Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sj?gren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.     

Diffuse large cell lymphoma in a patient with hairy cell leukemia: immunoglobulin gene analysis reveals separate clonal origins

A 75-year-old man with hairy cell leukemia (HCL) was found to have an immunoblastic lymphoma of the small bowel. Immunologic and genotypic characterization of these neoplasms revealed both the HCL and the immunoblastic lymphoma to be of the B cell lineage. The HCL expressed the HCL-associated antigens detected by the monoclonal antibodies HC-1 and HC-2, whereas the immunoblastic lymphoma failed to react with these antibodies. In addition, surface immunoglobulin light chains could not be accurately determined for the hairy cells, whereas the immunoblastic lymphoma was shown to express only kappa immunoglobulin light chains. These immunophenotype differences were compatible with either the clonal evolution of the HCL into the immunoblastic lymphoma or a separate clonal origin for these two neoplasms. An analysis of tumor DNA by Southern blot hybridization revealed different heavy-chain and kappa light-chain gene rearrangements in these two malignancies. Thus the occurrence of the large cell lymphoma most likely represents the emergence of a second clonally unrelated B cell malignancy.     

A case of systemic lupus erythematosus complicated with monoclonal CD5 + B cell proliferation suspected as chronic lymphocytic leukemia]

A case of systemic lupus erythematosus (SLE) complicated with monoclonal CD5 + B cell proliferation in peripheral blood and bone marrow is reported. A 59-year-old man suffering from left chest pain was admitted to the hospital because of thrombocytopenia (platelets 1.9 x 10(4)/mm3). The diagnosis of SLE was made from (1) pleuritis (2) autoimmune thrombocytopenia (3) positive anti-DNA antibodies, positive LE cell preparation (4) positive antinuclear antibodies. Prednisolone 60mg per day was started. From that time monoclonal CD5 + B cells began to increase in peripheral blood (maximum lymphocyte counts 11000/mm3, CD5 + B cells 77.6%) and bone marrow, and the complication of chronic lymphocytic leukemia (CLL) was suspected. It is said that patients of CLL often have various autoantibodies, and in about 15% of CLL patients complicate autoimmune hemolytic anemia, but those who develop collagen diseases are rare. And while lymphoid malignancies occur more often in the patients of SLE in comparison with normal subjects, the reports of the patients who complicate the proliferation of monoclonal CD5 + B cells like CLL are very few. But from many facts that indicate the relation between CD5 + B cell or its proliferation and the production of autoantibodies or autoimmune diseases, we consider this case worth to be reported.     

An improved clonal excess assay using flow cytometry and B-cell gating

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.     

Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia

Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T-cell receptor gamma-polymerase chain reaction (TCR gamma-PCR). Subsequently, in 6 patients, the CDR3 length and conformation from 22 different TCRBV subfamilies were analyzed after PCR amplification of cDNA using TCRBV subfamily-specific primers. T cells from 15 of 25 HCL patients showed clonal excess by the TCR gamma-PCR. In fluorescence-activated cell sorted T-cell subsets, more clonal bands were observed than in the unseparated T cells, with most of these in CD8+ subsets, but also in CD4+, CD3+ gamma/delta+, and a double-negative CD3+ alpha/beta+ subset. In other B-cell malignancies, 6 of 16 samples showed oligoclonal T cells, whereas only 2 of 18 normal spleen and blood samples showed abnormal bands. Analysis of the TCRBV subfamilies disclosed in all 6 HCL patients a markedly abnormal pattern, with many clonal bands in 5 to 15 subfamilies, and absent or abnormal weak patterns in another 1 to 8 subfamilies. In comparison, 6 normal samples (2 spleens and 4 blood samples) showed in only 1 blood donor 1 clonal band. Two patients with active HCL but without infections or treatment were tested several times during the course of the disease and showed a complete identical skewed T-cell repertoire with the same oligoclonal T-cell populations. In conclusion, T cells in the blood and spleen of HCL patients show impressive abnormalities with many oligoclonal T-cell populations and a very restricted and skewed TCRBV repertoire.     

