Natural autoantibodies, tolerance, and autoimmunity.
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Animals↗
Biomedical subjects
G Dighiero
Publications and source records attributed to G Dighiero.
Structural and affinity studies of IgM polyreactive natural autoantibodies.
Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.
Amino Acid Sequence↗
Chronic lymphocytic leukemia treatment.
Three main question were addressed during this session: 1) Is early treatment better than deferring treatment until necessary for early stage patients?; 2) Is there still a place for chlorambucil (CB) in the treatment of CLL? and 3) Should we consider purine analogues for first line of therapy in CLL? The first question was discussed by Ph. Travade, who presented the long term results from two trials of the French Cooperative Group in previously untreated stage A CLL patients and by Sue Richards who submitted to meta-analysis the data from the American, British, French, and Spanish groups. Consensus was reached to accept that the different randomized trials and the meta-analysis results clearly demonstrate that conventional CB schedules do not prolong survival in these patients. Since deferring therapy until it is required by disease progression to stages B or C does not compromise survival of these patients, therapy should be deferred until progression is observed. It is widely admitted that CB treatment provides relief from symptoms for a large proportion of patients. However, results from different trials presented indicate that at least at conventional schedules, CB is unlikely to prolong survival in CLL. However, Jaksic presented favorable results with high dose (i.e., 10-15 mg/day) continuous CB schedule. The role of purine analogues was discussed by M. Keating and A. Saven, who presented long term results in non-randomized studies with fludarabine (FDB) and 2-chloro-deoxyadenosine (CDA), respectively. In addition the results from two randomized American and French Trials were presented by K. Rai and M. Leporrier. Long term results indicate that purine analogues are active agents in CLL. Both drugs obtain important overall response rates (> 75%) and about 25% complete remissions. Long term results from MD Anderson series indicate that the median progression free interval is 30 months and that, in previously untreated patients, FDB is well tolerated. However, opportunistic infections and autoimmune complications are frequently observed in previously treated patients. Results from the American and French trials confirm that FDB is probably the best single agent in terms of response, since it appears to induce higher response rates than CB and anthracycline containing regimens. No important toxicities were observed in these series including exclusively previously untreated patients. The long term results from MD Anderson show a median survival of 5 years for CLL patients receiving FDB, which is not different to the historical results with other treatment modalities and is confirmed by the present results of the the American and French trials showing no improvement of the overall survival. These results suggest that purine analogues should be included in the list of drugs with proven efficacy in the initial treatment of patients with CLL. Although these drugs produce better responses, they do not cure CLL. For young patients with poor prognosis CLL, future attempts should be made to improve complete remissions at molecular level, which may lead towards significantly increasing the overall survival. Combination of purine analogues with other drugs, autologous transplantation and/or biological response modifiers like monoclonal antibodies or anti-idiotypic vaccines deserve investigation.
Antineoplastic Agents, Alkylating↗
Minimal residual disease detection in B-cell malignancies by assessing IgH rearrangement.
In B-cell malignancies, the uniqueness of the immunoglobulin heavy chain locus (IgH) clonal rearrangement provides a useful marker for the detection of minimal residual disease (MRD) after treatment. During the last decade, several techniques have been proposed and used for detecting MRD. In this review, we report the current PCR based techniques dealing with amplification of the VDJ segment since the CDR3 region is unique to each IgH rearrangement. The sensitivity of these techniques varies considerably with a detection level of one tumoral cell in 10(-2) to 10(-6) normal cells. Accurate and sensitive assessment of MRD may have profound impact in the clinical management of patients with hematologic malignancies. Although, a majority of studies have shown a good correlation between the rapidity or extent of the reduction in the number of tumoral cells and the subsequent relapse, other studies demonstrated substained positivity of PCR in patients in long term remission. Thus, current clinical studies of MRD should establish whether MRD predicts relapse uniformly and, therefore, justifies intensification of therapy in positive cases, or whether it simply detects leukemic cell populations whose proliferative potential has been altered by chemotherapy.
