Restricted immunoglobulin variable region (Ig V) gene expression accompanies secondary rearrangements of light chain Ig V genes in mouse plasmacytomas.

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

Chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia is the commonest form of leukaemia in Europe and North America, and mainly, though not exclusively, affects older individuals. It has a very variable course, with survival ranging from months to decades. Major progress has been made in identification of molecular and cellular markers that could predict disease progression in patients with chronic lymphocytic leukaemia. In particular, the mutational profile of immunoglobulin genes and some cytogenetic abnormalities are important predictors of prognosis. However, these advances have raised new questions about the biology, prognosis, and management of chronic lymphocytic leukaemia, some of which are addressed here. In particular, we discuss how better understanding of the function of the B-cell receptor, the nature of genetic lesions, and the balance between proliferation and apoptosis have affected our ability to assess prognosis and to manage chronic lymphocytic leukaemia. Available treatments generally induce remission, although nearly all patients relapse, and chronic lymphocytic leukaemia remains an incurable disease. Advances in molecular biology have enhanced our understanding of the pathophysiology of the disease and, together with development of new therapeutic agents, have made management of chronic lymphocytic leukaemia more rational and more effective than previously. Unfortunately, we know of no way that chronic lymphocytic leukaemia can be prevented. Early detection is practised widely, but seemingly makes no difference to the patient's eventual outcome.

Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies?

Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.

Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors.

To better understand the stage(s) of differentiation reached by B-type chronic lymphocytic leukemia (B-CLL) cells and to gain insight into the potential role of antigenic stimulation in the development and diversification of these cells, we analyzed the rearranged VH genes expressed by 83 B-CLL cells (64 IgM+ and 19 non-IgM+). Our results confirm and extend the observations of a bias in the use of certain VH, D, and JH genes among B-CLL cells. In addition, they indicate that the VH genes of approximately 50% of the IgM+ B-CLL cells and approximately 75% of the non-IgM+ B-CLL cells can exhibit somatic mutations. The presence of mutation varies according to the VH family expressed by the B-CLL cell (VH3 expressers displaying more mutation than VH1 and VH4 expressers). In addition, the extent of mutation can be sizeable with approximately 32% of the IgM+ cases and approximately 68% of the non-IgM+ cases differing by > 5% from the most similar germline gene. Approximately 20% of the mutated VH genes display replacement mutations in a pattern consistent with antigen selection. However, CDR3 characteristics (D and JH gene use and association and HCDR3 length, composition, and charge) suggest that selection for distinct B cell receptors (BCR) occurs in many more B-CLL cells. Based on these data, we suggest three prototypic BCR, representing the VH genes most frequently encountered in our study. These data suggest that many B-CLL cells have been previously stimulated, placing them in the "experienced" or "memory" CD5(+) B cell subset.

What do somatic hypermutation and class switch recombination teach us about chronic lymphocytic leukaemia pathogenesis?

