α antibodies induced by immunization were a determinant of TNF-K efficacy. Forty-eight TTg mice received one of the following treatments: TNF-K immunization (TNF-K group); weekly IFX throughout the study duration (IFXw0-15); TNF-K plus weekly IFX for 4 weeks (TNF-K + IFX); and weekly IFX for 4 weeks (IFXw0-4); PBS. Animals were killed at week 16. Anti-hTNF-α antibody titres and clinical and histological scores were compared. All TNF-K immunized mice (TNF-K and TNF-K + IFX) produced anti-hTNF-α antibodies. Titres were higher in TNF-K versus TNF-K + IFX (P < 0·001) and correlated inversely with histological inflammation (R = -0·78; P = 0·0001) and destruction (R = -0·67; P = 0·001). TNF-K + IFX had higher histological inflammation and destruction versus TNF-K (P < 0·05). A receiver operating characteristic (ROC) analysis of anti-hTNF-α antibody titres identified the criterion cut-off value to discriminate most effectively between the TNF-K and TNF-K + IFX groups. Mice with high versus low titres had less histological inflammation and destruction (P < 0·05). In a model of TNF-α-dependent arthritis, protection from articular damage by TNF-K correlates with the titres of induced anti-hTNF-α antibodies. The co-administration of TNF-K and a short course of infliximab does not result in less articular damage versus solely TNF-K, due probably to lower anti-hTNF-α antibody production. These results are relevant for future development of active anti-TNF-α immunization in human disease.

Screening for latent tuberculosis in anti-TNF-α candidate patients in a high tuberculosis incidence setting.

BACKGROUND: Screening for latent tuberculosis infection (LTBI) using a protocol comprising chest X-ray and tuberculin skin test (TST) interpreted with medical history, Sc1, reduces LTBI reactivation on treatment with anti-tumour necrosis factor-alpha (anti-TNF-α). In the district of Seine-Saint-Denis, France, where tuberculosis (TB) incidence ranges from 30 to >100/100 000 person-years, however, Sc1 might be insensitive as a screening tool. We adopted another protocol, Sc2, comprising Sc1 plus two additional tests: the QuantiFERON(®)-TB Gold In-Tube (QFT-GIT) and chest computed tomography (CT). METHODS: We screened 123 consecutive patients with inflammatory rheumatic diseases (IRDs), candidates for anti-TNF-α treatment, and evaluated the impact of Sc2 vs. Sc1 on the prescription of prophylactic anti-tuberculosis treatment. RESULTS: Sc2 led to a diagnosis of LTBI in 69 patients vs. 59 when using Sc1: eight were QFT-GIT-positive. Diagnosis was based on CT findings in two patients. QFT-GIT had higher diagnostic accuracy than TST, but no single diagnostic test could detect all patients at high risk for LTBI reactivation (respectively 30.2% and 37.5% of patients positive with only TST or QFT-GIT). CT detected TB sequelae in 3/46 rheumatoid arthritis patients who were negative to all tests. CONCLUSIONS: Testing with both TST and QFT-GIT seems the safest strategy for detecting LTBI in patients with IRD from populations with high incidence of TB. Systematic screening with CT warrants further evaluation.

Early and long-lasting protection from arthritis in tumour necrosis factor alpha (TNFalpha) transgenic mice vaccinated against TNFalpha.

OBJECTIVE: To evaluate the effect in mice with arthritis of active anti-tumour necrosis factor (TNF)alpha immunotherapy based on a keyhole limpet haemocyanin-human TNFalpha heterocomplex (hTNFalpha kinoid or TNFK) adjuvanted in incomplete Freund adjuvant. Immunotherapy was evaluated also with methotrexate. METHODS: Human TNFalpha-transgenic mice received TNFK with or without methotrexate. Follow-up ranged from 6 weeks (short term) to 17 weeks (long term). Arthritis was evaluated clinically and histologically. Monitoring included titration of anti-hTNFalpha antibodies by ELISA and neutralisation assay. RESULTS: Vaccination with TNFK was associated with rapid-onset, long-lasting protection. Long-term results showed significantly milder arthritis in vaccinated animals than in control animals at the peak of the disease. Vaccination was followed by resolution of the clinical evidence of arthritis, contrasting with severe progressive arthritis in the control group. Histological improvements with decreased inflammation and destruction were noted in all immunised groups, even after the shortest follow-up (6 weeks). High titres of neutralising anti-hTNFalpha antibodies were detected as early as the fifth week post immunisation and persisted over time. Methotrexate given concomitantly with the vaccine did not influence either the effect on arthritis or the anti-hTNFalpha antibody titres. CONCLUSION: Anti-cytokine induction of autoimmune protection against chronic hTNFalpha overproduction is an efficient alternative to TNFalpha blockade in experimental arthritis and can be achieved using a TNFK vaccine.