B-Lymphocytes↗
A murine model of human cold agglutinin disease.
Progress has been limited in the treatment of cold agglutinin (CA) disease by the absence of an animal model. We have recently studied at the molecular level one CA displaying the rare anti-Sia-1b specificity (CAGAS), CAGAS displays strong CA activity and is able to haemolyse mouse RBC in the presence of complement, thus constituting a suitable Ab for creating a murine model of CA disease. In the present work we introduced CAGAS VH and VL domains into eukaryotic expression vectors and transfected them into the non-secreting mouse myeloma X63 cell line. Clones expressing complete engineered pentameric IgM kappa CAGAS (eCAGAS) recapitulating the characteristics of serum CA (sCAGAS) could be obtained. The i.p. injection of eCAGAS to normal BALB/c mice induced a typical haemolytic anaemia, as demonstrated by the presence of spontaneous cold agglutination of RBC, induction of anaemia and significant reticulocytosis. Of interest, conspicuous bilateral ear loss was observed in one of these animals. In addition, i.p. injection of X63 transfected line into BALB/c nude mice induced ascites, typical haemolytic anaemia, and shortening of the mean RBC survival. These findings validate the practical interest of constructing a transgenic mouse model expressing eCAGAS.
Anemia, Hemolytic, Autoimmune↗
Long-term clinical outcome of B-cell chronic lymphocytic leukaemia patients in clinical remission phase evaluated at phenotypic level.
This retrospective study aimed to evaluate the long-term prognostic impact of phenotypic remission in B-cell chronic lymphocytic leukaemia (CLL) patients who have achieved clinical, haematological and bone-marrow complete remission (CR) after conventional chemotherapy. The clinical and phenotypic data of 77 CLL patients in CR with a median follow-up from CR achievement of 54 months (range 5-127) were analysed. 32 patients (42%) displayed a normalized phenotype as evaluated by k:lambda ratio or by CD5+/CD19+ cell numbers. Patients with normalized phenotype demonstrated a significantly higher incidence of female sex, a lower relapse rate, a trend for higher prevalence of stage A and a lower occurrence of CLL-related deaths. The relapse-free survival of patients with normalized phenotype was significantly longer (P = 0.02), whereas no difference in overall survival was found between the two groups. Interestingly, Binet's stage at diagnosis was highly predictive of the overall survival following CR achievement. From the results of the present study we conclude that a phenotype normalization at CR obtained with conventional chemotherapy indicates a higher probability of a longer CR but it does not translate into prolonged survival. Clinical features at diagnosis, such as stage distribution, are apparently stronger predictors of the final outcome. These results emphasize, however, the need for a routine assessment of the quality of response since this information could be crucial in designing therapeutic strategies for young patients suffering from advanced CLL.
Adult↗
What do chronic B cell malignancies teach us about B cell subsets?
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Animals↗
VH gene expression in CD5 positive and CD5 negative B cell chronic lymphoid malignancies.
In this review, we report analyses of VH genes in mature B cell malignancies generally or occasionally bearing CD5 antigen such as B CLL, MCL, SLVL and PLL. In the majority of cases, B CLL and MCL use VH genes in germline configuration. However in some cases a higher rate of random mutations is observed. These differences are not related to CD5 expression but are accounted by Ig phenotype, since less mutations are observed in CLL cases expressing membrane mu delta, when compared to forms exclusively expressing membrane mu. PLL and SLVL cases display mutated V genes independently of CD5 expression. Although there is some evidence that CD5+ B cells constitute a separate lineage, the possibility that CD5 constitutes an activation marker cannot be ruled out. Indeed, CD5- B cells can be induced to differentiate into CD5+ B cells and VH gene analyses showed no significative differences between CD5+ and CD5- B cell lymphoproliferative disorders. In this review we have tried to examine B cell chronic malignancies on the basis of phenotype and VH gene usage. Thus we propose a tentative classification where these disorders are allocated according to these characteristics.
Animals↗
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.
Amino Acid Sequence↗
High frequency of somatic mutations in the VH genes expressed in prolymphocytic leukemia.