Characterization of the light chain-restricted clonal B cells in peripheral blood of HCV-positive patients

To investigate the association between hepatitis C virus (HCV) and B cell proliferation, we searched for the clonal B cells by flow cytometric analysis of the surface immunoglobulin kappa (κ):lambda (λ) light chain ratios of the circulating B (CD19+) cells in 240 HCV-positive patients and 150 negative controls with liver diseases. Clonal B cells with light chain restriction (κ:λ ratio >3:1 or <1:2) were analyzed for CD5 expression and the presence of monoclonal immunoglobulin heavy-chain (IGH) gene rearrangements and the t(14;18) chromosomal translocation. Clonal B cells were detected in 7 cases with HCV (2.9%), but was never detected in the controls (p < 0.05). Of the 7 cases, all had monoclonal IGH gene rearrangements and one had the t(14;18) chromosomal translocation. These HCV-related clonal B cells are not uniform in the intensity of CD5 expression and showed no increase in the frequencies of CD5+ population compared with non-clonal B cells. No “chronic lymphocytic leukemia-phenotype” cells were found. The loss of clonality was observed in 2 cases treated with interferon and in one case treated with splenectomy. The longitudinal study is required to determine whether these circulating clonal B cells progress to lymphoproliferative disorders in future or not.     

Light chain-restricted autoantibodies in chronic idiopathic thrombocytopenic purpura,but no evidence for circulating clonal B-lymphocytes

In chronic idiopathic thrombocytopenic purpura (ITP) platelet destruction is caused by antibodies directed against platelet membrane glycoproteins (GP), and the predominant autoantigens are known to be GPIb/IX and GPIIb/IIIa. In a recent study we reported that these antibodies frequently had a restricted light chain phenotype, thereby supporting a clonal origin. Similar findings and the presence of clonal B-cell populations in immune thrombocytopenias have been reported by others. In the present study we further explored the hypothesis of clonal B-cell expansions in chronic ITP. Twenty patients with chronic ITP were investigated. Antibodies were detected with an ELISA (MAIPA) specific for GPIb/IX and GPIIb/IIIa; circulating clonal B lymphocytes were assessed by flow-cytometric (FACE) clonal-excess analysis and by analyzing Ig-gene rearrangements (CDR3) with the PCR technique. Nine patients displayed a GP-specific antibody restricted to either kappa or lambda phenotype. However, FACS analysis and Ig-gene rearrangement studies did not disclose any circulating clonal B-cell population. Considering the sensitivity of the FACS analysis and Ig-gene rearrangement for detection of clonal B-cell populations, the hypothesis of clonally derived autoantibodies in ITP is still valid. Most probably, the clonal B-cell expansion responsible for the production of autoantibodies in ITP, if present, is below the detection limit for the techniques employed.     

Mechanism of autoimmune hemolytic anemia in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignant clonal expansion of CD5+B lymphocytes. The CD5+B lymphocytes have been postulated to produce autoantibodies. CLL patients may demonstrate features of autoimmunity including autoimmune hemolytic anemia. However, the origin of the autoantibodies causing the hemolysis is not clear. The present studies were performed to determine whether these autoantibodies are the products of the neoplastic B-CLL clones. Immunoglobulins (Ig) were eluted from washed red blood cells (RBC) obtained from two CLL patients at the time they had autoimmune (DAT-direct antiglobulin test - positive) hemolytic anemia. The light chain phenotypes of these eluted autoantibodies were determined and found to be monotypic with exact correlation to the light chain expressed on the surface of the B-CLL clones. Elutions from RBC of DAT negative patients or normal volunteers failed to demonstrate measurable amounts of Ig. In contrast, Ig eluted from RBC obtained from SLE patients with DAT positive hemolytic anemia found to be polyclonal autoantibodies exhibiting both light chain types. Furthermore, CD5+B lymphocytes obtained from the same two CLL patients (DAT+) produce, in vitro understimulation with phorbal myristate acetate (PMA), monoclonal antibodies which react and bind to RBC. Thus these studies provide direct evidence demonstrating that the antibodies causing the autoimmune hemolytic anemia in our two CLL patients are the products of the B-CLL neoplastic clones.     

A patient with paroxysmal nocturnal hemoglobinuria, T cell large granular lymphocyte clonal expansion, and monoclonal gammopathy of undetermined significance

Paroxysmal nocturnal hemoglobinuria (PNH) has been described in association separately with T cell large granular lymphocyte (LGL) clonal expansions and plasma cell dyscrasias. We describe a patient with anemia related to hemolytic PNH, with concurrent T cell LGL oligoclonal expansion and IgG lambda monoclonal gammopathy of undetermined significance. Peripheral blood flow cytometry revealed decreased expression of CD55 and CD59 on erythrocytes and decreased expression of CD55 and CD66 on neutrophils. An LGL population was present in the peripheral blood and was characterized as oligoclonal by polymerase chain reaction-based analysis of the T cell receptor gamma-chain variable region. Serum protein electrophoresis with immunofixation showed a low level IgG lambda monoclonal protein. We describe the diagnostic evaluation of this patient and provide a brief review of the reported associations among PNH, LGL clonal expansion, and monoclonal gammopathy.     

