Oct 23, 1978 - JAMES P. HALPEIR, SHU NMAN Fu, ALICE B. GOTTLIEB, ROBERT J. ...... Humphreys, R. W., J. M. McCune, L. Chess, H. D. Herr- man, D. J. ...
Poor Mixed Lymphocyte Reaction Stimulatory Capacity of Leukemic B Cells of Chronic Lymphocytic Leukemia Patients Despite the Presence of Ia Antigens JAMES P. HALPEIR, SHU NMAN Fu, ALICE B. GOTTLIEB, ROBERT J. WINCHESTER, and HENRY G. KUNKEL, The Rockefeller UniversityX, Net lYork 10021
A B S T R A C T The humana Ial-like aintigens, selectively expressed on B lymphocytes, are noow recognized to be closely associate(l with, or identical to, the genie prodlucts of the imaljor histocompatibility complex responsil)le for stimulation in the mixed lymphocyte reaction. The leuikemie B lymphocytes of patienits wvith chronie lylmphocytic leukemnia express these antigens very well. In the presenit stuidy they were readily dletected by several techniq(uies utilizing 1)oth allo- anid heteroanitiseral. However, the leukemiic B cells fromii mlost patienits were founid to be extremiiely poor stimiulatinig cells in the miiixed lymphocyte reaction. This wvas particulatrly apparenit when comiiparisons were mclade on a B-cell basis with isolated normal B lymphocytes. Leukemic cell death, abnormal kinetics of leukemnic cell-me(liated stimulation, anid serum or cellular suppressor factors do not appear to explain these findings. Studies conmparing cells from a leukemic patient with those of her HLA idenitical sibling and results of mixed lymnphocyte reactions b)etween inormal and leukemic subjects discordanit for D-region-associated la aintigenis rule(d otut genetic explanations for the differenees observed. Experiments with normal peripheral bloo(1 mononotuclear cells dlepleted of T cells and monocytes exclude the quiantitative (leficiency of monocytes wlhich is fouind in the peripheral blood of Imlost leukemiic patients as an explaiiation. The presenit res.ults with chroniic lymphocytic leutkemia cells indicate thatt the mere expressioni of the Ial-like antigens by cell populations does niot renidler Dr. Halper's present aiddress is Depairtimienit of Medlicilne Cancer Research, Columbia IUniversitv Coll'ge of Phlvsicians anid Surgeons, New York 100:32. At the timile these studlies were perforiiecl, Dr. Hailper was ai recipient of' a fellowship froi)i the Arthritis Founidationi. Dr. Fui is at Schiolati- of the Leukemiia Society of America. Dr. Winchester is the rec-ipienit of Researc-h Career Developument Awvard K04-AI 00216. Rcceivc(d for publication 23 October 1978 a(id in revised
alnd Institiute of
themi effective stinitilators. The accuinulated evidence ol)tained indicate that abnormalities, particularly of membrane fumnctioll and metalbolislnl, knowni to occur in chronic lymiphocytic- leukemiaiil lymphocytes may be involved in the poor- stimulatory c-apacity of the leukemic B cells. IN TRO D U CTI ON
Hlum1(an1 B lymphocytes selectively express alloantigens with a restricted tisisue distribu,tion. The ailloantitgenis' extenlsive polymorphismn, linikage to the major histocompatil)ility complex (\IHC)' andI two-chain glvcoproteini stru-tcture have indicate(d that they represenit the hulmlan e(qtuivalent of the murl-inie Ila antigenis. They are (letected by alloantibodies (1) fountd in the sera of mlultiparous women aind transplant recipients andl)b heterosera (2-4) prepared by injectinig rabbits with Blymphocyte meml)rane fractions. These antigents have usuallvl been terme(d "Il-like"; for simplicity, they wvill be referred to here as Il antigens. Evidence hats been obtained that la alloantibodies will block a one-wav mixed lymphocyte reaction (NMLR) wlheni directed against (leterminciants expresse(d oni the stimuitllator cell populatioins (5) aind that treatmiienit of' the stimulahtor poptlattion with heteroaniitiscra is also effective (6). In adidlition, a close relattionship exists between .ILR typing usinlg homozygoius typing cells and(I serological typilng usinig alloantibodies clirected atgainist Il (7). Tlhuis, it hats been conieluded that Ila antigens are gene prodlImets of the NIHC regioni (IILA-D) responsible for MILR stimnulationi, or atre intimately associated vith these gene products. For this reatson certain Ila aItigenls lAbbreviations used in this paper: CLL, chronic lymphocytic leuikenmia; Dr, HLA-D region associated; E, sheep erythrocyte; Ml HC, major histocompatibilitv comliplex; \ILR, inixedi lymphocyte reaction; PBMI, )eripheral hlood m11on1onuclear cells.
