Is HLA class II allogenotyping universally applicable?
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DNA Probes↗
Biomedical subjects
D G Haegert
Publications and source records attributed to D G Haegert.
Phenotypic and genotypic heterogeneity of peripheral T-cell lymphoma.
A series of 21 phenotypically characterised T-cell lymphomas histologically defined as lymphocytic, lymphoblastic, immunoblastic, AILD type, pleomorphic, T-zone and Lennert's T-cell lymphoma, were investigated for T-cell receptor (TcR) and immunoglobulin (Ig) gene rearrangements. Phenotypic analyses of frozen sections and cell suspensions were heterogeneous and in many cases no single T-cell marker recognised all of the malignant cells. Data derived by staining with antibodies reactive with antigens in paraffin embedded tissue were consistent with T NHL in all cases except lymphoblastic lymphoma. TcR gene rearrangements were observed in lymphocytic, lymphoblastic and immunoblastic lymphoma, however, in the remaining 14 phenotypically and histologically defined peripheral T-cell lymphomas, 2 showed rearrangement of TcR gamma and beta genes consistent with T NHL and 2 showed Ig JH rearrangements only, suggestive of either reactive T-cell populations masking cryptic disease or presence of tumour populations with aberrant gene rearrangement and expression of T lineage antigens. No Ig or TcR gene rearrangements were found in the remaining 10 cases, in which morphologically identifiable tumour cells comprised 10-90% of the cell population. In 3/6 cases tested some CD3 positive cells failed to stain with WT31 or beta F1, monoclonal antibodies that recognise determinants on combined TcR gamma beta or TcR beta chains respectively. Whether these cases represent tumours arising from an undetermined cell of origin or polyclonal expansions of T-cells remains to be determined. Our results confirm the phenotypic heterogeneity of histologically defined peripheral T-cell lymphoma and indicate that in these particular histological subtypes gene rearrangement analysis can also yield heterogeneous results which may be unhelpful in determining cell lineage and clonality.
DNA Probes↗
CD3+ CD8+ T cell lymphocytosis masking B cell leukaemia.
A patient with CD3, CD8 positive lymphocytosis presented with features consistent with T cell chronic lymphocytic leukaemia/proliferations of large granular lymphocytes. The marrow and blood lymphoid populations (19.4 x 10(9)/l) contained more than 80% CD3 and CD8 positive cells with no evidence of a monotypic B cell population. A biopsy specimen of a vasculitic rash showed a diffuse infiltrate of CD3, CD8 positive cells into the upper dermis, consistent with T cell lymphocytic disease. After follow up for two years without treatment the blood lymphocyte count was 53 x 10(9)/l and was composed of cytologically small lymphocytes. A monoclonal SIg M D k lymphoid population (more than 90%) was demonstrable in sample blood and marrow aspirate. Gene rearrangement studies carried out on DNA extracted from peripheral blood lymphocytes at presentation and at two year follow up exhibited JH and Ck immunoglobulin gene rearrangement but no rearrangement of T cell receptor TcR gamma and beta genes. It is thought that this is the first well documented case of an aggressive CD8 positive lymphocytosis preceding, or in response to, an underlying B cell neoplasm.
Antigens, Differentiation, T-Lymphocyte↗
Co-expression of T3 and surface immunoglobulin in neoplasms of 'early' B cells: a report of 2 cases.
Chronic lymphocytic leukaemia (CLL) and well-differentiated lymphocytic lymphoma (WDLL) characteristically express surface membrane immunoglobulin (SIg) and react with monoclonal antibodies (Mabs) directed against a marker present on most normal T cells, namely T1. These SIg-positive B cells are usually negative for other T cell markers including T3 (CD3) which is expressed in all normal T cells. We report 2 cases, 1 CLL and 1 WDLL, in which the peripheral lymphocytes not only expressed monotypic SIg but also reacted with the T3-specific Mabs UCHT1 and OKT3 by indirect immunofluorescence or immunoperoxidase staining. False-positive staining by both UCHT1 and OKT3 was excluded by showing that lymphocytes sensitized with an irrelevant mouse Mab did not stain with second layer antibodies and that lymphocytes stained with second layer antibodies alone were always completely unreactive. Also, in 1 case the determinants demonstrated by both anti-Ig and UCHT1 were re-expressed after capping and shedding, i.e. appeared to be endogenous. The B cell origin of the CLL lymphocytes was supported by the finding of CK gene rearrangement in the absence of T beta gene rearrangement. It seems therefore that neoplasms of 'early' B cells may show bidirectional differentiation and co-express markers that are believed to be T-specific.
