Scattd. J. Imtntmol. 10, 127-133, 1979
Covariances of Mitogenic Responses in Leukaemic Blood Lymphocytes: a Functional Marker System for the Human B Lymphocyte Differentiation K.-H. ROBERT & B. NILSSON Department of Clinical Imtnunology and Section of Oncology and Haematology, Department of Medicine. Karolinska Institute, Huddinge Hospital, Huddinge, Sweden
Robert, K.-H. & Nilsson. B. Covarianees of Mitogenic Responses in teukaemic Blood Lymphocytes: a Fiinciional Marker System lor ihc Human B Lymphocyte Ditlereniiaiion, Scand. J, Immunol. 10. 127 1.13, 1979. Various polycional activating substances have been shown lo stimulate human chronic lymphntic leukaemia (CLLl cells lo undergo blast transformalion, to divide and lo secrete monoclonal immunoglobulin. CLL eells from dilTerent patients show distinct response palterns to these ligaiids. We have statistically analysed these response patterns and found that responses to certain ligands demonstrate covariance: Ihat is. a high response to one ligand is statistically associated with a high response to another ligand. A factor analysis of ihese data on the basis of responses of CLL cell.s from tweniy-onc patients and from the use of five diflcrent ligands in three dilTerent concentrations has shown that as few as two factors can account for as much as 63"mptoms. and a monoclonal B cell dominance in iheir peripheral blood. Culture procedure and assay far DNA synthesis. As enrlier described in detail [17]. microcullures of lymphocytes were stimulated by three dtHcrent conLcmraiions ol' ihc mitogens: phytohaemagglutinin (PHA) (I'UKK). l i l K l . I'lO). DxS (5. 50. 5(X) [Ag/ml). LPS (0.25. 2.5. 25 [ig.ml). anii-fi^m tl/40. IIO, 1/4), and PPD (2.5. 25. 250 iig.'mll. Cultures were harvested on day .^ and tested for increased DNA synthesis by 'H-thymidine incorporation, added 24 h before harvest. A.f.say for 'B' cells. Isoliited !ymphi>cytc> were incubated in balanced salt solution (BSS) for .10 mln at .n C. After three washes in .17 C BSS. cells were incubtited with liuorcscein conjugated l-(ab)^, Tragmenls (Kemila PrepEiriit AB. Kullcsmd, Minn.) of goal antihuman kappu und lambda sera and ihereufier washed in iee-cold BSS ihree times |12i. Cells were inspected for surface membrane fluorescence, and the dominance of either kappa or lambda tight chain expression on Ihe cells was used as a criterion for Ihe monoclonal B cell expansion in the disease. A.t.\ay for T celh. The capacity of normal T cells to form spontaneous rosettes with sheep erythrocytes (E-R FC) 18] was used as a marker Tor T cells. .Separation of T-cells. fc-Rf-C rosetled celh were separated from non-rosetted eells on a hicoll Isopaque gradient and red cells lyseJ by incubation with fresh human AB serum. Statistical analysis. Datu were analysed as means of triplicate cultures in terms of net cpm —thai is. subtraction of the spontaneous cpm from the experimental cpm. The SEM values of the triplicate cultures were within 10",, of the means. Correlation analysis. Correlation analysis was performed according to Spearman's rank order correlation |9] which IS nonparametric. In contrast to parametric tests such us ihe /-test in regression analysis, this test is independent of whether or noi the observations arc normally tlistribuied. Spearman's correliition is recommended when the observations are continuous- that is. not characterized by a large number of ties ai each rank, in this investigation (he observations (net cpm) were not normally distributed, but eontinuou.s with no or Tew ties at the different ranks. The correlation coefficients were regarded as significant if the probability values in a one-sided lest were less ihan 0.025. tactor analysis. The most distinctive characteristic of factor analysis 15] is its data reduction capability. Given an array of correlation eoefllcients for a set ofvariables. factor-analytic techniques enables conclusions as lo underlying putierns of relationships, so that data may
Covariances of Mitogenic Responses be 'rearranged' or "reduced' to a smaller sei of factors or components. These may then be regarded as a source of variables accounting for the observed interrelations in the data. In the factor-analysis used here, the hypothetical influence of only two factors on ihe lotal variance of responses to fifteen dilTerent mitogenic concentrations was tested. Hereby the two factors were represented by two axes in a coordinate system. The responses to each mitogenic concentration are (hen represented by one pair of coordinates, determining the relative influence of the two factors on the responses to this mitogenic concentration. The validity of (he postulated factors can be determined by the rate of the total variance of observations which can be explained by the faetors.