Intra-articular electrotransfer of plasmid encoding soluble TNF receptor variants in normal and arthritic mice.

BACKGROUND: Anti-inflammatory gene therapy is promising in inflammatory diseases such as rheumatoid arthritis (RA). We have previously demonstrated that intra-muscular (i.m.) electrotransfer (ET) of plasmids encoding three different human tumor necrosis factor-alpha-soluble receptor I variants (hTNFR-Is) exert protective effects in an experimental RA model. However, such a systemic approach could be responsible for side effects. The present study aimed at performing an intra-articular (i.a.) gene therapy by electrotransfer using the hTNFR-Is plasmids. METHODS AND RESULTS: We evaluated targeting of mice joints by CCD optical imaging after i.a. ET of a luciferase-encoding plasmid and we showed that ET led to strongly increased transgene expression in a plasmid dose-dependent manner. Moreover, articular and seric hTNFR-Is was detectable for 2 weeks. As expected, systemic hTNFR-Is rates were lower after i.a. ET than after i.m. ET. A longer protein secretion could be achieved with several i.a. ETs. Also, we observed that hTNFR-Is expression within arthritic joints was slightly higher than in normal joints. CONCLUSIONS: In collagen-induced arthritis (CIA), a mouse model for RA, we demonstrated that hTNFR-Is/mIgG1-encoding plasmid i.a. ET decreased joint destruction in the ankles. In conclusion, our results suggest that local TNFR-Is gene therapy may play a role in decreasing joint destruction in CIA.

Synoviocyte infection with adeno-associated virus (AAV) is neutralized by human synovial fluid from arthritis patients and depends on AAV serotype.

Intraarticular gene transfer with adeno-associated viral (AAV) vectors may allow efficient therapeutic transgene expression within the joint in diseases such as rheumatoid arthritis (RA), allowing high expression of the protein within the joint, preventing both systemic diffusion and side effects. However, humans demonstrate antibodies against AAV, which can influence gene transfer. To better understand critical obstacles to intraarticular gene therapy with AAV, we have previously shown that synovial fluid (SF) contains IgG to AAV that neutralizes chondrocyte infection in vitro. Our objective was therefore to compare neutralization exerted by SF from RA patients for four different AAV serotypes (AAV serotypes 1, 2, 5, and 8) on human primary synoviocytes. Serotype 2 infected synoviocytes most efficiently followed, in decreasing order, by serotypes 1, 5, and 8. SF from all patients partially inhibited infection of synoviocytes by at least one of the four serotypes. Infection with serotypes 1 and 2 was the most inhibited by SF, whereas inhibition was weak for serotypes 5 and 8. Last, we have shown that inhibition of AAV1/interleukin (IL)-4 infection of synoviocytes by SF could be reversed by increasing the number of AAV1/IL-4 particles, with a dose-dependent effect. We conclude that the most infectious AAV serotypes (1 and 2) in synoviocytes are also the serotypes most neutralized by SF. Thus, serotype 5 seems to demonstrate the best infection efficiency:immunogenicity ratio for local use in articular diseases. These data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

