Lymphocyte Surface Immunoglobulin Research Articles

Chemo-Immunotherapy With Hyper-CVAD/MA and Rituximab For B-Cell Lymphomas Leads To Prolonged Immune Suppression Compared To Conventional Treatment With R-CHOP

BackgroundFractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone (hyper-CVAD) alternating with high-dose methotrexate and cytarabine (MA) therapy is a widely used combination chemotherapy for lymphoid malignancies. Its use in combination with Ribuximab (R) has been shown to be safe and effective for Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL). Acute toxicities of the treatment are mostly hematologic, leading to cases with neutropenic fever and infection. However, long-term toxicities in terms of patients' immune function have not been fully elucidated. PurposeTo evaluate the long-term immune function after R-hyper-CVAD/MA therapy, and to compared it with those after R-CHOP therapy. MethodPatients who have received R-hyper-CVAD/MA therapy or R-CHOP therapy for more than 6 cycles for B-cell lymphomas (diffuse large B cell lymphoma (DLBCL), BL and MCL) between January 2007 and December 2012 were identified. The patients' immune functions were assessed in regards to white blood cell (WBC) count, lymphocyte count, lymphocyte surface marker analysis and immunoglobulin (Ig) measurement. ResultAltogether, 21 patients underwent evaluation. 11 patients received R-CHOP therapy, and 10 patients received R-hyper-CVAD/MA therapy. The mean age of the patients was 57 and 53.3 years-old, respectively. All patients in the R-CHOP group had DLBCL, whereas patients who received R-hyper-CVAD/MA were either treated for BL in 4, MCL in 1 and DLBCL in 5 patients. The median time from the initiation of treatment to the assessment of immune function was 1060 (range 434 – 2475) and 947.5 (range 448 – 1966) days, respectively. In terms of immune function, IgG level, the proportion of CD4, CD8 positive (+) cells, CD4/8 ratio were significantly different between those who received R-CHOP therapy and R-hyper-CVAD/MA therapy. The median IgG level was 910 mg/dL in those who received R-CHOP therapy as opposed to 484 mg/dL in those who received R-hyper-CVAD/MA therapy. 4 patients in the R-CHOP group presented with IgG level below normal range, whereas 9 patients in the R-hyper-CVAD/MA group had an IgG level below normal range (p = 0.011). The median proportion of CD4 and CD8 (+) cells were 32.7%, 24.8% in the R-CHOP group and 23.35%, 41.35% in the R-hyper-CVAD/MA group, leading to the median CD4/8 ratio of 1.22 and 0.5, respectively. The total WBC count, lymphocyte count, proportion of CD19 (+) cells, IgM or IgA levels were within normal limit, and did not significantly differ between groups. Of the patients in the R-hyper-CVAD/MA group, 3 patients required regular immunoglobulin replacement, and 1 patient had 2 episodes of hospitalization within 2 years of last treatment, one due to viral meningitis and another due to pneumonia. None of the patients in the R-CHOP group presented with infection episodes that required hospitalization. ConclusionOur results suggest that patients with aggressive B-cell lymphoma treated with R-hyper-CVAD/MA therapy remain at higher risk of developing hypo-gammaglobulinemia, even after 5 years out of treatment. Despite the recovery of B-lymphocytes, the proportion of CD4 (+) cells remained low in those who received R-hyper-CVAD/MA therapy, leading to an increased proportion of CD8 (+) cells and decreased CD4/8 ratio. Patients who have received R-hyper-CVAD/MA therapy should be closely monitored for their immune function for a longer period due to prolonged hypogammaglobulinemia.Table 1R-CHOP (mean +/- SD)R-hyper-CVAD/MA (mean +/- SD)p-valueWBC (/mm3)4590 +/- 12864660 +/- 12470.902Lymphocyte (/mm3)1537 +/- 5911463 +/- 5580.772CD4 (%)34.1 +/- 13.024.7 +/- 7.50.049CD8 (%)23.9 +/- 10.042.5 +/- 17.80.011CD4/81.69 +/- 1.070.74 +/- 0.500.009CD19 (%)22.3 +/- 10.317.7 +/- 13.80.406IgG (mg/dL)1013 +/- 282551 +/- 3460.003IgA (mg/dL)205 +/- 77125 +/- 1130.11IgM (mg/dL)71 +/- 2496 +/- 910.396 Disclosures:Okamoto:Novartis, BMS, GSK, Kirin, Chugai, Alexion: Honoraria, Research Funding.

