Characterizing the BCR repertoire during lymphocyte reduction and recovery mediated by cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

Leukopenia is a common manifestation of many diseases, including global outbreak SAS-CoV-2 infection. Granulocyte-macrophage colony-stimulating factor (GM -CSF) has been proved to be effective in promoting lymphocyte regeneration, but adverse immunological effects have also emerged. This study aim to investigate the effect of GM -CSF on BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration. Cyclophosphamide (CTX) and GM -CSF were used to inhibit and stimulate bone marrow hematopoiesis, respectively. High throughput sequencing was applied to detect the characteristics of BCR CDR3 repertoire in controls, CTX group and GM -CSF group. The white blood cells (WBCs) were quickly reduced (P < 0.05) with lymphocytes decreasing causing by CTX, and the WBCs and lymphocytes returned to the level of controls after GM -CSF treatment. The diversity of BCR heavy chain CDR3 repertoire was also significantly decreased in CTX group. Although there is still a big gap from the controls, the diversity was picked up after GM -CSF treatment. The expression of IGHD01-01, IGHD02-14 and IGHJ04-01 with high-frequency usage regularly and significantly changed in three groups, and many genes with low-frequency usage lost in CTX group and did not reappear in GM -CSF group. Moreover, two shared sequences and accounted for the highest proportion in GM -CSF group have been detected in animal model of chronic lymphocytic leukemia. These results revealed that GM -CSF can partially restore changes in the BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration, but it may also lead to rearrangement, proliferation and activation of abnormal B cells, which can provide a basis for further study on the adverse immunological effects and mechanism of GM -CSF treatment.

Safety and efficacy of obinutuzumab alone or with chemotherapy in previously untreated or relapsed/refractory chronic lymphocytic leukaemia patients: Final analysis of the Phase IIIb GREEN study.

The manageable toxicity profile of obinutuzumab (GA101; G) alone or with chemotherapy in first-line (1L; fit and non-fit) and relapsed/refractory (R/R) patients with chronic lymphocytic leukaemia (CLL) was established in the primary analysis of the Phase IIIb GREEN trial (Clinicaltrials.gov: NCT01905943). The final analysis (cut-off, 31 January 2019) is reported here. Patients received G (1000 mg) alone (G-mono; fit and non-fit patients) or with chemotherapy [fludarabine and cyclophosphamide (FC; fit patients); chlorambucil (non-fit patients); bendamustine (any patient)]. Study endpoints were safety (primary) and efficacy (secondary). Subgroup analyses were performed on prognostic biomarkers in 1L CLL. Overall, 630 patients received 1L and 341 received R/R CLL treatment. At the final analysis, no new safety signals were observed [Grade ≥ 3 adverse events (AEs): 1L 82·7%, R/R 84·5%; serious AEs: 1L 58·1%, R/R 62·5%]. Neutropenia (1L 50·5%, R/R 53·4%) and thrombocytopenia (1L 14·6%, R/R 19·1%) were the most common Grade 3-5 AEs. G-mono-, G-bendamustine and G-FC-treated patients with unmutated immunoglobulin heavy chain trended towards shorter progression-free survival. Achievement of minimal residual disease negativity was greatest in 1L patients treated with G-FC. In this final analysis of the GREEN trial, the safety profile of G was consistent with current risk management strategies. Biomarker analyses supported efficacy in the specific subgroups.

Advanced glycation end products upregulate the endoplasmic reticulum stress in human periodontal ligament cells.

BACKGROUND: The accumulation of advanced glycation end products (AGEs) appears to be the main factor responsible for modulating periodontal inflammation in diabetes. The aim of this study is to examine the effects of AGEs on inflammation in human periodontal ligament cells and to investigate the mechanism with a specific emphasis on the role of endoplasmic reticulum (ER) stress-induced nuclear factor-kappa B (NF-κB) pathway. METHODS: Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The protein expressions of ER markers and NF-κB were examined by Western blot analysis. The translocation of NF-κB was observed by immunofluorescence assay. Proinflammatory chemokine production was determined by enzyme-linked immunosorbent assay. RESULTS: Treatment with AGEs reduced cell viability in a concentration- and time-dependent manner. AGEs induced ER stress, as evidenced by survival molecules, such as glucose-regulated protein 78 (GRP78), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF-6), and apoptotic molecules, such as CCAAT/enhancer binding protein homologous protein (CHOP) and caspase 12. AGEs upregulated the nucleoprotein expression of NF-κB, enhanced translocation of NF-κB from the cytoplasm to the nucleus, and increased the production of proinflammatory chemokines interleukin-6 and interleukin-8. CONCLUSION: AGEs mediate inflammation of human periodontal ligament cells via the ER stress-induced NF-κB pathway.

