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Epstein Barr Virus Transformation of Peripheral Blood B Cells Secreting Antibodies Reactive with Cell Surface Antigens Marshall R. Posner, Hillary S. Elboim & Marea B. Tumber To cite this article: Marshall R. Posner, Hillary S. Elboim & Marea B. Tumber (1990) Epstein Barr Virus Transformation of Peripheral Blood B Cells Secreting Antibodies Reactive with Cell Surface Antigens, Autoimmunity, 8:2, 149-158, DOI: 10.3109/08916939008995733 To link to this article:

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0 1990 Harwood Academic Publishers GmbH

Autoimmunity, 1990, Vol. 8, pp. 149-158 Reprints available directly from the publisher Photocopying permitted by license only

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From ihr Division of HeniatologylO~icology and ~ I I CDeparinieni of Medicine. ilie Roger Williams Cancer Center, and Brown University Medical School, Providence. RI,02908

(Received July 9 , 1990) EBV transformable peripheral blood B cells secreting antibodies reactive with cell surface antigens present on two indicator human leukemia cell lines, NALM 1 and U937, were studied. Oligoclonal EBV transformants from patients with a variety of diseases were frequently found to produce cell surface reactive antibodies. Antibody secreting transformants could also, although less frequently, be readily cultured from the PBM of normal volunteers, and represented, by limiting dilution, I out of 113 transformable B cells. CD8 antibody had no effect on the frequency of antibody producing B cells, but depletion of CD8 + cells by immunomagnetic methods prior to transformation significantly ( P i0.05) increased the recovery of antibody secreting B cells to 1/33. Readdition of magnetically depleted cells did not significantly inhibit the transformation of these B cells. During the acute and recovery phases of some infections increasing numbers of these transformable antibody producing B cells appear in the circulation. The majority of antibodies produced were of the IgM class, although IgG antibodies were also detected. IgM antibody producing transformants were tested and some were found to react with autologous and allogeneic normal lymphocytes. These results lend support to the notion that B cells capable of secreting cell surface reactive antibodies, a proportion of which are autoreactive, are present in the normal repertoire of healthy adults, and that these cells are under active regulation by C D 8 + cells.

KEY WORDS: Epstein Barr Virus, T cells, CD8, B cells, autoantibody, cell surface antigens. ABBREVIATIONS: HuMoAb, Human Monoclonal Antibody; PSG, Puck's Saline G; PSG/WO, Puck's Saline G without calcium or magnesium

INTRODUCTION Serum antibodies reactive with cell surface and intracellular antigens may appear spontane~usly'-~, as a consequence of autoimmune diseases4-', as a result of infection^^-^ or subsequent to malignant transformation of selected B cell populations'".''. B cells capable of producing these antibodies are normally present in the repertoire of neonatal mice, but in humans the study of cell surface reactive antibodies has previously been limited to those available from the sera of patients with autoimmune d i ~ e a s e s ~ * 'Recently, ~-'~. however, human monoclonal antibodies (HuMAbs) have been used to study the production of autoantibodies and cell surface reactive To obtain human monoclonal antibodies, many investigators have attempted to expand selected or unselected populations of B cells by EBV transformaAddress correspondence to: Marshall R Posner, MD, Director, Human Monoclonal Antibody Laboratory, Roger Williams General Hospital, 825 Chalkstone Avenue, Providence, RI 02908. 149

tion, secondary selection for antibody production, and subsequent fusion16-2'. Importantly, antibody screening has frequently been directed towards soluble autoantigens, e.g. t h y r o g l ~ b u l i n ' ~intracellular .~~; autoantigens, e.g. ~ytokeratins'~.'~; and infection related antigens, e.g. pseudomonas endotoxin25,26. Antibodies reactive with cell surface antigens, particularly those expressed on neoplastic cells, have been substantially more difficult to ~ b t a i n ' ~ . ' ~ , ~ ' - * ' . Because of our interest in human neoplasia, we have developed HuMAbs from patients with leukemia which are reactive with cell surface antigens expressed on human leukemia cells and cell While some of these antibodies appear to be highly specific for discrete populations of malignant cells, others are reactive with broadly expressed antigen(s) and appear to be "(auto)antibodies", cell surface autoantigen reactive antibodies of uncertain relation to normal or pathologic conditions. Some of these antibodies may represent true autoantibodies; others may represent promiscuous autoreactive antibodies; and some may be reactive with "neoantigens" expressed on the