Detection of thoracotomy-induced alterations in cell- and humoral-mediated immune response.

It is well known that thoracotomy leads to several complications. In this study, effects of thoracotomy on cellular and humoral immunities have been investigated. Leukocyte counts and lymphocyte counts of 100 patients operated by thoracotomy have been determined preoperatively and on the postoperative 3rd hour, and 1st, 2nd, 3rd, 5th days. Also lymphocyte surface markers (CD3, CD4, CD8, CD4/CD8, CD19, CD16/56) and immunoglobulin levels (IgG, IgA, IgM, IgE) in 40 out of 100 patients in the preoperative period and postoperatively twice on 7th day and then in the 3rd week have been detected. For the methodology hemocounter, flow cytometer, immunoprecipitation, and enzyme-linked immunosorbent assay were used. A marked increase in leukocyte count while a marked decrease in lymphocyte count has been observed after thoracotomy (P<0.001). There was not any significant alteration in levels of lymphocyte surface markers and immunoglobulins in the postoperative period (P>0.2). According to these results, leucocytosis occurred but lymphocyte count decreased in the early postoperative period. Immunoglobulin levels and subpopulation of lymphocytes were not affected from the operative stress.

Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway.

Ligation of a B lymphocyte surface immunoglobulin (sIg) antigen receptor (AgR) by its specific Ag ligand initiates a signaling pathway that culminates in B cell activation. However, many events of this pathway have not been elucidated. Here we present three novel findings that demonstrate directly that AgR-mediated signaling in B cells functions by the p21ras/ras.GAP-dependent pathway. First, stimulation of TA3 7.9 Ag-specific murine B lymphoma cells for 2 min with either Ag or F(ab')2 anti-IgM induces p21ras activation as measured by an increase in the GTP/GDP ratio of its bound nucleotides. This activation of p21ras does not occur via a change in its guanine nucleotide exchange rate. Second, Ag stimulation results in the inhibition of activity of p120 ras.GAP, a protein that regulates p21ras activation. Tyrosine phosphorylation of ras.GAP occurs within 1 min after Ag stimulation but is no longer detectable at 20 min after stimulation, at which time ras.GAP activity remains inhibited. Thus, tyrosine phosphorylation of ras.GAP is not required for the inhibition of its activity. Third, despite the role proposed for a ras.GAP-associated p190 protein in the control of ras.GAP activity in B cells, p190 was not detectable either in anti-ras.GAP immunoprecipitates of [35S]methionine labeled lysates of Ag-stimulated or -unstimulated 7.9 cells or as a tyrosine phosphoprotein in Western blots of anti-ras.GAP immunoprecipitates of Ag-stimulated 7.9 cell lysates. Inasmuch as the TA3 7.9 B lymphoma is representative of a mature, sIgM-bearing B cell, our observations raise the intriguing possibility that the capacity of p190 to associate with ras.GAP and regulate the activities of ras.GAP and p21ras in a B cell is dependent on the stage of differentiation of the B cell.