RESULTS T cell fraction in CLL hlood tympbocyte preparations The proportion of T cells, determined as E-RFC, in the cell cultures from the patients, varied between 0.5% and 22",,. However. 80",, of the cell cultures contained less than 10'/;, E-RFC positive cells. All CLL cell cultures demonstrated a typical monoclonal B cell dominance as indicated by kappa or lamhda light chain restriction on surface membratie Ig positive (Snilg) cells.
PPD 25fjg/ml p=0.003
Covatiatiee of responses to mitogens in CLL blood tymphocytes Blood lymphocytes from twenty-one individual patients with CLL were cultured in vitro in the presence orfive different activating ligands. each in three diflerent concentrations. Data from studies of cells from twelve individual patterns were included in an earlier report [17], which however lacks a statistical evaluation of the data such as is presented here. Thus the data used for the eovariance determinations are derived from a very large number of individual CLL cell cultures. Though dominated by the leukaemic cells, the citltures also contained a few normal ceils. In order to minimize the risk o\' an expansion of these cells, ciilitires were harvested already on day .1. Correlations were either absent or positive for all mitogens tested (Fig. 1). Unlike the other mitogens, the DxS and anti-fiom correlations were strongly discordant with regard to different concentrations, and for these mitogens the lowest and the highest concentrations are presented in Fig. I. Data can be simimarized as follows. Responses to the lower DxS concentration and LPS were significantly correlated {/'l
DxS SOOfjg/ml
p=0.243
p=o,ni
p=0.206
p=( 1.003
p^0,023
p=0.]77
p=0.401
p.O.l.ll
p^O.014
P=0.n27
p=0,095
p=().O78
p=0.479
p
Covariances of Mitogenic Responses in Leukaemic Blood Lymphocytes: a Functional Marker System for the Human B Lymphocyte Differentiation K.-H. ROBERT & B. NILSSON Department of Clinical Imtnunology and Section of Oncology and Haematology, Department of Medicine. Karolinska Institute, Huddinge Hospital, Huddinge, Sweden
Robert, K.-H. & Nilsson. B. Covarianees of Mitogenic Responses in teukaemic Blood Lymphocytes: a Fiinciional Marker System lor ihc Human B Lymphocyte Ditlereniiaiion, Scand. J, Immunol. 10. 127 1.13, 1979. Various polycional activating substances have been shown lo stimulate human chronic lymphntic leukaemia (CLLl cells lo undergo blast transformalion, to divide and lo secrete monoclonal immunoglobulin. CLL eells from dilTerent patients show distinct response palterns to these ligaiids. We have statistically analysed these response patterns and found that responses to certain ligands demonstrate covariance: Ihat is. a high response to one ligand is statistically associated with a high response to another ligand. A factor analysis of ihese data on the basis of responses of CLL cell.s from tweniy-onc patients and from the use of five diflcrent ligands in three dilTerent concentrations has shown that as few as two factors can account for as much as 63"mptoms. and a monoclonal B cell dominance in iheir peripheral blood. Culture procedure and assay far DNA synthesis. As enrlier described in detail [17]. microcullures of lymphocytes were stimulated by three dtHcrent conLcmraiions ol' ihc mitogens: phytohaemagglutinin (PHA) (I'UKK). l i l K l . I'lO). DxS (5. 50. 5(X) [Ag/ml). LPS (0.25. 2.5. 25 [ig.ml). anii-fi^m tl/40. IIO, 1/4), and PPD (2.5. 25. 250 iig.'mll. Cultures were harvested on day .^ and tested for increased DNA synthesis by 'H-thymidine incorporation, added 24 h before harvest. A.f.say for 'B' cells. Isoliited !ymphi>cytc> were incubated in balanced salt solution (BSS) for .10 mln at .n C. After three washes in .17 C BSS. cells were incubtited with liuorcscein conjugated l-(ab)^, Tragmenls (Kemila PrepEiriit AB. Kullcsmd, Minn.) of goal antihuman kappu und lambda sera and ihereufier washed in iee-cold BSS ihree times |12i. Cells were inspected for surface membrane fluorescence, and the dominance of either kappa or lambda tight chain expression on Ihe cells was used as a criterion for Ihe monoclonal B cell expansion in the disease. A.t.