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malignant leukemic cell lines, as may be found during (CRL 8014, ATCC, Rockville, MD). The origins of HTLV 1 infection, or as a result of malignancy the leukemic cell lines used in these studies have been NALMI, a lymphoid CML cell line was induced derepression of certain e n ~ y m e s " ~ ' ~.~ ' ~ ~ described". ~~~'' In the present study, we describe the frequency and kindly supplied by Dr. J. Pesando (Biomembrane investigate the T cell mediated regulation of EBV Institute, Seattle, WA) and U937, a histiocytic/ transformation of B cells secreting antibodies reactive monocytic lymphoma cell line by Dr. R. Todd (Ann with cell surface antigens present on two indicator Arbor, MI). Murine monoclonal antibodies were obtained for human leukemia cell lines, NALM I , a lymphoid CML cell line, and U937, a monocytic cell line. Such analysis of lymphocyte populations and included antibody secreting, transformed B cells could be fluorescein labeled T8 (CD8) and TI1 (CD2), and readily obtained from the PBM of normal volunteers. phycoerythrin labeled TI (CD5) and T4 (CD4) as well Transformation and outgrowth of these B cells is as appropriately labeled negative controls (Coulter, regulated by CD8+ cells and some of these IgM Hialeah, FL). antibodies react with autologous and allogeneic peripheral blood mononuclear cells. Furthermore, during the acute phase of some infections, the Cell Separation and EBV Transformation regulation, in peripheral blood, appears to be severely After informed consent, PBM, were obtained by affected with the result that increasing numbers of venipuncture in preservative free heparin and these cells are transformed. separated away from contaminating cells by density gradient separation as reported previously". Initially, PBM were selectively depleted of CD8 positive cells by MATERIALS AND METHODS antibody mediated complement lysis. Cells were suspended at a density of 10 x 10h/ml in media and Cell Culture 1OOpl of culture supernatant from an OKT8 culture Cell lines and established hybridomas were grown in were added. The cells were incubated for 30 minutes at Alpha-MEM, lacking nucleosides with the following room temperature. Rabbit complement (Pel Freeze, additives: 1 mM sodium pyruvate, 2 mM I-glutamine, Brown Deer, WI) was added at a final concentration I % (v/v) non-essential amino acids, 10% (v/v) fetal of 1 :5, and the cells incubated for I hour at 37°C. The bovine serum (high cloning efficiency and growth cells were then washed 3 times with Puck's Saline G promotion) (GIBCO, Grand Island, NY) 0.22% (w/v) (PSG) without calcium or magnesium (PSG/WO). sodium bicarbonate, and 50 ,ugm/ml gentamicin. EBV This removed approximately 90% of the T8 positive cultures were established and maintained in the same population with yields of PBM ranging from 25-50% media containing 20% fetal bovine serum. Other of the starting population. additives were included as indicated. Cultures growing For panning, cells were treated with OKT8 as in flasks were sealed and maintained at 37°C after above, or OKT8 and OKTl 1 and incubated at 4OC for gassing with a 5% CO,/air (v/v) mixture. Repeated 20 minutes, washed three times with cold PSG, resusgassings after initiation were performed as needed. pended in a minimum of 7 ml of cold media and placed Cultures in 96-well microtiter plates or 24-well in sterile l00mm bacterial plates which had been multiwells were incubated in a 5% CO, atmosphere at precoated with goat anti-mouse immunoglobulins 37°C in a humidified incubator'h.20.32. (Tago, Burlingame, CA) diluted at 15 pgm/ml in 10 ml of PSGfWO and subsequently treated as described by Wysocki and Cell depletion by panning Cell Lines, Hyhridomas, and Monoclonal Antibodies resulted in 25%0-30% yields with < 10% CD8 positive The IgG human monoclonal antibodies HLI, HL2, cells in the depleted population. HL3, FIIDE2 and FIICD9 and the IgM HuMAb Magnetic bead depletion was performed using HL4.1 and F5C9 were derived by fusion of EBV magnetic beads coated with goat anti-mouse inimunotransformants with the HMMA2.1 lTG/O cell line, a globulins at 150pgm/ml of beads, according to the non-secreting human mouse myeloma analogue (H B manufacturer's specifications (Tosyl Activated Dynal 9583, ATCC, Rockville, MD) and are previously Beads, Robbins Scientific, Mountain View, CA). Cells describedlh?I) ?x 7 0 . The B95-8 marmoset cell line was were mixed with the appropriate antibody as used as a source of EBV for cell transformation. B95-8 described above. After 3 washes, cells were rcsuspendsupernatant was collected as previously described and ed at 2-5 x 107cells/ml in cold media and lop1 of stored at - 70°C in aliquots until use". Transform- beads/ 10' cells added. Cells and beads were rotated in ation efficiency of stored lots of EBV did not vary the cold for 20 minutes, resuspended in 4-10ml of significantly over time or between lots. The OKT8 additional media, and depleted using a magnetic field. (CD8) hybridoma was obtained from the ATCC In some experiments antibody positive cells plus beads