Diverse chemicals including aryl hydrocarbon receptor ligands modulate transcriptional activity of the 3'immunoglobulin heavy chain regulatory region.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a known disruptor of B-cell differentiation and a ligand for the aryl hydrocarbon receptor (AhR), induces binding of the AhR to dioxin responsive elements (DRE) in sensitive genes. The Ig heavy chain (IgH) gene is a sensitive target of TCDD and may be transcriptionally inhibited by TCDD through inhibition of the 3'IgH transcriptional regulatory region (3'IgHRR). While the 3'IgHRR contains binding sites for several transcription factors, two DRE motifs were also identified which may be responsible for TCDD-induced inhibition of 3'IgHRR activation and may implicate the AhR as an important regulator of IgH expression. The objectives of the present study were to determine if 3'IgHRR modulation is limited to TCDD or if structurally diverse chemicals (AhR ligands and non-AhR ligands) from environmental, industrial, dietary or pharmaceutical origin are also capable of modulating the 3'IgHRR and to verify a correlation between effects on a stable 3'IgHRR reporter and the endogenous IgH protein. Utilizing a CH12.LX mouse B-cell line that stably expresses a 3'IgHRR-regulated transgene, we identified an inhibition of both 3'IgHRR activation and IgH protein expression by the non-dioxin AhR activators indolo(3,2-b)carbazole, primaquine, carbaryl, and omeprazole which followed a rank order potency for AhR activation supporting a role of the AhR in the transcriptional regulation of the 3'IgHRR and IgH expression. However, modulation of the 3'IgHRR and IgH expression was not limited to AhR activators or to suppressive effects. Hydrogen peroxide and terbutaline had an activating effect and benzyl isothiocyanate was inhibitory. These chemicals are not known to influence the AhR signaling pathway but have been previously shown to modulate humoral immunity and/or transcription factors that regulate the 3'IgHRR. Taken together these results implicate the 3'IgHRR as a sensitive immunological target and are the first to identify altered 3'IgHRR activation by a diverse range of chemicals.

Suppression of immunoglobulin production by a novel dihydroorotate dehydrogenase inhibitor, S-2678.

We discovered a novel dihydroorotate dehydrogenase (DHO-DH) inhibitor, S-2678 ([2-fluoro-2',5'-dimethyl-4'-[6-(3-methyl-2-butenyloxy) pyridin-3-yl] biphenyl-4-yl]-(3-methyl-2-butenyl) amine). Its inhibitory activity against DHO-DH was more potent than that of A77 1726, an active metabolite of the anti-rheumatic drug leflunomide. S-2678 suppressed immunoglobulin production in mouse B cells and human peripheral blood mononuclear cells in vitro, with little or no inhibition of cell proliferation, probably through inhibition of class switch recombination in the immunoglobulin heavy chain loci in B cells. In vivo antibody production induced by systemic immunization with ovalbumin was dramatically suppressed by oral administration of S-2678, without any toxicological signs. However, S-2678 did not affect T-cell activation in vitro, and cytokine production induced by intravenous anti-CD3 antibody in mice. S-2678 did not affect host defense in a mouse model of Candida infection, whereas leflunomide severely impaired it. In conclusion, S-2678 selectively acts on B cells, resulting in antibody production, which suggests that it is useful for the treatment of humoral immunity-related diseases.

Transitional B cells lose their ability to receptor edit but retain their potential for positive and negative selection.

Ligation of B cell receptors on immature bone marrow B cells, either by an endogenous Ag or by an anti-B cell receptor Ab induces secondary V(D)J gene rearrangements, termed receptor editing. Whether the same signal induces receptor editing in transitional B cells is not clear. In this study, we examined the responses of immature and transitional B cells from V(H)12Vkappa1A Ig transgenic mice to stimulation with an anti-Igbeta Ab. Our results demonstrated that immature B cells stimulated with a low concentration of anti-Igbeta Ab, mimicking Ag stimulation, underwent receptor editing both in vivo and in vitro, as evidenced by the detection of dsDNA breaks at Jkappa recombination signal sequences, whereas transitional B cells did not. The lack of dsDNA breaks in transitional B cells contrasts with their increased expression of RAG1 and RAG2, suggesting a novel mechanism that may prevent rearrangements. Furthermore, treatment of transitional B cells with high concentrations of anti-Igbeta Abs induced apoptosis, whereas low concentrations induced differentiation. Our results support the idea that transitional B cells lose the capacity to edit, but are sensitive to positive and negative selection.