B-Lymphocytes↗
Co-expression of surface immunoglobulin and T3 on hairy cells.
Whereas it is generally believed that most cases of hairy cell leukaemia (HCL) represent B cell neoplasms, it is reported here that 4 of 14 cases of HCL not only express monotypic surface immunoglobulin (SIg) but also react with the T cell-specific monoclonal antibody (Mab) UCHT1 by indirect immunofluorescence or immunoperoxidase staining. Although both UCHT1 and OKT3 recognize the T3 molecule, which is expressed in all normal T cells, UCHT1+ hairy cells (HCs) were consistently OKT3-. Spurious reactivity of UCHT1 antibodies with SIg+ HCs was excluded by showing that lymphocytes sensitized with an irrelevant mouse Mab did not stain with second layer antibodies and lymphocytes sensitized with second layer antibodies alone were always completely unreactive. Moreover, in 1 case the determinants demonstrable by both UCHT1 and anti-Ig were strongly re-expressed after capping and shedding, i.e. appeared to be endogenous. It is concluded that HCs with a hybrid T-B surface phenotype are more common than hitherto reported, that HCs may express T3 molecules as well as SIg but that determinants recognized by OKT3 may be cryptic or buried in the HC membrane.
Antibodies, Monoclonal↗
Demonstration of T4, T8, M1 and B7 determinants on human T cells with a rosette test: implications for the assay specificity of monoclonal antibodies.
Under optimal test conditions significantly more freshly isolated human T cells reacted with OKT4, OKT8, OKM1 and OKB7 monoclonal antibodies (Mabs) in the indirect antiglobulin rosetting reaction (IARR) than by indirect immunofluorescence. Rabbit erythrocytes (E) coated with anti-mouse immunoglobulin were more sensitive indicator cells in the IARR than similarly coated sheep E. Treatment of T cells with neuraminidase further enhanced T cell reactivity in the IARR with each Mab so that an average of 60% or more of T cells were T4+, T8+ and M1+ and at least 40% had the T4+ T8+ phenotype. The various findings suggest that the rosette assay detects determinants on T cells that are expressed below the detection threshold of immunofluorescence. Moreover, these findings indicate that the cellular specificities of a particular Mab may change when one assay system is substituted for another or when the protocol of a particular assay is altered.
Animals↗
Expression of T3 and T11 determinants on human peripheral lymphocytes as analysed by the indirect antiglobulin rosetting reaction (IARR) and indirect immunofluorescence.
Whereas by indirect immunofluorescence approximately 80% of freshly isolated human peripheral blood lymphocytes bound T3 (Leu 4)-specific and T11-specific monoclonal antibodies, by a more sensitive technique--the indirect antiglobulin rosetting reaction--more than 90% of these lymphocytes were T3+ (Leu 4+) and T11+; this finding suggested that some non-T cells may also express T3 and T11 determinants. In double labelling experiments most normal surface membrane immunoglobulin (smIg)-bearing lymphocytes, ie, B cells, were shown to express T3 and T11 determinants at low density. The determinants were re-expressed after stripping with trypsin. Moreover, greater than 90% of neoplastic lymphoid cells expressing monotypic smIg from six donors were T3+ and/or T11+. Together these results provide new data on the cell distribution of 'T cell markers'. These findings also indicate that the rosette test is too sensitive to distinguish between biologically distinct lymphocyte population, ie between T and B cells.
Antibodies, Monoclonal↗
Direct and mixed antiglobulin rosetting reaction.
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Antibodies, Anti-Idiotypic↗
Expression of immunoglobulin-cross-reactive molecules by neoplastic human T cells. I. Surface detection and isolation of molecules reactive with chicken anti-F(ab')2, anti-alpha and anti-mu antibodies.