TNF-alpha antibodies and osteoprotegerin decrease systemic bone loss associated with inflammation through distinct mechanisms in collagen-induced arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is associated with focal and systemic bone loss involving cytokines such as RANKL and TNF-alpha. RANK-L promotes focal and systemic osteoporosis, whereas osteoprotegerin (OPG) inhibits bone resorption. Although anti-TNF-alpha antibodies (anti-TNF-alpha Ab) decrease joint inflammation and bone erosions, their effects on bone loss are unknown. The aim of this study was to evaluate the effects of OPG and anti-TNF-alpha Ab, separately or in combination, on inflammation and bone remodeling in collagen-induced arthritis (CIA), a model of RA. METHODS: DBA/1 mice (n=28) were immunized with bovine type II collagen and treated with OPG-Fc or anti-TNF-alpha Ab or both, or saline. One group of mice (n=7) was not immunized (naive group). Urinary deoxypyridinoline (D-pyr) and whole-body bone mineral density (BMD) were measured at baseline and at sacrifice. Histomorphometric parameters were evaluated at the femoral metaphysis. RESULTS: Anti-TNF-alpha Ab, but not OPG, decreased the clinical arthritis score (P<0.02 vs. saline) and the histological score of inflammation. The BMD change from baseline to sacrifice (DeltaBMD) was significantly smaller in CIA mice than naive mice. OPG and anti-TNF-alpha Ab significantly increased DeltaBMD versus saline, and the effect was greater with OPG (P<0.003). DeltaD-pyr decreased by 65% with OPG and 13% with anti-TNF-alpha Ab. Compared with saline, OPG increased trabecular bone volume (BV/TV) (P<0.02), decreased trabecular separation (P<0.02), and decreased the bone formation rate (BFR) (P<0.01). Anti-TNF-alpha Ab produced no significant changes in bone volume or trabecular separation but increased trabecular thickness (P<0.02 vs. saline) to a value close to that in naive mice, suggesting preservation of bone formation. No additive effects of OPG and anti-TNF-alpha Ab were found. CONCLUSIONS: Systemic OPG and anti-TNF-alpha Ab therapy prevented bone loss in CIA mice through distinct mechanisms involving decreased bone resorption and preserved bone formation. Combining these two agents might help to prevent bone loss in inflammatory diseases.

Immune responses to gene therapy vectors: influence on vector function and effector mechanisms.

Circumventing the immune response to the vector is a major challenge with all vector types. Viral vectors are the most likely to induce an immune response, especially those, like adenovirus and AAV, which express immunogenic epitopes within the organism. The first immune response occurring after vector transfer emerges from the innate immune system, mainly consisting in a rapid (few hours) inflammatory cytokines and chemokines secretion around the administration site. This reaction is high with adenoviral vectors and almost null with AAV. It is noteworthy that plasmid DNA vectors, because of CpG stimulatory islets, also stimulate the innate immunity via the stimulation of TLR receptors on leukocytes. Specific immune response leading to antibodies production and T lymphocytes activation also occurs within a few days after vector introduction. Capsid antigens are mostly responsible for specific immunity toward adenoviruses, and are also involved in the response against AAV. In the former case only, however, viral gene-encoded proteins can also be immunogenic. The pre-existing humoral immunity coming from early infections with wild-type AAV or adenovirus can prevent efficient gene transfer with the corresponding vectors. In all cases, some parameters like route of administration, dose, or promoter type have been extensively described as critical factors influencing vector immunity. Strategies to fight against vector-induced immunity can come from the immunology field, since tolerance induction or immunosuppression are a possibility. Alterations to vector structure have also been extensively performed to circumvent the immune system and thus enhance gene transfer efficiency and safety.

Active immunization against murine TNFalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection.

New lines of treatment targeting cytokines have been successfully developed recently and are now widely used in therapy. They are based on passive administration of cytokine inhibitors either soluble receptors or mAbs and the major example is TNFalpha in rheumatoid arthritis (RA). Since a few years, our group has developed a novel alternative approach targeting cytokines by using active immunization against biologically inactive but immunogenic cytokine derivatives. In the present work, we present a new aspect of this research, based on immunization against specific cytokine peptides chosen by molecular modelling. We could elicit a significant humoral response against four TNFalpha peptides by active immunization, and show that the Abs generated cross-reacted with the native cytokine with good titers as determined by ELISA. Interestingly, during coimmunization experiments with couples of peptides, one showed a clear immunodominant effect over the other. Overall, we could not show the neutralization of TNFalpha biological activity in vitro by the immunized sera, but it seems that it is not a prerequisite to observe clinical efficacy. Indeed, using the LPS/galactosamine-induced shock, we could demonstrate that one of the four peptides tested conferred a clinical protection. These results validate the feasibility and efficacy of active immunization against cytokine peptides, and confirm that active immunization against cytokines could represent in the future an alternative to passive immunization in many diseases.