Immunohistochemical studies on small intestinal mucosa of Kawasaki disease

The expression of immunoglobulin b locus (k chain) allotypes on the surface of rabbit peripheral blood lymphocytes (PBL's) is examined using an indirect double immunoelectron microscopic labeling technique. Ferritin and whelk hemocyanin individually conjugated to allotypically specific IgG are used as ultrastructurally identifiable molecular markers. These indicators are coupled to lymphocyte surface immunoglobulin (Ig) allotypic determinants by an antiallotype antibody linkage. Human red blood cells, conjugated with IgG of a specific allotype and used as test cells, demonstrate the absolute specificity and high efficiency of the ultrastructural labeling technique. Specific labeling on rabbit PBL's shows that 65–75% of the cells are positive for surface Ig. Lymphocytes from homozygous donors (b4b4 or b6b6) are labeled specifically with only the appropriate allotypic labeling system. Thirty-three percent of the PBL's from heterozygous donors (b4b6) express both allotypes (allelic inclusion) on the cell surface; the remaining proportion of Ig-bearing cells have only one detectable allotype present (allelic exclusion). We conclude that approximately 50% of the Ig-bearing PBL's demonstrate allelic inclusion for the b locus allotypes. On allelically included heterozygous lymphocytes, both allotypic determinants can undergo specific endocytosis. Endocytosis of one allotype on heterozygous cells can be induced by stimulation with antiallotypic serum without affecting the surface appearance of the other allelic marker (separate endocytosis).

Humoral and cellular parameters of the immune system of Cebus apella monkeys. Cross reactivity between monkey and human immunoglobulins

The humoral and cellular immunological parameters of the New World non-human primate Cebus apella were analysed. The study included: serum protein immunoelectrophoretic analysis; cross reactivity between monkey and human immunoglobulins by immunoprecipitation, ELISA and indirect immunofluorescence tests; immunoglobulin quantitation by radial immunodiffusion; and assays with peripheral blood lymphocytes involving tests for E and EAC rosettes and detection of surface markers (surface immunoglobulins and CD4-CD8 antigens). The results obtained showed that (a) at least three immunoglobulins with electrophoretic mobility corresponding to IgG, IgA and IgM which showed cross reactivity with the human ones were present in serum; (b) it was possible to evaluate the relative monkey immunoglobulin concentration using specific antibodies against human immunoglobulins and to obtain absolute values using adequate conversion factors; (c) lymphocytes forming E and EAC rosettes were found in peripheral blood in a similar proportion to that reported in man; (d) lymphocyte surface immunoglobulins were detected using anti-human immunoglobulin serum; (e) it was not possible to demonstrate the presence of T helper and T suppressor/cytotoxic lymphocytes using OK T4 and OK T8 monoclonal antibodies.

Crosslinkage of B lymphocyte surface immunoglobulin by anti-Ig or antigen induces prolonged oscillation of intracellular ionized calcium.

Our results indicate that B lymphocytes stimulated with anti-Ig or antigen exhibit repetitive [Ca2+]i transients which persist for hours. The magnitude of these transients favors an important and ongoing role for [Ca2+]i elevation in antigen driven B cell activation. Repetitive Ca2+ transients may prove to be a prevalent mechanism of Ca2+ signaling. In preliminary experiments (with L. E. Samelson and R. D. Klausner), we have observed Ca2+ transients in cloned T cells stimulated with antigen. Woods et al. have described repetitive free Ca2+ transients in hepatocytes stimulated with extracellular ligands promoting glycogenolysis, and suggest that the intervals of base-line [Ca2+]i levels explain the absence of mitochondrial overload in chronically stimulated cells. These considerations apply equally to B lymphocytes and recommend caution in delineating the range of Ca2+-mediated functions by prolonged coculture of cells with Ca2+ ionophores. Our experiments were done in a simple recording chamber with one cell type. No cell interactions were observed. Given the variety of indicator dyes now available, the technical approach we present, augmented by a more sophisticated recording chamber, is a potentially powerful tool for examining the intrinsic, and T- or accessory cell-dependent, physiology of B cell differentiation.

Small-Cell Lymphoma and Sjögren's Syndrome

To our knowledge, the association of the plasmacytoid variety of small-cell lymphoma with primary Sjögren's syndrome has not been reported. We describe a patient with SS who developed plasmacytoid small-cell lymphoma. As opposed to the commonly detected IgM, the lymphocyte surface immunoglobulins contained monoclonal IgA/kappa light chains. In addition to the unique immunopathologic features, the impressive response to chemotherapy and the importance of surface immunoglobulin markers in the diagnosis of malignancy in Sjögren's syndrome are discussed.

High expression of lymphocyte surface immunoglobulin in chronic lymphocytic leukaemia may be a bad prognostic sign.