Glycoprotein V-specific platelet-associated antibodies in thrombocytopenic patients.

In autoimmune thrombocytopenia, platelet-associated IgG (PA-IgG) frequently displays specificity against glycoprotein (GP) IIbIIIa and/or GP IbIX. Because in a high proportion of patients positive PA-IgG may not be explained by these GP specificities, studies on other target proteins are needed. We studied the presence of GP V-specific PA-IgG by direct monoclonal antibody-specific immobilization of platelet antigens (MAIPA) with the monoclonal antibody SW16. We focused on 69 consecutive random patients with histories of thrombocytopenia who were strongly positive for PA-IgG detected by the direct platelet immunofluorescence test (PIFT). PA-IgG against GP V (ratio > or = 1.5) was noted in 15 (22%) patients. The degree of PA-IgG measured by PIFT, and of GP IIbIIIa-and/or GP IbIX-specific PA-IgG measured by direct MAIPA, correlated directly with the GP V-specific PA-IgG (P < 0.001). In one patient, GP V-specific antibodies were associated with quinidine-induced thrombocytopenia. Although this patient had strongly positive GP V-specific PA-IgG, she remained negative in GP IIbIIIa- and GP IbIX-specific direct MAIPA. Two patients studied because of thrombocytopenia associated with gold therapy had strongly positive GP V-specific PA-IgG. In one patient with rheumatoid arthritis and severe gold-induced thrombocytopenia, the amount of GP V-specific PA-IgG decreased during the recovery phase. Thus, GP V may represent an important target antigen in autoimmune-mediated thrombocytopenia, especially in drug-induced thrombocytopenia.     

Aberrant expression of immunoglobulin light chain isotype in B lymphocytes from patients with monoclonal gammopathies: isotypic discordance and clonal B-cell excess

A study was made of 29 patients with monoclonal gammopathies to detect aberrations in immunoglobulin (Ig) light chain isotype expression in lymphocytes at various levels of B-cell differentiation, namely, circulating surface Ig positive (SIg+) cells, Ig-secreting cells (plaque forming cells, PFC) and mitogen-induced PFC. By using kappa-lambda analysis, two major phenotypes of aberrant Ig light chain isotype expression were found in circulating B cells at these three levels of differentiation: an absolute increase in B cells bearing the same Ig light chain isotype as that of monoclonal protein (clonal B-cell excess), and a relative decrease in those B cells (isotypic discordance). Isotypic discordance (ID) was found to be essentially negative in patients with monoclonal gammopathy of undetermined significance (MGUS) provided that they were in a stable condition. In myeloma patients, ID was found only in stage I, except for a remission case of stage III (4/7 in stage I, 0/8 in stage II, and 1/6 in stage III). ID was not restricted to a circulating SIg+ cell level but was also demonstrable at a spontaneous or pokeweed mitogen-induced PFC level. However, ID was negative at a PFC level induced by Staphylococcus aureus Cowan I. Clonal B-cell excess (CE) was frequently found in patients with active myeloma but not in stable patients (0/8 in MGUS, 1/7 in stage I, 8/8 in stage II, and 4/6 in stage III). CE was positive not only at a circulating SIg+ cell level but also at a circulating PFC level. Furthermore, patients with CE at a PFC level were found to have a higher proliferating capacity, defined as a percentage labelling index of marrow myeloma cells, than those without CE at a PFC level (P less than 0.02). ID and CE can therefore be considered as useful markers for discriminating between MGUS and myeloma, evaluating the clinical stability and predicting the clinical course.     

Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease

Immunotherapy with unconjugated CD20 monoclonal antibodies has proven effective for treating non-Hodgkin's lymphoma and autoimmune disease. CD20 immunotherapy depletes mature B cells but does not effectively deplete pre-B or immature B cells, some B cell subpopulations, antibody-producing cells, or their malignant counterparts. Because CD19 is expressed earlier during B cell development, a therapeutic strategy for the treatment of early lymphoblastic leukemias/lymphomas was developed by using CD19-specific monoclonal antibodies in a transgenic mouse expressing human CD19. Pre-B cells and their malignant counterparts were depleted as well as antibody- and autoantibody-producing cells. These results demonstrate clinical utility for the treatment of diverse B cell malignancies, autoimmune disease, and humoral transplant rejection.     