Prolymphocytic leukemia (PLL) is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed lg heavy chain variable region (VH) genes in eleven cases of de novo PLL Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%), with preferential use of the V3-23 gene (50% of the VH3 genes). All sequences from expressed VH genes diverged from their putative germline counterpart, and in eight cases the divergence was greater than 5%. In seven cases, which expressed the V3-23 gene and VH4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B-PLL cells display a skewed repertoire of lg VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.
Adult↗
In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals.
Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3-mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N-acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.
Acetylcysteine↗
CD4+ lymphocytes from HIV-infected patients display impaired CD45-associated tyrosine phosphatase activity which is enhanced by anti-oxidants.
It has been proposed that signal transduction defects may, at least partially, account for the functional impairment of CD4+ lymphocytes during HIV-1 infection. Recently, we have demonstrated that unresponsive CD4+ lymphocytes from these patients had reduced protein tyrosine phosphorylation after CD3 engagement, and that this defect was associated with constitutively altered levels of p56lck and p59fyn kinases. Since CD45 is essential for T cell receptor (TCR) and CD2-mediated activation of protein tyrosine kinases, we study here CD45-associated tyrosine phosphatase activity in resting and activated CD4 T cells from HIV-infected patients. We found a significant decrease in the basal and post-activation phosphatase activity of CD45 which correlated well with impairment of proliferative responses. In addition, decreased levels of cellular thiols observed in resting CD4+ lymphocytes from these patients suggested a disturbed redox status. Although expression levels of CD45 were decreased in most patients, a significant recovery of phosphatase activity and proliferative responses was observed in most patients by preincubating cells with N-acetyl-L-cysteine and beta2-mercaptoethanol. In some patients, anti-oxidant treatment failed to significantly enhance phosphatase activity and proliferative responses. The low responses of purified CD4+ lymphocytes from these patients were associated with a high ratio of apoptotic cell death which did not appear to be influenced by anti-oxidant treatment.
Apoptosis↗
Expression and production of cytokines by heterohybrids and their parental B cells in CLL.
Three hybrids derived from CD5+ B cell chronic lymphocytic leukemia (B-CLL) and their parental B cells were studied for phenotypic evolution, immunoglobulin (Ig), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) secretion. When phenotypic evolution was examined, hybrids showed the loss of classical B cell markers, indicating that they follow the same pattern of phenotypic differentiation as normal B cells. Hybrids displayed spontaneous high Ig secretion, which did not appear to be modified through stimulation by phorbol 12-myristate 13-acetate (PMA), recombinant interferon-gamma (rIFN-gamma) and Staphylococcus aureus Cowan I (SAC). Parental cells secreted minimal amounts of Ig spontaneously or through IFN-gamma and SAC stimulation, whereas PMA succeeded in increasing this secretion. An opposite pattern was observed when TNF-alpha and IL-6 secretion an expression at the mRNA level were assessed in hybrids and parental cells. TNF-alpha and IL-6 were spontaneously secreted by parental cells and this secretion was increased after PMA and SAC stimulation, both cytokine secretion and expression at the mRNA level were negative in hybrid cells. The absence of expression of these cytokines could be explained either by chromosomal loss or by down regulation. These results indicate that when parental CLL cells are induced to differentiate in the heterohybrid model, they acquire high spontaneous secretion of Ig, lose the classical B cell phenotypic markers and down regulate the expression of the cytokines studied.
B-Lymphocytes↗
Complete nucleotide sequence of Ig V genes in three cases of Burkitt lymphoma associated with AIDS.