Lymphocytes from 27 patients with B-type chronic lymphocytic leukaemia (CLL) were tested with immunofluorescence for expression of surface membrane immunoglobulin (SmIg) and the B cell antigen 7420. When age at time of testing, sex, clinical stage according to Binet, disease progression according to Levin and percentage of SmIg and 7420 antigen-expressing cells were used as explanatory variables for survival in a linear logistic regression model, only the SmIg variable was significant (p less than 0.025). A high proportion of SmIg bearing cells (greater than 61%) was associated with short survival, large tumour mass and aggressive disease. The tentative conclusion is drawn that the differentiation stage of the tumour cells in CLL determines the clinical course of the disease and is inversely correlated to the SmIg expression.

Altered expression of B lymphocyte surface immunoglobulins in minimal change nephrotic syndrome and focal glomerulosclerosis.

In 20 patients with nephrotic syndrome (10 minimal change glomerulonephritis, MCN; 10 focal glomerulosclerosis, FGS) the peripheral blood lymphocytes showed a statistically significant increase in IgG-bearing cells (SIgG-C) in comparison with 30 patients with other histological types of primary glomerulonephritis with and without nephrotic syndrome (14 and 16, respectively). In the same MCN and FGS patients the serum IgG levels were slightly decreased. Furthermore, 5 cases of MCN in sustained remission for 1 year after steroid therapy showed normalization of the SIgG-C and the serum IgG levels. The possible significance of these phenomena in the pathogenesis of the hypo-IgG-globulinemia in MCN and FGS is discussed.

Changing behavior of surface immunoglobulin on mouse B lymphocytes during immune development: I. LPS-induced changes in the lateral mobility of B lymphocyte surface immunoglobulins on two populations which express different surface immunoglobulin phenotypes

The redistribution of surface membrane immunoglobulin molecules (sIg) was studied in two functionally distinct populations of mouse splenic B lymphocytes, namely, those bearing membrane IgM(IgG −) and those bearing IgG. Brief exposure to mitogenic doses of bacterial lipopolysaccharide (LPS) produced direct but differential effects on the subsequent ability of specific antibodies to induce this redistribution on each cell type. Studied as a function of temperature, antibody-induced redistribution of sIgM on cells previously exposed to LPS was observed to occur at temperatures lower than the temperatures required for similar sIgM redistribution on lymphocytes not exposed to LPS. In contrast, mitogen-treated sIgG + cells demonstrated an opposite and long-lasting effect (at least 40 hr), requiring higher temperatures to allow sIgG movement comparable to that seen on untreated sIgG-bearing lymphocytes. Thus, we conclude that LPS interacts with both IgM +(IgG −) and IgG + lymphocytes, but that such interactions produced different membrane effects on each B-cell subset. This membrane change can therefore be useful as a quasi-functional differentiation marker. Furthermore, differences in sensitivity to cellular activation by LPS seen between sIgM-bearing (sIgG −) and sIgG-bearing B cells may be a reflection of such direct, although different, membrane effects.

Dimethyl sulfoxide inhibits capping of surface receptors

The microfilaments of cellular slime mold cells are known to be dislocated from their attachment to the plasma membrane by 10% dimethyl sulfoxide (DMSO). We report here that 10% DMSO inhibited capping of lentil lectin receptors on the surface of vegetative cells of the cellular slime mold, Dictyostelium mucoroides When the DMSO was washed from the cells, capping took place. Capping of surface immunoglobulin of mouse lymphocytes was also reversibly inhibited by 10% DMSO.

Inhibition of the mobility of mouse lymphocyte surface immunoglobulins by locally bound concanavalin A.