Therapeutic gene transfer for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF-alpha or IL-1 blocking agents (such as anti-TNF-alpha monoclonal antibodies, soluble TNF-alpha receptor, type II soluble receptor of IL-1, IL-1 receptor antagonist), anti-inflammatory cytokines (such as IL-4, IL-10, IL-1), growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra articular injection) or a systemic treatment (extra articular injection). All the main vectors has been used in experimental models, including the more recent lentivirus and adeno-associated virus. Ex vivo gene transfer was done with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogenic origin. In vivo gene therapy is simpler, although less controlled method. Clinical trials in human RA has started with ex vivo retrovirus expressing IL-1 receptor antagonist and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive researches have to be conducted in pre-clinical studies.

Collagen II-pulsed antigen-presenting cells genetically modified to secrete IL-4 down-regulate collagen-induced arthritis.

We explored the possibility that pulsed antigen-presenting cells (APC) provide a model vector system for site-specific delivery of immunosuppressive proteins during collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. Thus, mice were treated with either B cells or macrophages engineered to secrete IL-4 and loaded (or not) with type II collagen (CII). Systemic injection of an IL-4-producing B cell hybridoma resulted in a reduction of arthritis severity which was further improved when APC were incubated with CII before their transfer. Unmanipulated B cells loaded with CII also exerted a potent suppressive effect. Likely, clinical amelioration was observed in mice given at priming syngeneic bone marrow-derived macrophages producing IL-4 and pulsed with CII in comparison to the other groups. When the same dose of cells was transferred at disease onset, a moderate beneficial effect was observed. Whatever the APC inoculated, the beneficial effect did not rely upon an IL-4-driven shift towards Th2 phenotype. Systemic administration of fluorescent dye labeled macrophages to arthritic mice has shown that some of these cells rapidly migrate to joints. Moreover, IL-4 transfected macrophages retained their potent capacity to present CII peptides to T cells. These findings validate the use of CII peptide-loaded engineered APC as therapeutic vector cells in CIA and allow consideration of this strategy for the administration of various anti-inflammatory proteins.

The type II decoy receptor of IL-1 inhibits murine collagen-induced arthritis.

IL-1 is a key cytokine involved in the inflammatory response. The type II receptor of IL-1 (IL-1RII) acts as a decoy receptor, binding and inhibiting the effect of IL-1. This study was undertaken to establish whether IL-1RII can ameliorate collagen-induced arthritis, a model of inflammatory arthritis in mice. We used human keratinocytes transfected with the human (h)IL-1 RII gene as a source of hIL-1 RII protein. We showed that these cells expressed both the membrane and soluble form of receptor. In vitro, IL-1-stimulated murine macrophage cells showed a decreased expression of TNF-alpha in the presence of hIL-1 RII. We engrafted the hIL-1RII-transfected cells in the back of mice developing collagen-induced arthritis. We found that clinical and histological parameters of arthritis were significantly decreased in mice treated with cells producing hIL-1RII. In addition, hIL-1RII administration was able to reduce the expression of mRNA for IL-6 and myeloperoxidase in the joints of treated animals. These data show that hIL-1 RII anti-inflammatory properties in the model of collagen-induced arthritis in mice and could have a regulatory role in rheumatoid arthritis.

Adeno-associated virus-mediated delivery of IL-4 prevents collagen-induced arthritis.

Immunomodulation of autoimmune inflammatory diseases like rheumatoid arthritis can be achieved by anti-inflammatory T2 cytokines such as interleukin (IL)-4 administered by gene therapy. In this study we investigated the efficiency of adeno-associated viruses (AAV) vectors in collagen-induced arthritis (CIA). After injection of AAV-LacZ in the tarsus area of mice, the expression of the transgene was localized in the deep muscles cells near the bone. LacZ expression was found in liver, heart and lung after i.m. injection of AAV-LacZ, showing a spread of the vector over the body. Anti-AAV neutralizing antibodies were detected in the serum after i.m. injection of AAV-LacZ, but they did not alter the transgene expression after re-administration of AAV-LacZ. Long-term IL-4 expression persisted 129 days after intra-muscular injection of 3.7 x 10(10) or 11.2 x 10(10) AAV-IL-4 p.p. (average 7.7 or 17.5 pg IL-4/mg proteins, respectively). More importantly, the treatment of CIA with AAV-IL-4 vector in mice produced a therapeutic benefit, since we show a diminished prevalence of the disease, a significant reduction in paw swelling, attenuated histological synovitis and a 10 days delayed onset of arthritis. This is the first evidence that AAV vector-mediated gene therapy using a T2 cytokine is efficient in an animal model of rheumatoid arthritis.