To investigate the role of polyclonal stimulation and antigen driven selection in the pathogenesis of acquired immunodeficiency syndrome (AIDS) related lymphomas, we studied the variable region nucleotide sequence of heavy (VH) and light (VL) chains expressed by 3 Burkitt lymphomas (BL) associated with HIV infection. Two cases expressed the VH3-30P1 gene with 88.6% and 86.7% homology when compared to their germinal counterpart, whereas the VH4-18 was rearranged in the third one (89% identity). All these genes displayed high numbers of mutations (27, 22, 28 respectively), predominating in CDR regions. The encoded light chain genes determined for cases 1 and 2 expressed the same V kappa I-018 gene. These results indicate that: 1) Although, it is difficult to address the issue of VH usage based on the limited number of cases studied, Burkitt's lymphoma associated with AIDS may use a restricted repertoire of Ig genes. 2) Mutations and/or replacements predominated in CDR regions, which might suggest the occurrence of an antigen driven selection process, at least in some AIDS associated lymphomas. However, the high ratio of mutations observed in framework (FW) regions also favors the possibility that the antigen selection process is associated with polyclonal B cell stimulation.
Acquired Immunodeficiency Syndrome↗
What is the CLL B-lymphocyte?
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Amino Acid Sequence↗
Autoimmunity and B-cell malignancies.
There is evidence indicating that autoreactive B cells constitute a substantial part of the B-cell repertoire. This autoreactive repertoire secrete the so called natural autoantibodies characterized by their broad reactivity mainly directed against very well conserved public epitopes. They fulfill the definition of an autoantibody since they are self-reactive, but they are not self-specific. As yet, NAA directed against determinants of polymorphism have not been reported. 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, we can assume that 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, it can be alternatively postulated, that overexpression of certain genes reflect what happens during ontogeny, since V genes expression is a developmentally regulated phenomenon and not all V genes are expressed during fetal life. Some of the genes that are recurrently expressed by these malignancies are also over-expressed in fetal repertoires and even in the adult normal B cell repertoire. We do not know whether it is the challenge by self-antigens or whether alternatively this over-expression simply reflects what happens with the fetal repertoire which could have selective advantages for malignization.
Adult↗
Chronic lymphocytic leukemia.
B-cell chronic lymphocytic leukemia (B-CLL), the leukemia with highest incidence in Western Countries, is a neoplastic disease characterized by a progressive accumulation of functionally incompetent, long-lived small mature lymphocytes in the blood, bone marrow, and lymphoid tissues. B-CLL lymphocytes express characteristic low amounts of surface immunoglobulin and CD5 antigen. They are resistant to Epstein Barr Virus (EBV) transformation; are in prolonged G0 phase, express high amounts of bcl-2 and display a defective response to stimulation through the antigen receptor. Despite the presence of many genetic abnormalities, in CLL, there are no typical chromosomal aberrations or oncogenic abnormalities associated with this disease.
Animals↗
Adverse and beneficial immunological effects of purine nucleoside analogues.
Purine nucleoside analogues are a new class of drugs with activity against non-dividing lymphocytes. They should thus play a major role in the treatment of low grade lymphoid malignancies. These drugs have been shown to have strong effect in DNA synthesis on actively dividing cells, mainly through interference with DNA polymerases and ribonucleotide reductase. However, the cell cycle kinetics of low grade lymphocytic lymphomas is characterized by the presence of very low growth fractions. Hence, the action of these drugs in slowly progressing lymphoid malignancies cannot be accounted by the same mechanism observed in actively proliferating tumors and needs to be explained through activity against quiescent resting lymphocytes. Recent work has stressed the role of purine analogues in inducing programmed cell death of quiescent lymphocytes, which could be explained through the induction of accelerated DNA strand breaks. This process leads to consumption of NAD for poly(ADP-ribose) synthesis, which could induce critical depletion of ATP. As this action extends to normal resting lymphocytes deleterious effects related to their immunosuppressive action are also observed, i.e. prolonged lymphopenia predominating in T cells and especially in CD4 subset, increased frequency of opportunistic infections and perhaps increase in autoimmune complications like autoimmune hemolytic anemia. Nevertheless, beneficial effects of this immunosuppressive action have also been reported in the prevention of graft-versus-host disease, graft rejection and in some autoimmune diseases like multiple sclerosis. Work needs to be carried out to define better the mechanisms of action of these drugs on the different immunological effectors, as well as studies in animal models of transplantation and autoimmune diseases.
Animals↗