B-CLL cells express CD5 and IgM/IgD and thus have a mantle zone-like phenotype of naive cells, which, in normal conditions express unmutated Ig genes. However, recent studies have shown that 50%-70% of CLL harbour somatic mutations of VH genes, as if they had matured in a lymphoid follicle. Interestingly, the presence or absence of somatic hypermutation (SHM) process is associated with the use of particular VH genes. Particular alleles of the VH1-69 gene and the VH4-39 gene are preferentially expressed in an unmutated form, while VH4-34 or the majority of VH3 family genes frequently contain somatic mutations. The fact that some genes like VH1-69 and VH3-07 recombine this VH segment to particular JH segments and the restricted use of CDR3 sequences by CLLs expressing the VH4-39 gene suggest that the observed differences in BCR structure in B-CLL could result from selection by distinct antigenic epitopes. It is currently unclear whether this putative antigen-driven process could occur prior to leukaemic transformation and/or that the precursors were transformed into leukaemic cells at distinct maturational stages. The mutational profile of Ig genes has been shown to be associated with disease prognosis. These results could favour the idea that CLL could correspond to two different diseases that look alike in morphologic and phenotypic terms. In CLL with mutated Ig genes, the proliferating B cell may have transited through germinal centres, the physiologic site of hypermutation, whereas in CLL with unmutated Ig genes the malignant B cell may derive from a pre-germinal centre naïve B cell. Despite these clinical and molecular differences, recent studies on gene expression profiling of B-CLL cells showed that CLL is characterized by a common gene expression signature that is irrespective of Ig mutational status and differs from other lymphoid cancers and normal lymphoid subpopulations, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Activation induced cytidine deaminase (AID) plays a key role in SHM and class switch recombination (CSR). However, the mechanisms accounting for AID action and control of its expression remain unclear. In a recent work we have shown that in contrast to normal circulating B-cells, AID transcripts are expressed constitutively in CLL patients undergoing active CSR, but interestingly this expression occurs predominately in unmutated CLL B-cells. These data favour the view that AID protein may act differentially on CSR and SHM pathways, but the role-played by AID in both processes remains to be elucidated. Recent work indicates that AID is expressed in a small fraction of tumoral cells, which could suggest that this small fraction of cells may correspond to B-CLL cells that would have recently experienced an AID-inducing stimulus occurring in a specific microenvironment.

Recent progress in chronic lymphocytic leukemia. International Workshop on chronic Lymphocytic Leukemia.

The data we discuss indicate substantial recent progress in understanding and treating CLL. However, despite considerable new information, many of the intriguing issues we posed at previous IWCLL meetings remain unanswered. Prominent among these are the questions of what causes CLL, what is the relation between CLL and normal B-cell development, are T-cell abnormalities a cause of consequence of CLL, why are auto-immune features so prominent and how is CLL best treated? Although these gaps in our knowledge are unfortunate, they give us the opportunity for yet another IWCLL meeting: 1996 in Greece. More to follow.

Predictive value of serum thymidine kinase level for Ig-V mutational status in B-CLL.

In B-CLL IgV(H) genes mutational status is a major prognostic factor. Since sequencing of IgV(H) genes is not available in most laboratories, an easily performed surrogate assay is desirable. To identify the best surrogate assay, and to better discriminate prognostic subgroups we analyzed clinical and biological data from 58 typical CLL cases. A higher serum thymidine kinase level (>15 U/l) proved to be a strong predictor of mutational status, and the only independent one among the studied parameters. To further identify prognostic subgroups, cluster analysis was employed on 38 cases on which all data were available, which segregated two groups including 25 and 13 patients, respectively. These two clusters differed by their proliferative potential and appeared to discriminate patients with very different clinical course and outcome. s-TK was strikingly different among these two clusters, suggesting that s-TK level could be used routinely to identify patients at risk of progression.

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells.

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete V(H) nucleotide sequence and the presence of RNA transcripts from different C(H) domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions.

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

Immunochemical studies of polyspecific natural autoantibodies: charge, lipid reactivity, Fab'2 fragments activity and complement fixation.

Polyspecific natural autoantibodies (NAAb) are antibodies present in normal unimmunized animals and are able to react with very dissimilar antigens (Ag). To better delineate the characteristics of polyspecificity, we subjected monoclonal NAAb to four different immunochemical studies: (1) Two-dimensional gel electrophoresis performed on eight NAAb did not reveal any obvious relationship between charge and antigen specificities; (2) NAAb widely polyspecific on proteins and nucleic acid were reactive with lipids bearing either phosphate, sulfate or carboxyl polar groups; (3) pepsin digestion of polyspecific IgM NAAb yielded Fab'2 fragments which maintained their multireactivities, but exhibited a decrease in reactivity as compared to that seen with monospecific mAb (induced); (4) two different assays were used to analyse the complement fixation ability of IgM NAAb. While very weak or no complement fixation was observed with a classical complement fixation test (fluid phase), when a complement enzyme immunoassay was used where Ag is immobilized on a solid phase, polyspecific NAAb fixed reproducible and easily detectable amounts of complement.