Light chain phenotypes of HLA antibodies in cases with suspected neonatal alloimmune thrombocytopenia

BACKGROUND AND OBJECTIVES: Platelet-specific antibodies are detectable in only about 30% in suspected neonatal alloimmune thrombocytopenia (NAIT). Human leucocyte antigen (HLA) class I antibodies are often detectable, and as platelets express the corresponding antigens, it has been suggested that these antibodies can be responsible for NAIT. Light chain phenotyping may assist in the diagnosis of HLA antibodies-induced NAIT. MATERIALS AND METHODS: We determined light chain phenotypes of platelet reactive HLA antibodies in 17 sera from mothers who delivered offspring with suspected NAIT. Ten sera also contained platelet-specific antibodies: HPA-1a (n = 5, one with HPA-15b), HPA-5b (n = 4) and autoantibodies (n = 1). Sera were tested for kappa or lambda restriction by monoclonal antibody-specific immobilization of platelet antigens (MAIPA). RESULTS: The cross-match with paternal platelets was positive in all cases due to HLA antibodies. We identified 5, 3 and 3 sera to contain lymphocytotoxic antibodies of single, two or multiple specificities, respectively. In six cases, HLA antibodies were only detectable by MAIPA. Light chain restriction (n = 9) was not associated with HPA containing antibodies or to any pattern of the HLA antibodies. Similarly, polyclonal antibodies (n = 8) were seen in all categories. CONCLUSION: We show that pregnancy-associated HLA antibodies can be clonal or polyclonal, irrespective of a diagnosis of NAIT. ONE-SENTENCE SUMMARY: Pregnancy associated HLA antibodies can be clonal or polyclonal, irrespective a diagnosis of NAIT.     

Common clonal origin of chronic lymphocytic leukemia and high-grade lymphoma of Richter's syndrome

Patients with B-cell chronic lymphocytic leukemia (CLL) infrequently may develop high-grade B-cell lymphoma, or Richter's syndrome lymphoma (RS lymphoma). Such lymphomas differ from the original leukemia in both histology and clinical behavior. Studies seeking to define the clonal relationship between the cells of the two malignancies in any one patient have yielded conflicting reports. We examined the clonal relationship between the early and late neoplastic cells of a patient who underwent Richter's transformation. In contrast to the original leukemia cells, the secondary high-grade lymphoma was CD5-. However, both the leukemia cells and the evolved RS lymphoma expressed surface IgM lambda reactive with Lc1, a murine monoclonal antibody specific for a supratypic cross-reactive idiotype encoded by a subset of human Ig variable region genes of the VH4 subgroup. Nucleic acid sequence analyses of the heavy and light chain variable region genes expressed by both leukemia and lymphoma cells show that the CD5- B-cell lymphoma constitutes a clonal expansion of mutant cells derived from the original CD5+ B-cell leukemia. Moreover, certain sets of somatic mutations distinguish the Ig variable region genes used by RS lymphoma from those expressed by the CLL B cells. This is the first study to establish the clonal relationship between CLL and RS lymphoma through primary structural analyses of the expressed Ig genes.     

Can drugs cause autoimmune thrombocytopenic purpura?

A wide range of medications can cause life-threatening immune thrombocytopenia (ITP), hemolytic anemia, or neutropenia in sensitive individuals. The antibodies associated with these conditions usually require soluble drug to be present in order to react with the cell membrane glycoproteins for which they are specific. However, some patients make drug-independent antibodies (autoantibodies) as well. Occasionally, only autoantibodies are produced following exposure to a drug. Although drugs and other small molecules can become conjugated to proteins in vivo, which may induce an immune response, only fragmentary information is available to explain how exogenous substances sometimes perturb the immune system in such a way that antibodies capable of causing immune cytopenia are produced. Platelets are affected by drug-induced antibodies more often than any other blood element. For many drug-induced thrombocytopenias, the targeted membrane glycoproteins are readily accessible for laboratory investigation and methods for detecting the responsible antibodies are well developed. Techniques for studying cellular aspects of the immune response induced by drugs through in vitro manipulation of T and B lymphocytes are also advancing rapidly. Studies of drug-induced ITP may provide clues to the general mechanisms whereby drugs and other xenobiotics induce immune diseases. Clinicians should consider the possibility of an exogenous trigger in patients who present with apparent autoimmune thrombocytopenia.     