The IgE repertoire in PBMCs of atopic patients is characterized by individual rearrangements without variable region of the heavy immunoglobulin chain bias.

BACKGROUND: Patients with atopic diseases are characterized by high levels of specific IgE production. However, little is known about the composition of their B-cell repertoires. OBJECTIVES: We sought to analyze the complete PBMC-derived IgE repertoire and to compare clonal expansions between different patients. METHODS: We have analyzed the IgE-bearing B-cell receptor repertoire in highly atopic patients (>1000 IU/mL) using quantitative RT-PCR, complementarity determining region 3 spectratyping, and sequence analysis. Three representative patients were additionally followed during anti-IgE therapy. RESULTS: Atopic patients exhibited 100 to 1000 times more IgE-specific transcripts than control individuals. These patients used a variable region of the heavy immunoglobulin chain (VH) epsilon repertoire highly similar to their IgM and IgG repertoires, with preference of VH3b, VH4, VH3a, and VH1 segments. Each patient harbored individual clonal expansions, most probably as correlation of allergen-specific IgE production. Common expansions within the complementary determining region 3 shared by several individuals with similar sensitization patterns were found in spectratyping analysis. However, these antigen-driven expansions showed differences on the sequence level. In omalizumab-treated patients the clinical improvement was paralleled by a clear increase in the ratio of IgG/IgE transcripts. CONCLUSION: The IgE repertoire in atopic patients follows the VH use patterns seen for other immunoglobulins and seems to preferentially recruit individual rearrangements rather than public expansions. CLINICAL IMPLICATIONS: The detailed analysis of the IgE B-cell repertoire is highly suitable to follow changes in IgE uses during different therapy modalities.

A novel domain antibody rationally designed against TNF-alpha using variable region of human heavy chain antibody as scaffolds to display antagonistic peptides.

Neutralizing of TNF-alpha has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-alpha and it's neutralizing monoclonal antibody. On the basis of the structural character and physical-chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-alpha (named as ATD5) was designed as TNF-alpha antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-alpha was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-alpha and inhibit the binding of TNF-alpha to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-alpha-mediated cytotoxicity and inhibit TNF-alpha-mediated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-alpha inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-alpha using in vitro analysis systems in combination with the computer-aided modeling method.

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines.

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.

Intraclonal variability of VH genes in follicular lymphoma patients who have received anti-idiotypic immunotherapy.

Subclonal heterogeneity can affect idiotypic determinants present in the clonotypic immunoglobulin of B-cell follicular lymphomas (FLs) and may limit the effect of antilymphoma treatments performed by immunization of patients with their own tumor-associated idiotypic immunoglobulin. Idiotype-secreting hybridomas were obtained by fusion of tumor cells from 5 patients with FL, and the K6H6/B5 human heteromyeloma and rearranged VH genes from tumor samples and hybridomas were amplified, cloned, and sequenced. Sequences were aligned with germline genes and somatic mutations, intraclonal heterogeneity and genealogic relations of the B-cell clones in the different biopsy specimens were determined. The VH sequence of the progenitor clone was determined in samples of the tumoral population. Further diversification resulted in the presence of 2 to 6 subclones in 4 of the 5 samples studied. Only in 1 patient did the hypermutation mechanism introduce differences among most of the potential idiotopes present in individual subclones. The VH sequence of the hybridoma that provided the idiotypic-vaccine was identified in one of the tumor subclones in all cases. No relapse has been demonstrated in 3 of the 4 vaccinated patients (follow-up: 29-103 months). We conclude that despite potential differences in the idiotypic region expressed by individual tumor cells, at least some potential idiotopes may be preserved among all the tumor subclones in most cases studied. All vaccinated patients developed immune responses against the autologous tumor idiotypic immunoglobulin. Polyclonal anti-idiotypic immune responses induced with a vaccine obtained from 1 hybridoma may be effective against all the idiotypic variants present in the tumor population.

The effect of antiviral therapy on t(14;18) translocation and immunoglobulin gene rearrangement in patients with chronic hepatitis C virus infection.