Evidence for an antigen-driven selection process in human autoantibodies against acetylcholine receptor.

Autoantibodies to the nicotinic acetylcholine receptor (AChR) play a central role in the neurological symptoms associated with myasthenia gravis (MG). A better knowledge of the structural organization and of the mechanisms leading to the production of these antibodies may help in understanding the pathogenesis of the disease. To achieve this, four IgG anti-AChR monoclonal autoantibodies obtained in a previous work were derived from lymphoid cells of MG patients. Two of them (MH1 and MH6) were capable of modulating in vitro the expression of AChR at the surface of TE-671 cells. We report here the complete nucleotide sequence of the heavy and light chains of these four antibodies. Although it is difficult to address the issue of VH gene usage in anti-AChR autoantibodies because of the limited number of clones studied, our results associated with others which have appeared in the literature point to non-stochastic usage by anti-AChR antibody of some defined VH genes belonging to VH2 and VH5 minifamilies overexpressed in the fetal repertoire. The second and major aim of this work was to assess the role of an antigen-driven selection process in the production of anti-AChR autoantibodies. When comparing the expressed sequences to their closest germline counterparts, it appeared that all four studied clones displayed numerous mutations in VH regions. In particular, MH1 and MH6, characterized by their AChR modulating capacity, displayed a higher than expected number of mutations and replacements occurring in CDR regions. These data point to an antigen-driven selection process. On the contrary, the mutational process observed in the MH% clone was borderline and that of MH7 was compatible with a random process. Interestingly, when comparing mutations in heavy and light chains, a significantly lower number of mutations were expressed in light chains for the four clones.

Activation of the PI3K/AKT pathway by microRNA-22 results in CLL B-cell proliferation.

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal B cells arrested in G0/G1 stages that coexist, in different proportions, with proliferative B cells. Understanding the crosstalk between the proliferative subsets and their milieu could provide clues on CLL biology. We previously identified one of these subpopulations in the peripheral blood from unmutated patients that appears to be a hallmark of a progressive disease. Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mRNA and microRNA expression of this leukemic subpopulation, and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microRNA-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that miR-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-Kip1) and overexpression of Survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors.

Basic biology of autoimmune phenomena in chronic lymphocytic leukemia.

There is evidence indicating that autoreactive B cells constitute a substantial part of the B cell repertoire. This autoreactive repertoire secretes the so-called natural autoantibodies characterized by their broad reactivity mainly directed against well-conserved public epitopes. Their germinal origin is suggested by their early appearance during ontogeny, their expression of cross-reactive idiotopes, and structural studies of their sequence. As for the physiological role of the repertoire, it may play a major role as a first barrier of defense. It is presently unknown whether these polyreactive B cells could constitute a pre-immune template which, through an antigen-driven process, may be involved in the production of immune high-affinity antibodies. This autoreactive B cell repertoire frequently undergoes malignant transformation, although there is controversy concerning the reasons accounting for this. It has been postulated that the continuous challenge of this autoreactive repertoire by self-antigens could create propitious conditions for malignant transformation to occur. However, this hypothesis still needs to be substantiated. Chronic lymphocytic leukemia (CLL), the most frequent form of leukemia in western countries, is characterized by constant expression of the CD5 marker and low expression of surface membrane immunoglobulin (Ig) in B lymphocytes. CLL B lymphocyte is frequently committed for natural autoantibody secretion. Despite expressing the Epstein-Barr virus (EBV) receptor CLL B cells cannot be infected by the EBV virus, they overexpress the bcl-2 protein and they are unable to adequately respond when stimulated through the B cell receptor pathway. Autoimmune-associated phenomena are frequently observed in B-cell CLL. These autotoxic manifestations are mainly directed against hematopoietic cells. In most cases, autoantibodies against red blood cells are warm reactive polyclonal IgG. Immune thrombocytopenia is observed in about 2% of cases, but higher frequencies of increased platelet associated Igs have been reported. Pure red cell aplasia and autoantibodies against neutrophils are only rarely observed. This pattern is similar to that observed in primary immunodeficiency syndromes, in which immune thrombocytopenia, autoimmune hemolytic anemia, and pure red cell aplasia are frequently observed. The potential role of T cell defects in inducing autoimmune complications in B-cell CLL has been stressed by recent publications showing increased frequency of autoimmune hemolytic anemia in patients treated with purine nucleoside analogues. However, evidence is presently scarce concerning a functional impairment of T cells after administration of these drugs.