Phenotypic analysis by flow cytometry of surface immunoglobulin light chains and B and T cell antigens in lymph nodes involved with non-Hodgkin's lymphoma

The objective of this study was to demonstrate the diagnostic usefulness of flow cytometric analysis of surface membrane immunoglobulin light chain and monoclonal antibody reactivities in B cell non-Hodgkin's lymphoma. For this purpose, lymph node cell suspensions from 80 patients (20 normal lymph nodes, 11 lymph nodes with benign lymphoid hyperplasia, and 47 lymph nodes with B cell non-Hodgkin's lymphoma) were studied to detect the expression of surface B and T cell differentiation antigens recognized by a panel of monoclonal antibodies (anti-Leu-1, anti-Leu-5, anti-HLA-DR, J-5, anti-BL-1, anti-BL-2, and anti-BL-7). The clonal excess calculation, percent kappa-positive minus percent lambda-positive/percent kappa-positive plus percent lambda-positive cells per discrete level of fluorescence intensity, was used to study the clonality of surface membrane immunoglobulin light chain expression. Among the BL surface antigens, BL-7 proved to be most consistently expressed in B cell non-Hodgkin's lymphoma (79 percent). It was also present in 57 percent of lymph nodes with benign hyperplasia. No significant relationships were detected between the patterns of reactivity with the anti-BL monoclonal antibodies and histologic subtypes, although the small number of cases tested in each category precludes any definitive conclusions. Immunophenotypic heterogeneity within subgroups was also observed with expression of the other antigens examined. Monoclonal expression of surface membrane immunoglobulin light chain was seen in 43 of 47 (91 percent) of lymph nodes with non-Hodgkin's lymphoma, three of 11 (27 percent) hyperplastic lymph nodes, and one of 22 (4 percent) normal lymph nodes. When the presence of BL-7 and clonal excess was examined as a panel, 83 percent of B cell non-Hodgkin's lymphomas were positively identified, whereas one normal lymph node and no hyperplastic lymph nodes gave positive results. The simultaneous presence of clonal excess and BL-7 can be a useful diagnostic aid in the differentiation of lymphomatous from hyperplastic lymph nodes. Cytofluorimetry provides a rapid, objective, and reproducible technology to confirm the diagnosis of lymph node involvement in B cell non-Hodgkin's lymphoma.     

Multiple hepatitis attacks with immunothrombocytopenia in a heroin addict

A 25-year-old heroin-addict had four episodes of acute hepatitis, each of them associated with thrombocytopenia. Serologically, hepatitis A and B as well as cytomegalo- or Epstein-Barr virus infections were excluded. High levels of circulating immune complexes and antibodies reacting with aminophospholipids, a component of both liver cell and platelet membranes, were detected during the last two attacks of hepatitis, but were absent during remission. Platelet-associated antibodies of the IgM class paralleled the 'anti-phospholipid' antibodies during the fourth attack. These findings demonstrate an immunological basis of the thrombocytopenia and point to the possibility of a direct association between liver cell injury and peripheral platelet destruction in certain patients.     

Platelet surface-bound IgG in patients with immune and nonimmune thrombocytopenia

We quantitated the amount of platelet surface-bound IgG using an 125I monoclonal anti-IgG assay in 149 patients with thrombocytopenia and 260 normal donors. The normal subjects had 122 +/- 5 molecules of IgG/platelet (mean +/- SE). Fifty-five patients with nonimmune thrombocytopenia had 338 +/- 37 molecules of IgG/platelet, whereas 67 patients with immune thrombocytopenia studied at the time of their initial evaluation had 4,120 +/- 494 molecules of IgG/platelet. An analysis of the distribution of values in these two groups indicated that 90% of the patients with immune thrombocytopenia had greater than 800 molecules of IgG/platelet, whereas only 7% of patients with nonimmune thrombocytopenia exceeded this amount. The immune thrombocytopenia patients included 39 idiopathic, 14 secondary, and 14 drug-induced disorders, and they did not significantly differ in their distribution of values for platelet IgG. The nonimmune thrombocytopenic patients included 12 cases with a platelet destructive mechanism; their platelet-bound IgG was similar to that of the other nonimmune patients. Twenty-seven patients with treatment resistant immune thrombocytopenia were also studied they had 2,100 +/- 670 molecules of IgG/platelet. Their values were significantly greater than those of the nonimmune thrombocytopenic patients and not significantly different from those of immune thrombocytopenic group. Their distribution of values was much broader, however, with 33% of patients having less than 800 molecules of IgG/platelet, suggesting possible alternate mechanisms in their thrombocytopenia. Thus, patients with immune thrombocytopenia have a high frequency of elevated IgG on the platelet surface which reflects the pathophysiology of this disorder. Quantitation of platelet-bound IgG provides a useful laboratory tool in the differential between immune and nonimmune thrombocytopenia.     