Expression of Fas ligand improves the effect of IL-4 in collagen-induced arthritis.

The present study was aimed at investigating whether the expression of Fas ligand (FasL) by CHO cells transfected with IL-4 (CHO/IL-4) or IL-10 (CHO/IL-10) genes would improve the effect of the cytokine. DBA/ 1 mice immunized with type II collagen were treated with suboptimal doses of transfected CHO cells (a single s. c. injection of 2 x 10(5) cells) around onset of arthritis. Severe collagen-induced arthritis (CIA) developed in the control groups injected with PBS, CHO /beta-galactosidase/FasL, CHO/IL-4 or CHO/IL-10 cells. In contrast, administration of CHO/IL-4/FasL, but not CHO/IL-10/FasL, cells significantly reduced the clinical severity and resulted in rapid and sustained suppressive effect. Amelioration of CIA was not due to a prolonged in vivo secretion of IL-4 since expression of FasL by CHO cells shortened the in vivo survival of the xenogeneic cells. In fact, administration of FasL(+) cells was associated with a decreased proportion of Mac1(+) neutrophils in the blood and an increased expression of myeloperoxidase at the site of engineered cell engraftment. These findings suggest that the mechanism underlying the beneficial effect of IL-4 delivered by cells expressing FasL involves the combination of the anti-inflammatory properties of IL-4 and the apoptosis of Fas(+) Mac1(+) granulocytes participating in the pathogenic process.

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-alpha)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13.

TNF-alpha is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-alpha transgene modified with the 3' untranslated region of beta-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-alpha-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4weeks old. Control groups of transgenic mice were engrafted with beta-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-alpha transgene, endogenous mouse TNF-alpha and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3weeks of treatment (P<0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-alpha transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.

Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.

The anti-inflammatory effects of the recently identified cytokine interleukin (IL)-13 on collagen-induced arthritis (CIA) was explored and compared to those of IL-4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/I mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL-13 or IL-4 genes were inoculated subcutaneously on days 10 and 25 post-priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animal. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL-13 and IL-4 gene-transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase-polymerase chain reaction at different times after immunization. Our data show that IL-13-induced suppression of CIA coincided with a decreased TNF-alpha mRNA expression in the spleen of treated animals. This may explain at least partially the anti-inflammatory effects of IL-13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/TH2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.

T cell regulation of collagen-induced arthritis in mice. III. Is T cell vaccination a valuable therapy?

Since T cells play a critical role in collagen-induced arthritis (CIA), CD4+ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy-1-2+, CD4+, CD8-, T cell receptor (TcR) alpha beta + and produced interleukin-2 in response to CII peptides presented by I-Aq molecules. The clones were collagen type-specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide-cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical V alpha and J alpha (V alpha BMB, J alpha 20) gene segments and two distinct V beta (V beta 1 and V beta 4) associated with the J beta 2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both alpha and beta chains of the TcR. Inoculation of irradiated anti-CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long-lasting suppression or an exacerbation of CIA whereas a control CD4+ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti-CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen.

Injection of native type II collagen (CII) to susceptible strains of mice (H-2q) induces a rheumatoid arthritis-like disease. To study the role of CD8+ T cells in the collagen-induced arthritis (CIA), we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H. Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CII-pulsed macrophages and recognize different antigenic epitopes in association with Kq molecules. When these T cell clones were irradiated and inoculated into (C3H.Q x AKR)F1 mice 21 days prior to priming with native CII/complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.

[Acute renal insufficiency associated with lymphoma. A new case]. / Insuffisance rénale aiguë d'origine lymphomateuse. Une nouvelle observation.

We report on a case of acute renal failure secondary to relapse of a centrocytocentroblastic non-Hodgkin's lymphoma. Twenty-seven cases of acute renal failure due to lymphomatous infiltration of the kidneys have been so far reported. The clinical presentation is non-specific; the size of the kidneys appears normal or enlarged; diagnosis is confirmed by renal biopsy and/or CT scan examination. Chemotherapy and/or radiotherapy sometimes lead to dramatic improvement of renal function. However, prognosis is poor, mostly due to lymphomatous evolution.