Flow cytometric analysis of protein-tyrosine phosphorylation in peripheral T cell subsets. Application to healthy and HIV-seropositive subjects.

This paper describes an improved method for detecting tyrosine phosphorylation levels in T cell subsets by flow cytometry early after CD3 crosslinking stimulation. It consists in introducing gentle paraformaldehyde fixation between CD3 crosslinking in cold conditions and the shift to 37 degrees C, which activates downstream signalling machinery. We used the combined properties of monoclonal antibodies for stimulating cells and for detecting surface markers to analyze protein-tyrosine phosphorylation levels in T cells subsets following stimulations which mimic physiological activation. Overall data obtained in healthy subjects, using two- or three-color immunofluorescence, indicated that: (1) most CD3 positive cells phosphorylate tyrosine substrates following CD3 crosslinking stimulation and (2) helper cells phosphorylate tyrosine to a slightly better extent than cytotoxic cells after CD3 crosslinking. Nevertheless, the two subsets follow similar kinetic patterns and tend to retain a homogeneous profile. Processing of samples from HIV-seropositive patients showed heterogeneous phosphorylation levels in both subsets, when compared to normal donors. This assay should, in the future, lead to easy and rapid exploration of the signal transduction pathway in different subsets of T cells under normal and pathological conditions.

Investigation of a new parameter in chronic lymphocytic leukemia: the percentage of large peripheral lymphocytes determined by the Hemalog D. Prognostic significance.

The number of large peripheral lymphoid cells and the ratio of these large unstained cells to the total number of peripheral lymphocytes was determined by means of the Hemalog D in 57 patients with chronic lymphocytic leukemia (CLL) and in 100 control subjects. Although the absolute number of large unstained cells/mm3 is simply a reflection of peripheral lymphocytosis, the ratio large unstained cells to total lymphocyte count was shown to correlate with clinical staging. In control subjects, this ratio ranged from 3.2 per cent to 11¿per cent. In those with CLL it was less than 11.2 per cent in 43 patients and greater than 11.2 per cent in 14 patients. These 14 patients corresponded statistically to patients with advanced disease in our clinical staging system (stages III and IV). An increase in the large unstained cells to total lymphocyte ratio is therefore a statistical criterion of poor prognosis.

Molecular characterization of a monoclonal murine anti-blood group A antibody.

A mouse monoclonal anti-human blood group A antigen (AC12, mu, kappa) has been generated and sequenced in order to analyze the immunoglobulin genes used to generate antibodies with anti-human blood group A specificity. Mice were immunized with human type A RBC. Anti-A producing hybridomas were detected by agglutination against human type A RBC. Total cellular RNA was extracted from hybridomas cells. PCR amplification and sequencing of anti-A heavy and light chain cDNAs were performed. The VH and VK sequences of antibody AC12 were shown to be very homologous to that used by other antibodies recognizing carbohydrates as well as glycoproteins, peptides or haptens constituting self antigens as well as nonself antigens. The VH sequence of antibody AC12 presented important homology with a previously reported monoclonal anti-blood group B antibody. The antibody AC12 also presented homology with the VH and VK sequences of a previously reported human anti-blood group A antibody which contributes additional evidence in favor of a restricted usage of V segments by antibodies directed against red blood antigens.

Selective depletion of low-density CD8+, CD16+ lymphocytes during HIV infection.