Fluorescence photobleaching recovery was used to study directly and quantitatively the inhibition of the lateral mobility of surface immunoglobulins (sIg) on mouse lymphocytes by localized binding of concanavalin A (Con A) coupled to platelets. Up to a threshold occupancy of about 10% of the upper cell surface by Con A-platelets, the diffusion coefficient and mobile fraction of sIg remained as in untreated cells (5.3 X 10(-10) cm2/sec and 0.65, respectively). At higher surface occupancy, these values decreased to 8 X 10(-11) cm2/sec and 0.11. The magnitude of the effect was independent of the percentage occupancy above the threshold and of the distance from the bound Con A-platelets, indicating a cooperative and propagated phenomenon. Treatment with colchicine or cytochalasin B separately induced only partial reversal of the Con A-induced modulation. Treatment with both reversal of the Con A-induced modulation. Treatment with both drugs together was synergistic and fully reversed the mobility inhibition. The modulation was unaffected by NaN3 and 2-deoxyglucose, suggesting no dependence on metabolic energy. Con A-platelets did not affect the mobility of a lipid probe. Models for the Con A-induced modulation and the relationship between the effects of Con A on sIg mobility and patch formation are discussed.

Inhibition of antigen-induced and anti-immunoglobin-induced capping by hydrocortisone and propranolol

The redistribution of lymphocyte surface immunoglobulin (Ig) induced by anti-lg antibody or bound sheep erythrocyte antigen (capping) was inhibited reversibly by hydrocortisone succinate (2 × 10−4 M). Capping inhibition was also effected by cholesterol and progesterone, favoring the hypothesis that the effect was mediated through changes in the physical properties of the cell membrane rather than hormonal action. Capping was also inhibited by propranolol at concentrations where it may exert a local anesthetic as well as a β-adrenergic blocking effect (2 × 10−4 M). Since a fourfold excess of the β agonist isoproterenol failed to restore capping in the presence of propranolol, the local anesthetic mode of action is most likely. Nitroprusside, an inhibitor of contraction in vascular smooth muscle and platelets, failed to inhibit capping.

Isotypic discordance of paraproteins and lymphocyte surface immunoglobulins in myeloma

B lymphocyte surface immunoglobulins (Smlg) were studied in 24 patients with multiple myeloma by means of anti-isotypic antisera, and their heavy and light chain isotypes were compared in each patient with those of the paraprotein. In 21 patients, lymphocyte Smlg consisted of only one light chain type, and in 16 of only 1 heavy chain type. However, the Smlg and paraprotein heavy and light chain types were identical in only 5 patients while in 6 they differed in heavy and light chain types, in 7 in light chain type, and in 4 in heavy chain type. In 2 patients with light chain myeloma, Smlg light chains were isotypically the same as the paraprotein. Isotypic discordance between paraprotein and Smlg may signify the proliferation of a second malignant clone with failure to differentiate into secreting plasma cells. Alternatively, it is conceivable that the lymphocyte Smlg could have the same idiotypic specificity as the paraprotein despite the isotypic differences, but this will require further studies using anti-idiotypic antisera.

Isotypic Discordance of Paraproteins and Lymphocyte Surface Immunoglobulins in Myeloma

B lymphocyte surface immunoglobulins (Smlg) were studied in 24 patients with multiple myeloma by means of anti-isotypic antisera, and their heavy and light chain isotypes were compared in each patient with those of the paraprotein. In 21 patients, lymphocyte Smlg consisted of only one light chain type, and in 16 of only 1 heavy chain type. However, the Smlg and paraprotein heavy and light chain types were identical in only 5 patients while in 6 they differed in heavy and light chain types, in 7 in light chain type, and in 4 in heavy chain type. In 2 patients with light chain myeloma, Smlg light chains were isotypically the same as the paraprotein. Isotypic discordance between paraprotein and Smlg may signify the proliferation of a second malignant clone with failure to differentiate into secreting plasma cells. Alternatively, it is conceivable that the lymphocyte Smlg could have the same idiotypic specificity as the paraprotein despite the isotypic differences, but this will require further studies using anti-idiotypic antisera.