\ay for T celh. The capacity of normal T cells to form spontaneous rosettes with sheep erythrocytes (E-R FC) 18] was used as a marker Tor T cells. .Separation of T-cells. fc-Rf-C rosetled celh were separated from non-rosetted eells on a hicoll Isopaque gradient and red cells lyseJ by incubation with fresh human AB serum. Statistical analysis. Datu were analysed as means of triplicate cultures in terms of net cpm —thai is. subtraction of the spontaneous cpm from the experimental cpm. The SEM values of the triplicate cultures were within 10",, of the means. Correlation analysis. Correlation analysis was performed according to Spearman's rank order correlation |9] which IS nonparametric. In contrast to parametric tests such us ihe /-test in regression analysis, this test is independent of whether or noi the observations arc normally tlistribuied. Spearman's correliition is recommended when the observations are continuous- that is. not characterized by a large number of ties ai each rank, in this investigation (he observations (net cpm) were not normally distributed, but eontinuou.s with no or Tew ties at the different ranks. The correlation coefficients were regarded as significant if the probability values in a one-sided lest were less ihan 0.025. tactor analysis. The most distinctive characteristic of factor analysis 15] is its data reduction capability. Given an array of correlation eoefllcients for a set ofvariables. factor-analytic techniques enables conclusions as lo underlying putierns of relationships, so that data may
Covariances of Mitogenic Responses be 'rearranged' or "reduced' to a smaller sei of factors or components. These may then be regarded as a source of variables accounting for the observed interrelations in the data. In the factor-analysis used here, the hypothetical influence of only two factors on ihe lotal variance of responses to fifteen dilTerent mitogenic concentrations was tested. Hereby the two factors were represented by two axes in a coordinate system. The responses to each mitogenic concentration are (hen represented by one pair of coordinates, determining the relative influence of the two factors on the responses to this mitogenic concentration. The validity of (he postulated factors can be determined by the rate of the total variance of observations which can be explained by the faetors.
RESULTS T cell fraction in CLL hlood tympbocyte preparations The proportion of T cells, determined as E-RFC, in the cell cultures from the patients, varied between 0.5% and 22",,. However. 80",, of the cell cultures contained less than 10'/;, E-RFC positive cells. All CLL cell cultures demonstrated a typical monoclonal B cell dominance as indicated by kappa or lamhda light chain restriction on surface membratie Ig positive (Snilg) cells.
PPD 25fjg/ml p=0.003
Covatiatiee of responses to mitogens in CLL blood tymphocytes Blood lymphocytes from twenty-one individual patients with CLL were cultured in vitro in the presence orfive different activating ligands. each in three diflerent concentrations. Data from studies of cells from twelve individual patterns were included in an earlier report [17], which however lacks a statistical evaluation of the data such as is presented here. Thus the data used for the eovariance determinations are derived from a very large number of individual CLL cell cultures. Though dominated by the leukaemic cells, the citltures also contained a few normal ceils. In order to minimize the risk o\' an expansion of these cells, ciilitires were harvested already on day .1. Correlations were either absent or positive for all mitogens tested (Fig. 1). Unlike the other mitogens, the DxS and anti-fiom correlations were strongly discordant with regard to different concentrations, and for these mitogens the lowest and the highest concentrations are presented in Fig. I. Data can be simimarized as follows. Responses to the lower DxS concentration and LPS were significantly correlated {/'l
DxS SOOfjg/ml
p=0.243
p=o,ni
p=0.206
p=( 1.003
p^0,023
p=0.]77
p=0.401
p.O.l.ll
p^O.014
P=0.n27
p=0,095
p=().O78
p=0.479
p