C(liu. Invest. ©O Tle American .Societiy for Clitnicatl Inivestigatiout, Inc. Volumoe 64 November 197.9 1141 -1148
detected are designated as D-region-associated (Dr) antigens.
The expression of Ia antigens by B lymphocytes and results of experiments with isolated cell populations have led to the conclusion that B lymphocytes are capable of stimulating in the MLR (8-11). Most cases of chronic lymphocytic leukemia (CLL) represent a neoplastic proliferation of B lymphocytes (12) which, like their normal counterparts, express membrane immunoglobulin (Ig) and Ia antigens (2, 3, 13). Therefore, it was of interest to examine the stimulatory capacity of these cells which can be obtained readily in large a(mounts and in a highly purified state. METHODS Cell preparation and isolation of lymphocyte subpopulations. Heparinized peripheral blood samples were obtained from normal volunteers and patients with CLL. Mononuclear cells were isolated by standard Ficoll-Hypaque methods (Pharm-acia Fine Chemicals, Div. of Pharmacia Inc., Piscataway, N. J.). Cell yields were always >60% and consisted of >95% mononuclear cells; viability, as assessed by trypan blue exclusion, was always >95%. B-Cell purification was achieved by elimninating T cells forming sheep erythrocyte (E) rosettes with neuraminidase-treated E (14). The nonpelleting (interfiace cells) contained 10,000 lymphocytes/mm3 consisting predominantly of mature small lymphocytes and a clinical picture consistent with this diagnosis. In most instances the lymphocyte counts were >30,000. The patients were untreated or on -10 mgld of prednisone at the time their lymphocytes were studied. The surface markers of PBM obtained from representative leukemic patients are shown in Table I. All patients studied had 97% purity) contrasted with CLL cells, >90% of which expressed surface membrane IgNM. The normiial B cells give a very good stimulation that contrasts strikingly with the CLL cells. A number of compparisonis were also carried out with B lymphoblastoid cell line cells also consisting predominantly of Ia-positive cells bearing IgM. Here againi the poor reactivity of the CLL cells was evident. Relation of Ia anttigen expression to AILR stimulatory capacity. B lymphocytes fromii the leukem-ic patients appeared to stain as l)rightly as normal B lymphocytes with fluorochrome-coinjuigated anti-Ia heterosera and, usinig indirect fluorescence, with alloantibodies. The expressioin of these antigens by lymphocytes from patient Ei, which gave normal stimiulation in the MLR, and by those from J, which were poor stimulators, was investigated in more detail. These leukemic lymphocytes were stained in parallel with normal B lymphocytes, using serial dilutions of 180 r If)
_ 120 z t
STIMULATOR CELL CONCENTRATION x 10 6
FIGURE 4 Comparison between stimulatory capacity of highly purified normal B cells (-) and CLL B cells (A) in a typical MLR experiment. The whole normal PBM effect is also shown (0). Autologous counts wvere -1,200.