The turnover of surface immunoglobulins of small lymphocytes and blastic cells

We have previously shown that the maintenance of surface Ig by most B cells in culture is dependent on the continuous presence of polyclonal B-cell activators (PBA). We put forward the hypothesis that lymphocytes shed their surface Ig but replace it only if they are continuously stimulated i.e., that the turnover of surface Ig is a mitogen-dependent process. To demonstrate this hypothesis, rabbit spleen cells were labeled with 125I-Fab anti-light-chain allotype antibody and were incubated in cultures supplemented with normal rabbit serum of an allotype different from that present on the lymphocytes. We found that the surface Ig is released with a half-life of 2 hr whether a PBA was present or not. However, the surface Ig was regenerated only in the presence of the PBA. The decrease in the percentage of cells bearing surface Ig paralleled to a large extent the decrease in the total amount of surface Ig determined by 125I-Fab antibody. The surface Ig of blastic B cells induced in cultures with dextran sulfate had the same half-life of 2 hr, suggesting that the turnover of surface Ig of small and blastic lymphocytes is similar and that it is regulated without any relation to cell activation. The basic phenomena described here and previously for rabbit lymphocytes also occurred when human or mouse lymphoid cell suspensions were used. We concluded that (a) the turnover of surface Ig in vitro is a mitogen-dependent process with a half-life of 2 hr, (b) its regulation is not dependent on cell activation, and (c) it is similar in man, rabbit, and mouse.

Alternative hypotheses of lymphocyte surface immunoglobulin expression, B lymphocyte activation and B lymphocyte differentiation.

Alternative hypotheses of lymphocyte surface immunoglobulin expression, B lymphocyte activation and B lymphocyte differentiation.

The effect of local anesthetics on the lateral mobility of lymphocyte membrane proteins

In this study, we have measured by fluorescence photobleach recovery (FPR) the relative mobilities of two rat lymphocyte membrane proteins, surface immunoglobulin (SIg) and AgB (histocompatability antigens). The results of the FPR experiments showed that SIg was relatively restricted in its mobility as compared with AgB. Treatment of cells with local anesthetics (LA), agents which are assumed to act, in part, by severing the linkage between the cytoskeleton and integral membrane proteins increased the mobility of SIg to a mobility similar to that of AgB. This finding suggests that SIg is relatively restricted in its mobility due to a tethering by cytoskeletal elements. LA also had a small, but significant effect on the mobility of AgB.

Effect of molar ratio of fluorescent dye to IgG on the detection of lymphocyte surface immunoglobulins by immunofluorescence.

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S-Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained. 1. The percentage of S-Ig bearing cells increased markedly when labeled anti-human H- or L-chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S-Ig was caused by increased non-specific adsorption of the fraction of labeled antibody with a high F/P molar ratio. 2. This non-specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mcie showed less non-specific adsorption than those from humans. 3. It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.

Rhesus Monkey B Lymphocyte Surface Immunoglobulin: Analysis with a Fluorescence-Activated Cell Sorter

Abstract Rhesus monkey spleen, mesenteric lymph node, peripheral lymph node, blood, thymus, and bone marrow mononuclear cell suspensions were stained with fluorescein-conjugated F(ab′)2 fragments of affinity-purified rabbit anti-human Fab, rabbit anti-human γ, rabbit anti-human µ, rabbit anti-human δ, and/or rabbit anti-human α as well as rabbit anti-keyhole limpet hemocyanin (RaKLH) and analyzed for fluorescence intensity and frequency of positive cells with a fluorescence-activated cell sorter. Greater than 90% of surface (s)IgM-bearing spleen, blood, and lymph node lymphocytes bore sIgD, whereas only approximately 40% of sIgM-bearing bone marrow lymphocytes had sIgD. In all of these organs greater than 90% of sIgD-bearing cells also had sIgM. Substantially smaller numbers of lymphocytes bore sIgG than sIgM or sIgD, and most sIgG-bearing cells in all organs studied lacked sIgM and sIgD. Few if any cells in any organ studied bore detectable sIgA, and less than 1% of thymus cells had any sIg. B cell populations from spleen, peripheral lymph node, and mesenteric lymph node averaged similar quantities of sIgM and sIgD per B cell. Blood B cells averaged twice as much sIgM and sIgD as B cells from spleen and lymph node, and 9 times as much sIgD as sIgD+ bone marrow cells. sIgD+ bone marrow B lymphocytes have considerably more sIgM than sIgD- sIgM+ bone marrow B cells. Our data confirm that the sIgD+ sIgM+ cells are the predominant B lymphocyte in primates, and suggest that monkey B cells acquire sIgD in the marrow before migrating to other lymphoid organs.