There is evidence that the T-cell antigen receptor is immunoglobulin-related and that normal human T cells express surface determinants which cross-react with chicken anti-F(ab')2, anti-alpha and anti-mu antibodies. Surface marker study of six neoplastic human T cell lines suggested that F(ab')2- and mu-cross-reactive determinants may be expressed independently of alpha-cross-reactive determinants. F(ab')2-cross-reactive materials--F(ab')2-CRMs--were then isolated from several T-cell lines and analysed by reverse passive haemagglutination and in immunoprecipitation experiments by polyacrylamide disc gel electrophoresis in buffers containing sodium dodecyl sulphate (SDS-PAGE) under reducing conditions. It was shown that MOLT-4- and CCRF-CEM-derived F(ab')2-CRMs differ serologically and that alpha- are indeed expressed independently of F(ab')2- and mu-cross-reactive determinants. The F(ab')2-CRMs were shown to be T cell-derived, to express determinants similar to or identical with F(ab')2-cross-reactive determinants expressed in normal T-cell membranes and to correspond closely to T-cell antigen binding molecules. Evidence was also obtained that the two F(ab')2-CRMs differ from one another in molecular weight. SDS-PAGE under non-reducing conditions of MOLT-4- and CCRF-CEM-derived F(ab')2-CRMs identified major components of mol. wt. 135,000 and mol. wt. 330,000 respectively. Immunoprecipitation of 125I-labelled F(ab')2-CRMs by chicken anti-F(ab')2 followed by SDS-PAGE under reducing conditions identified major components of mol. wt. 80,000 and mol. wt. of 51,000 from CCRF-CEM-derived F(ab')2-CRMs and tentatively identified components of mol. wt. 52,000 and mol. wt. 23,000 from MOLT-4-derived F(ab')2-CRMs.
Animals↗
Test systems for detection of human T associated Ig-related surface determinants using chicken antiglobulin reagents.
In earlier experiments the mixed antiglobulin rosetting reaction (MARR) and the direct antiglobulin rosetting reaction (DARR) were found to be of similar sensitivity in demonstration of immunoglobulin (Ig)-bearing B lymphocytes. These test systems were used in the present study together with chicken anti-F(ab')2, anti-IgM and anti-IgA antibodies to detect Ig-related determinants on human peripheral blood T cells. Although for each antiglobulin reagent the reactivity of viable T cells in the MARR and the DARR appeared to be optimal after treatment of the lymphocytes with neuraminidase, the MARR was always more sensitive than the DARR. Further, when test conditions were established so as to avoid erroneous detection of Ig bound to T cell Fc receptors, the MARR was found to be more sensitive than indirect immunofluorescence. Under the most sensitive mixed antiglobulin rosetting conditions, the mean number of T cells expressing F(ab')2-, alpha- and Mu-related determinants was 99%, 50% and 39% respectively. The complete inhibition of the MARR by human IgM when anti-IgM antibodies are used and the more than 70% inhibition by IgA when anti-IgA antibodies are used, largely exclude false positive rosette formation due to contaminating specificities directed against non-Ig determinants on T cells. Trypsin treatment of T cells removed all determinants recognized by anti-IgM and anti-IgA antibodies and these cells reexpressed these determinants during in vitro culture; this indicates that these determinants are T cell products and do not represent adsorbed Ig molecules.
Animals↗
The mixed antiglobulin rosetting reaction (MARR) and the direct antiglobulin rosetting reaction (DARR): sensitive tests for demonstration of immunoglobulin-bearing B lymphocytes.
The technology of the mixed antiglobulin rosetting (MARR) and the direct antiglobulin rosetting reaction (DARR) is reviewed. These tests have been found to be more sensitive than the standard direct immunofluorescence (DIF) method for delineation of Ig-bearing B lymphocytes and to have practical advantages over DIF. Further, with these rosette tests, an important B lymphocyte population has been identified which is Ig-negative by DIF and functionally quite different from typical DIF-positive B lymphocytes but which expresses a low density of surface membrane Ig that is detectable by the MARR and the DARR.
B-Lymphocytes↗
Human peripheral blood T lymphocytes express alpha-, mu- and F(ab')2-related determinants in their surface membranes.
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Antibodies, Anti-Idiotypic↗
Are most "null" cells B cells?
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B-Lymphocytes↗
Demonstration of immunoglobulin-related molecules on human peripheral blood T lymphocytes using chicken anti-F(ab')2 and anti-IgM antibodies.