Poor Mixed Lymphocyte Reactiotn Capacity of Leukemic B Cells
TABLE II rhodamine-conjugated F(ab')2 fragments of anti-Ia heterosera. Percentage of positive cells and intensity of Stitnulatory Capacities of Mixtures of Normal and Leukemic Cells: CLL Lymphocytes Do Not Inhibit the MLR fluorescence at each dilution were scored by an indeStimulation Capacity of MHC Identical PBM pendent observer. The staining properties of the leukemic cells from the two patients were indistinStimtulationi (cprn x 10-3) Cell conicenltration guishable despite their dissimilar MLR stimulatory (cellsltl/i x 10-6) of Expected* Expected* capacities; both reacted at least as strongly as the froimi fromii Normal Leukeminc normal B cells. CalcuObPBM in leukemnic noralll.l PBM in The capacity of the above two leukemic B cells to mixture inixture PBM lated PB31 served absorb anti-Ia heterosera was also compared to that of 17 30 47 43 2.5 1.25 purified normal B lymphocytes. Graded numbers of 1.25 6 90 96 78 0.625 cells from each of the three cell preparations were 1 74 75 83 0.625 0.313 added to 100 gl of diluted fluorochrome-conjugated 56 95 26 F(ab')2 fragments of anti-Ia heterosera in U-bottomed 2.5 30 2.5 132 17 90 107 1.25 1.25 microplates. After 2 h at 4°C the plates were centri6 74 120 80 0.625 0.625 fuged and the supernates' ability to stain cells from a B-lymphoblastoid cell line and normal B cells was 68 2.5 49 30 79 5 measured. On the basis of the number of cells re(luired 116 99 26 90 1.25 2.5 to absorb out fluorescence activity, cells from Ei were 17 74 91 83 0.625 1.25 80-100% as efficient as normal B lymphocytes; those from J were 116-140% as efficient as normal * Calculated from dose-response curve (Fig. 3). Sum of stimulation expected from CLL and normal PBM. B lymphocytes. Stimulatory capacity of mlixtures of PBM isolated from normnal atnd leukemic subjects. To investigate the possibility that PBM isolated from leukemic increases with increasing stimulator concentrations up patients contained a population capable of inhibiting to 1-2 x 107 cells/ml. This deficient stimulatory capaca normal MLR or lacked an accessory cell required for ity was most striking when CLL cells were compared stimulation, the stimulatory capacities of mixtures on a cell to cell basis with normal B cells isolated by of normnal and leukemic cells were examined. In seven several techniques. The normal B cells gave a strong such experiments, using cells from different leukemic stimulation while the CLL B cells at similar concentrasubjects, the observed values were well within ±33% tions had little or no effect. The two leukemic subjects whose PBM showed near of those calculated with only occasional exceptions. Thus, there was no evidence of inhibition or reconstitu- normal behavior when used as stimulators in the MLR tion. The results obtained with a mixture of cells from are of special interest because the leukemic B cells the leukemic and normal HLA identical siblings are are perhaps more normal than those in other cases of shown in Table II and clearly demonstrate the absence CLL in that they have been shown to be capable of differentiating into plasma cells synithesizing monoof a suppressor effect of the leukemic population. In related experiments it was found that depletion of clonal macroglobulins in vivo and in vitro (20). Previous studies of the MLR stimulatory capacities residual T cells from leukemic PBM in two cases did not enhance the stimulatory capacity of the remaining of CLL lymphocytes have reported both normal (25-27) B lymphocytes. Hence, specific MLR suppressor T and decreased stimulatory capacity (28, 29). However, cells could not be demonstrated in the leukemic it is apparent in all of the studies that when the data are recalculated on a per B-cell basis, the MLR generated population. by CLL subjects are abnormal. On comparing the doseresponse curves obtained with isolated normal B DISCUSSION lymphocytes and those obtained with the PBM isolated When used as stimulators in the MLR in the system from most cases of CLL, it is clear that even a low described above, PBM prepared from normal subjects proportion of residual normal B lymphocytes in the give increasing stimulation with increasing stimulator leukemic PBM coould give rise to most of the observed concentration until maximal stimulation is reached at stimulation obtained at higher concentration of cells. 1.25-2.5 x 106 cells/ml (stimulator responder ratio Poor viability of leuikemic cells, increased sensitivity = 1 or 2). As stimulator cell concentration is further to irradiation, dissociation of thymidine uptake from increased, stimulation falls off. In conitrast, PBM iso- lblast transformation, increased age of leukemic sublated from most subjects with CLL give relatively low jects, kinetic difference between stimulation by leustimulation at the concentrations at which normal PBM kemic and normal cells and requirements for special generate maximal stimulation, and the stimulation plasma factors were ruled out as explanations for the