Two color cytofluorometric analyses of CD3-, CD16+, Leu 19+ natural killer cells (NK) were assessed in HIV seropositive patients, high-risk seronegative homosexuals, and healthy heterosexuals. A selective depletion of lymphocytes bearing NK phenotypes was found among HIV-positive infected patients. When the CD16+ lymphocyte compartment was further dissected, lymphoid cells bearing simultaneously low cell-surface density CD8 and CD16 (Leu 11a or Leu 11c) or Leu 19 epitopes were selectively and significantly decreased. This important depletion of CD8+ NK cells, which in most cases are CD3-, accounts for the decline in low-density CD8+ lymphocytes in HIV positive group, while a significant increase occurs in their CTL pool. Furthermore, in HIV negative high-risk homosexuals, a less profound but significant reduction of this lymphocyte subset was also found. Whether the involvement of the NK compartment, especially NK cells expressing the CD8 marker, may influence the outcome of individuals infected with HIV is still an open question.

Serum levels of IL-2, IL-1 alpha, TNF-alpha, and soluble receptor of IL-2 in HIV-1-infected patients.

Serum levels of the interleukins (IL-1 alpha, IL-2), tumor necrosis factor-alpha (TNF-alpha), and soluble receptor of IL-2 (sIL-2R) were studied by enzyme-linked immunosorbent assay (ELISA) in 12 normal healthy controls and 52 HIV-1 seropositive patients. Results indicated that: (1) sIL-2R levels were significantly increased in most HIV-1 seropositive patients. This increase appeared to be correlated with low CD4 cell counts and with the presence of detectable levels of p25 antigen. Furthermore, initially high levels of sIL-2R appeared to be correlated with progression of disease. (2) IL-2 levels were found to be increased in about 43% of asymptomatic carriers (ASY) and subjects with lymphoadenopathy-associated syndrome (LAS) compared with 12% in the case of AIDS-related complex (ARC) and AIDS patients. (3) There was a positive correlation between serum levels of TNF-alpha and IL-1 alpha in nearly all patients. Detectable levels of both cytokines were found in 34% of ASY and LAS patients and only rarely were detectable in ARC and AIDS patients. (4) Sixteen patients in whom progression of disease was observed were studied initially and at the moment they upstaged. No significant modification of serum levels of the three cytokines and sIL-2R studied could be evidenced. It was concluded that sIL-2R could be a useful marker of disease activity and progression, though a prospective study is necessary. For IL-2, IL-1 alpha, and TNF-alpha, this study indicated the presence of variable alterations in serum levels in HIV-1-infected patients.

Platelet antibodies in serum of patients with human immunodeficiency virus (HIV) infection.

Between 10 and 15% of human immunodeficiency virus (HIV) seropositive individuals develop an immune thrombocytopenic purpura; however, the mechanism involved in platelet destruction is not yet established. In the present work, we have analyzed 208 sera from HIV seropositive individuals, including 85 thrombocytopenic patients, for the presence of autoantibodies against platelet proteins by using the Western blot technique. Our results indicate that: (1) antibodies against platelet proteins were found in 8 of 123 (6.5%) nonthrombocytopenic patients, as compared with 17 of 85 (20%) of thrombocytopenic patients (p less than 0.03); (2) these antibodies appeared to be more frequently found in advanced stages of disease (p less than 0.02); (3) the reactivity of positive sera with antigenic determinants implicated several distinct platelet proteins; (4) antigens thus recognized are unrelated to the major membrane glycoproteins IIb and IIIa, as well as absent in vero cells and trypsin-sensitive cells. Such results underscore the difficulties in establishing the mechanisms involved in platelet destruction during HIV infection.

Potential immunological action of purine nucleoside analogues.

Purine nucleoside analogues are a new class of drugs with activity against nondividing lymphocytes; thus, they should play a major role in the treatment of low grade lymphoid malignancies. As these drugs are active against resting lymphocytes, harmful effects related to this action were expected and have been reported. However, the toxic effects on resting lymphocytes observed during treatment of lymphoid malignancies may potentially be of some benefit in patients with autoimmune diseases. To substantiate this possibility, a considerable amount of work needs to be carried out in order to better define the mechanism of action of these drugs, as well as their potential activity on different immunological effectors. Also, studies in animal models of autoimmune disease should be undertaken.