A mean of 98% of lymphocytes in T-enriched preparations of human peripheral blood bound purified chicken anti-human F(ab')2 antibodies to their surface membranes as demonstrated by the mixed antiglobulin rosetting reaction (MARR). By reverse passive hemagglutination these antibodies reacted strongly with kappa light chains and with Fd gamma but gel diffusion analyses, absorption on affinity columns and inhibition experiments established that the anti-F(ab')2 antibodies were not isotype specific and that there was extensive serological cross-reactivity between Fd gamma and kappa and probably also between Fd gamma and mu chains. The antigenic determinants recognized on the T-cells surfaces were not shared by Ig fractions from several other mammalian species. Trypsin treatment of lymphocytes removed all determinants recognized by anti-F(ab')2 antibodies from approximately two-thirds of the cells but these cells almost completely re-expressed these determinants during in vitro culture; this indicates that the T-cell determinants seen by anti-F(ab')2 antibodies are T cell products and do not represent adsorbed Ig. Complete inhibition of the MARR by human F(ab')2 excludes false positive rosette formation due to contaminating specificities directed against non-Ig molecules on T cells. Together the various findings are consistent with the conclusion that most human peripheral blood T cells express Ig or Ig-related molecules in their surface membranes. A mean of 13% of lymphocytes in T-enriched preparations were reactive with mu-chain specific chicken antibodies; it may be that a minority of T cells express mu chain determinants in addition to those recognized by anti-F(ab')2 antibodies.
Animals↗
Do human B and null lymphocytes form a single immunoglobulin-bearing population?
Human "null" lymphocytes have been recognised because they lack T-lymphocyte membrane markers and the surface membrane immunoglobulin (smIg), detectable by direct immunofluorescence, which conventionally characterises B lymphocytes. But almost all null lymphocytes express smIg determinants which are demonstrable by the direct antiglobulin and mixed antiglobulin rosette tests. It is argued that the definition of a B lymphocyte should be extended to include the null cells which react in these tests. If the heterogeneity of the B-lymphocyte population is more widely recognized, the precise nature of human natural and antibody-dependent killer cells may become clearer.
Antibody Affinity↗
Delineation of IgM-receptor bearing human T and B lymphocytes using a direct plaque forming cell (PFC) assay.
Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4 degrees C, added to SRBC monolayers then incubated at 37 degrees C with guinea pig complement to develop the plaques. The PFC assay has methodological advantages over rosetting methods which demonstrate IgM receptors, and under certain conditions is more sensitive than these rosette techniques. A mean of 17% of freshly isolated uncultured lymphocytes, enriched for B cells, formed direct plaques while a mean of 3% of T-enriched preparations formed direct plaques. However, if the lymphocytes were preincubated with vibrio cholerae neuraminidase (VCN) these figures increased to 46% and 35% respectively. The specificity of plaque formation by VCN-treated lymphocytes was established. SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent failed to bind to VCN-treated lymphocytes, inclusion of IgM, but not other Ig molecules in the test medium, inhibited plaque formation, and, most important, plaque formation by T and B cells was inhibited by F(c)5 mu but not by Fab mu fragments. These results indicate that T and B lymphocytes express IgM-class specific membrane receptors, that these receptors may be hidden on normal lymphocytes but are revealed by treatment with VCN and that the IgM receptor on VCN-treated lymphocytes is F(c)mu specific. These findings are discussed briefly with regard to other and partly contradictory data obtained after overnight in vitro lymphocyte culture. As demonstrated by direct PFC assay, the B cell IgM receptor is trypsin sensitive.
B-Lymphocytes↗
Demonstration of IgM receptors on human peripheral blood monocytes using a direct plaque forming cell (PFC) assay.
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Antibodies↗
Demonstration of surface membrane immunoglobulin on L lymphocytes by the mixed antiglobulin rosetting reaction (MARR) and by the direct antiglobulin rosetting reaction (DARR).
Various preparations of human peripheral blood lymphocytes were examined for the presence of membrane-incorporated immunoglobulin (Ig) molecules using the direct antiglobulin rosetting reaction (DARR) and/or the mixed antiglobulin rosetting reaction (MARR). In an initial study, 97% of lymphocytes depleted of sheep erythrocyte (E) rosette-forming cells were found to be Ig positive by the MARR. This suggests that the majority of E rosette-negative lymphocytes are Ig-bearing B lymphocytes. Since most investigators divide E rosette-negative lymphocytes into an Ig-positive or B-cell population and an Ig-negative or null-cell population, it was decided to study further a well defined null-cell subset. Lymphocytes with membrane-labile IgG markers or L lymphocytes are reported to be E rosette negative, Ig negative by direct immunofluorescence (DIF), C3 receptor negative and Fc receptor positive. L lymphocytes were separated from DIF-positive B cells then tested for the presence of surface Ig, approximately 80% of L lymphocytes were found to be Ig positive with both the MARR and the DARR. Comparison of gamma, alpha and mu heavy chain determinants on both DIF-positive and L lymphocytes indicated that Ig isotypes are similar on both cell types. These various findings suggest that most L lymphocytes are of B-cell lineage.
Antibodies, Anti-Idiotypic↗