The mechanism of lymphomagenesis of hepatitis C virus (HCV)-related B-cell lymphoma is unknown. Recently, it has been suggested that HCV may induce B-cell clonal proliferation and t(14;18) translocation in patients chronically infected with the virus. Thus, this study investigated the effect of antiviral treatment on immunoglobulin heavy-chain gene (IgH) rearrangement and t(14;18) translocation in HCV infected patients. Twenty-nine patients with chronic HCV infection were studied in whom IgH rearrangement and/or t(14;18) translocation were previously detected. The IgH rearrangement (FR3/JH) and t(14;18) translocation (MBR bcl2-JH) were detected in peripheral blood mononuclear cells by polymerase chain reaction. Fifteen of 29 patients (8 with IgH rearrangement, 6 with t(14;18) translocation, and 1 with both) were treated with either interferon-alpha or by combination therapy with interferon and ribavirin for 6 to 12 months. IgH rearrangement became negative in 7 of 9 treated patients compared with only 1 of 8 of nontreated patients (P <.02). The t(14;18) translocation became negative in 6 of 7 treated patients compared with 1 of 6 nontreated patients (P =.03). Disappearance of IgH rearrangement or t(14;18) translocation was strongly associated with virologic response to treatment. Two t(14;18)+ patients developed B-cell lymphoma during follow-up. Antiviral treatment appears to be effective in eliminating the clonal proliferation of B cells in patients with chronic HCV infection and may prevent the subsequent development of lymphoma. The mechanism can be related to a direct effect of interferon-alpha on the proliferating clone or to an indirect effect by eradicating the antigenic stimulus.

Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms.

The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor-IgG (TNFR-IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR-IgG is a chimeric IgG fusion protein bearing one N-linked glycosylation site in the Fc region and three complex-type N-glycans in the TNF-receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH(4)Cl. The amount of galactose (Gal) and N-acetylneuraminic acid (NANA) residues on TNFR-IgG correlated in a dose-dependent manner with the ammonium concentration under which the N-linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable beta-galactosidase and sialidase activity, which increased throughout the culture. The beta-galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N-glycans was observed in incubation studies using beta-galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans-Golgi) as well as intracellular nucleotide sugar pools (increases UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). Ammonium might also affect the expression rates of beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP-GNAc pool [sum of UDP-GlcNAc and UDP-GalNAc]). The ammonium effect on TNFR-IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N-glycosylation were found in the product synthesized in the presence of glucosamine. No differences in beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH(4)Cl. pH titration of endogenous CHO alpha2,3-ST and beta-1,4-GT revealed a sharp optimum at pH 6.5, the reported trans-Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans-Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR-IgG by a pH-mediated effect on glycosyltransferase activity.

NFE, a new transcriptional activator that facilitates p50 and c-Rel-dependent IgH 3' enhancer activity.

The induction of immunoglobulin heavy chain (IgH) 3' enhancer activity has been coupled to ligand/receptor-dependent activation of resting B cells. To search for transcriptional target sites that account for this induction, extracts from lipopolysaccharide (LPS)-stimulated B cells and cell lines were used. Here we describe, by gel-retardation analysis, the identification of an NF-kappaB site and an adjacent nuclear factor ets-like (NFE) site in the 3' enhancer. The NFE motif binds four protein complexes in resting B cell extracts, of which two are down-regulated upon LPS stimulation. Gel shift-shift experiments of the NF-kappaB complexes with specific antibodies identified p50 and c-Rel proteins to be the predominant factors in primary LPS-stimulated cell extracts. Site-directed mutagenesis of these motifs demonstrates that they contribute to part of the enhancer activity in plasma cells. One copy of the NFkappaB/NFE motifs, linked to a heterologous reporter construct, displays lymphoid-restricted reporter gene activity in transient transfection assays. Mutation of either site abrogates all promoter activity. Complementation experiments demonstrate that although p50 and c-Rel expression vectors reconstitute transcription of an intact NF-kappaB/NFE reporter construct in a dose-dependent manner, mutation of the NFE site or the NF-kappaB site abrogates essentially all transcriptional activity in both plasma cells and in COS cells. Taken together, we provide evidence for the existence of an activator, NFE, which in combination with the p50 and c-Rel proteins, are part of the transcription factor machinery that regulates 3' enhancer activity, and thus the control of the IgH locus in late B lymphocyte development.

Early B-cell factor (EBF) down-regulates immunoglobulin heavy chain intron enhancer function in a plasmacytoma cell line.

The immunoglobulin heavy chain intron enhancer contains two potential binding sites for early B-cell factor (EBF). To investigate the functional properties of these, EBF was expressed in the EBF non-expressing S194 plasmacytoma cell line and found to down-regulate the activity of a co-transfected immunoglobulin heavy chain intron enhancer reporter construct. The expression of an unrelated reporter construct was unaltered. Dividing the immunoglobulin heavy chain intron enhancer into two subregions showed that the EBF mediated down-regulation of expression was mediated by at least two independent sites. These data indicate a role for EBF in the regulation of immunoglobulin gene expression.

Staphylococcal enterotoxin D functions as a human B cell superantigen by rescuing VH4-expressing B cells from apoptosis.