Halper, Fu, Gottlieb, Winchester, and Kunkel
poor stimulatory capacity of the leukemic PBNM. No evidence for cells capable of suppressinig the MLR was found in the leukemic PBM. PBM isolated from patients with CLL contain a decreased proportion of monocytes, cells which are required, in smiall numbers, to support the MLR (21, 22). However, normal B cells depleted of monocytes by a variety of techni(lues so that they contained a percentage of moniocytes similar to or lower than that found in the leukemnic PBM1 were excellent stimulators. Monocyte depletion, however, did lead to an attenuation of the decrease in stimulation at high stimulator concentrations seen with normal but not leukemic PBM . In a recenit study, Steinman and Witmer (24) reported that for mouse spleen the primary stimulatory cells in the MLR is an Ia-positive dendritic cell, and they brought into (luestion a significant role for B cells. Previous work (8-11) appearing to demonstrate the strong stimulatory capacity of the B cell in the human MLR did not utilize B cells purified sufficiently to eliminate minor populations of active contaminating cells, such as the dendritic cells. However, the new techni(lue of isolating B cells using anti-IgM-coated erythrocytes allowed isolation of B cells free of contaminating Ia-positive non-B cells. Conclusive evidence for the strong stimulatory capacity of normal B cells was obtained,2 ruling out the B-cell nature of CLL lymphocytes as the explanation for their poor stimulatorv capacity. Because of the identity of, or close relationship between, Ia antigens and the MHC determinants (LD) responsil)le for stimulation in the MLR, the expression of these antigens by the leukemic cells was examined. These cells wNere found to express Ia antigens as strongly as normal B lymphocytes as assessed by fluoreseent staining and absorption studies. This is consistent with previous results using anti-Ia heterosera and the fluorescence-activated cell sorter (13). The alloantigenic portions of the Ia molecules were also found to be expressed normally on CLL B cells in other studies (2, 30), and the bimolecular complexes precipitated from normal and leukemic B cells shown to be identical (30). Recently, several diseases have been found to be associated with Ia and/or LD antigens (31), and this group may include CLL (32). Results with the MHC identical siblings and the failure of the leukemic cells to stimulate any of the large number of genetically heterogenous normal responders, soime of whom lacked Dr antigens expressed by cells of the leukemic subjects they were tested against, argue against a genetic explanation for the decreased stimulatory capacity of leuikemic cells. To serve as a stimulator in the MILR, it is likely that a cell must meet certain metabolic and membrane
functional re(quirements in addition to possessing the Dlocus antigens (33, 34). Many abnormalities in these parameters have been documented in CLL (35). Of particular interest is the recent finding that CLL B cells are permeable to merocyanine a compound which is excluded by normal lymphocytes (36). The cells from one of the two subjects mentioned above which were capal)le of giving a more normal MLR and differentiating in vitro were also unique in not being permeable to merocyanine; the other similar patient was not available for testing.3 Further investigation will be required to determine whether such membrane or other metabolic defects explain the results of this study. At present, however, it can be concluded that leukemic B cells, despite their strong expression of Ia antigens, differ from their normal counterparts by being poor stimulators in the MLR. ACKNOWLE DG MIE NTS We would like to thank Dr. NM. Fotino of the New York Blood Center for performing HLA typing. This study was supported by U. S. Public Health Service grants RR 102, Al 10811, CA 20107, and the National Leukemia Association.
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