Patients with high-risk myelodysplastic syndrome can have polyclonal or clonal haemopoiesis in complete haematological remission

The clonality of mature peripheral blood-derived myeloid and lymphoid cells and bone marrow haemopoietic progenitors from 18 females with myelodysplasia (MDS) (five refractory anaemia, RA; one RA with ringed sideroblasts, RARS; three chronic myelomonocytic leukaemia, CMML; four RA with excess of blasts, RAEB; five RAEB in transformation, RAEB-t) was studied by X-chromosome inactivation analysis. Using the human androgen-receptor (HUMARA) assay, we analysed the clonal patterns of highly purified immature CD34⁺38^? and committed CD34⁺38⁺ marrow-derived progenitors, and CD16⁺14^? granulocytes, CD14⁺ monocytes, CD3⁺ T and CD19⁺ B lymphocytes from peripheral blood. In high-risk patients (RAEB, RAEB-t), clonality analysis was performed before and after intensive remission-induction treatment. All patients, except one with RA, had predominance of a single clone in their granulocytes and monocytes. The same clonal pattern was found in CD34⁺ progenitor cells. In contrast, CD3⁺ T lymphocytes were polyclonal or oligoclonal in 14/18 patients. X-chromosome inactivation patterns of CD19⁺B cells were highly concordant with CD3⁺ T cells except for two patients (one RA, one CMML) with monoclonal B and polyclonal T lymphocytes, therefore suggesting a clonal mutation in a progenitor common to the myeloid and B-lymphoid lineages or the coexistence of MDS and a B-cell disorder in these particular patients. After high-dose non-myeloablative chemotherapy, polyclonal haemopoiesis was reinstalled in the mature myeloid cells and immature and committed marrow progenitors in three of four patients achieving complete haematological remission. Therefore we conclude that most haematological remissions in MDS are associated with restoration of polyclonal haemopoiesis.     

Restricted VH3 gene usage in phage-displayed Fab that are selected by intravenous immunoglobulin

OBJECTIVE: To perform a comparative analysis of 1) intravenous Ig (IVIG)-bound Fab fragments from a patient with autoimmune thrombocytopenia that had progressed to systemic lupus erythematosus (SLE) and 2) IVIG-selected Fabs from an SLE patient without thrombocytopenia. METHODS: IVIG preparations have been successfully used to treat certain cases of autoimmune thrombocytopenia and SLE. Specific interactions of IVIG with the components of the immune system are not well characterized. To investigate these, we had previously cloned a large number of phage-displayed IgG Fab fragments, derived from 3 patients with autoimmune thrombocytopenia, that were specifically bound by IVIG molecules during panning. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used VH germline gene segments of all IVIG-bound Fabs were 3-23 and 3-30/3-30.5. One patient's autoimmune thrombocytopenia had progressed to SLE. Using the same cloning and panning procedures, we performed a comparative analysis of this patient's IVIG-bound Fab fragments and the IVIG-selected Fabs from an SLE patient without thrombocytopenia. RESULTS: We observed an exclusive selection of antibodies derived from 3-23 and 3-30/3-30.5 germline segments. In contrast to the Fab fragments from the autoimmune thrombocytopenia patient who developed SLE, none of the IVIG-selected Fabs from the SLE patient without thrombocytopenia bound to thrombocytes. CONCLUSION: Our results suggest a preferential interaction of a subfraction of IVIG-representative of normal Ig repertoires-with antibodies and B cell receptors derived from these 2 gene segments. Importantly, these are the most frequently rearranged VH germline genes among human B cells. This kind of interaction is characteristic of a B cell superantigen, since light chains, antigen specificity, and the high variation in the third complementarity-determining region 3 showed little influence on the selection of 3-23- or 3-30/3-30.5-derived Fabs by IVIG. However, at least some of the contact residues on Fabs for IVIG appear to be different from those for staphylococcal protein A and human immunodeficiency virus gp 120. The IVIG-selected Fabs may now be used to clone antibodies representative of this IVIG subfraction to study their possible regulatory influence on the B cell repertoire during normal development and disease.