Staphylococcal enterotoxins are potent superantigens, in that they activate T cells bearing specific V beta-chain gene segments. In this study, we analyzed the capacity of staphylococcal enterotoxin D (SED) to function as a B cell superantigen. SED induced T cell-dependent polyclonal proliferation and differentiation of B cells. In the absence of T cells, SED induced survival of B cells uniquely expressing VH4 containing IgM. The mechanism of survival of VH4-expressing B cells appeared to relate to the countering of apoptosis initiated by the engagement of HLA-DR by SED. Analysis of the VH4 gene products expressed by SED-stimulated B cells revealed the usage of six of the known functional VH4 genes with a variety of different CDR3 regions, employing different DH and JH gene segments. Moreover, the sequence analysis identified a possible site for SED binding of VH4 that includes the solvent-exposed surfaces of 3' CDR2/FR3 and/or FR1. Thus, SED appears to function as a unique B cell superantigen by inducing survival of VH4-expressing B cells.

Structure and expression of mouse germline immunoglobulin gamma 3 heavy chain transcripts induced by the mitogen lipopolysaccharide.

Germline immunoglobulin heavy chain gene transcription is though to direct isotype switching by modulating the accessibility of specific switch regions to a recombinase. In this study, cloned cDNA copies of mouse germline Igh-8 RNAs have been used to characterize the Igh-8 transcription unit. The 5' end of these transcripts are derived from an exon denoted Ig3, located 1 kilobase 5' of the Igh-8 switch region. Sequence analysis of cDNA and genomic clones reveals that these RNAs are noncoding. In splenic B cell cultures treated with lipopolysaccharide (LPS), germline Igh-8 transcript levels are upregulated after 8 h due to increased transcription. This induction is consistent with the identification of a putative binding site for the LPS inducible transcription factor NF-kappa B approximately 150 nucleotides upstream of the sites of transcript initiation. Furthermore, nucleotide sequence comparisons reveal that the region encompassing the site of germline Igh-8 transcription initiation is highly homologous to part of the Ig2b exon, and is also conserved upstream of the Igh-1 switch region. The implications of these findings for the control of germline Igh-8 transcription is discussed.

Immune function in patients treated with phenytoin.

Multiple immunologic side effects have been ascribed to phenytoin. Numerous reports in the literature discuss the possible cellular and humoral abnormalities that appear to be present in patients given phenytoin. The most consistent finding is a reduction in serum IgA found in up to 20% of patients. To resolve some of the conflicting studies on cellular immune status, 191 patients taking phenytoin were evaluated initially with an serum IgA determination, and then further immune studies were done on the 11% with IgA values lower than two standard deviations below the mean. Data collected included total lymphocyte counts, lymphocyte population studies, and responses to in vitro mitogen stimulation. Only 2 of 191 patients had serum IgA values less than 5 mg/dL, which is an incidence not significantly different than that in the population at large. The patients with decreased serum IgA values did not have an increased incidence of autoimmune phenomena, allergic disorders, gastrointestinal manifestations, or recurrent upper respiratory tract infections. Their cellular immune status showed no significant variations from control values. Thus it appears that routine monitoring of patients on phenytoin with serum IgA determinations is of limited value, and the immunologic side effects of phenytoin are not expressed as a cellular abnormality.

Rearrangement and expression of T cell antigen receptor genes in B cell chronic lymphocytic leukemia.

Fifty-nine patients with B cell chronic lymphocytic leukemia (B-CLL) were screened for clonal rearrangement of T cell receptor (TCR) beta and gamma chain genes. Four were found with rearranged TCR beta genes, but none had detectable rearrangement of TCR gamma genes. One typical patient with B-CLL had a TCR beta gene structure consistent with a variable-diversity-joining rearrangement into the C beta 2 gene on one allele. An apparently identical rearrangement pattern was seen in a second patient, which suggested that there may be a restriction on the repertoire of possible TCR beta gene recombinations in mature B cells. Two further patients had a simple deletion of sequences, consistent with a diversity-joining rearrangement into C beta 2 on one allele. All four patients had rearrangements of immunoglobulin heavy- and light-chain genes typical of mature B cell malignancies. However, on review of clinical, morphological, and immunophenotype data, two had features consistent with B cell prolymphocytic leukemia or B lymphoma, and a third had progressed to a prolymphocytic transformation. Low-level expression of a predominantly 1.0- to 1.2-kilobase germ line TCR beta gene transcript was detected in several B-CLLs and at a comparable level in the four with rearranged TCR beta genes. This, together with the low frequency of TCR gene rearrangement, suggests that most B-CLL cases arise at a developmental stage when factors required for TCR gene activity are not operative.