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were added back to depleted populations based on the fraction removed. Magnetic depletion resulted in a 90-95% removal of antibody positive cells and 4075% of the predicted cell yield. Because of the high yield and easy recovery, this method was used in all add back experiments. PBM, CD8 depleted PBM, CD8 antibody exposed PBM, and CD8 depleted PBM plus CD8 positive cells were transformed by resuspension in media containing a 1:10 dilution of B95-8 supernatant. Cells were then aliquoted into 24-well multiwells or 96-well flat bottomed microtiter plates at relatively low densities resulting in oligoclonal EBV transformants. Cultures in 24-well multiwells were initiated at 12.0 x IO5cells/well in 1 ml. For 96-well microtiter plates, serial two fold limiting dilutions were performed as previously described and were initiated with 1.0-2.0 x 104cells/well in the first row3*. The initial rows and subsequent dilutions were in loop1 of media with a 1 : l O dilution of B95-8 supernatant. Cultures were fed at 1 week intervals for the first three weeks by the addition of 0.5ml of media or 5011 of media to multiwells and microtiter wells respectively. At three weeks microtiter wells to be tested for antibody were transferred to multiwells containing 1 ml of media and fed as needed. Multiwells were fed by removal of media and refeeding prior to the first screening to dilute antibody resulting from B cell stimulation and secretion rather than transformation. Positives were rescreened after further feeding to establish that antibody was being secreted by transformants. EBV transformation efficiencies were determined by evaluation of serially diluted microtiter plates at 3-4 weeks. Growing wells were enumerated and a transformation efficiency calculated’*. Transformation Efficiency (TE) = l/X, where:

x =

1/{ eln

?(well%w i t h growth/wells per dilutton)-(In 2/21 -0.578

IN) and N = number of potential transformants in the starting dilution} Specific antibody producing B cell transformation efficiency was determined by the formula: I / [ ( N x TE)/FP], where N = the number of cells seeded/well or density, TE = the EBV transformation efficiency, and FP = the fraction of wells antibody positive. This result describes the number of B cells that must be transformed in the culture to get one positive antibody producer. Indirect Inzniunc?fluorescenceof Cell Surfuce Antigens

Indirect immunofluorescence (IF) for the detection of human antibodies reactive with cell surface antigens was performed as reported previously with some modifications“. In brief. live cells from cell lines or PBM were washed with PBS. To block Fc binding, 10 x lo6 U937 or PBM ceHs/ml were incubated in a

I : 50 dilution of a mixture of nonspecific mouse ascites and sera for 15 minutes at 4°C and then lOOpI/test were transferred to test tubes. NALMI cells were resuspended without blocking and transferred. One hundred microliters of culture fluid containing test supernatant or monoclonal antibody were added to the cells and incubated at 4°C for 30 minutes. The cells were then washed twice with PBS, followed by the addition of 100 pl of fluorescein conjugated F(ah’)? goat anti-human IgG, goat anti-human IgM, or goat IgG (Tago, Inc., Burlingame, anti-human IgM CA) diluted in PSG with 2.5% fetal bovine serum. The sample was incubated for 30 minutes at 4°C and then washed once in PBS. Following this, the pellet of live cells was resuspended in 500p1 of 1% (v/v) formaldehyde in PBS to fix the sample. The fixed cells were resuspended and stored up to five days at 4°C until analysis. Analysis was performed on an Epics C cell sorter (Coulter, Hialeah, FL). Negative controls were used in all assays, and a positive was considered to be > 5% above the negative control. All positives were tested for isotype after dilutional feeding and growth of the culture.


RESULTS Cell Surfuce Reuctive Antibodies ure Reudily Produced by EBV Trun.Fformants,from PBM

EBV transformation in 24 well multiwells of PBM from normal volunteers and patients resulted in multiple culture producing antibodies reactive with cell surface determinants on one or both of two indicator leukemic cell lines used for detection (Table I ) . Initial experiments utilized PBM from patients with leukemia in remission. Several IgG and IgM antibody producing cultures were obtained. Six of these have been captured as HuMAb and have been described 1h.28. To determine if the presence of transformable B cells capable of secreting cell surface reactive antibodies were a general phenomenon, normal Table 1 EBV Transformed peripheral blood B cell cultures produce cell surface reactive antibodies.

Normal volunteers Acute viral infection Leukemia HIV Infection


1 I6

11.8 & 3.1



83.3 f 10.1



17.5 f 2.1



23.8 & 2.5

Supcrnalants from EBV tran5formanis were icsrcd Ibr antibody reacting with cell surljce

a n t i g n a o n the human lcukcmia cell lines NALMI and lJV37





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Figure 1 EBV transformation during acute infections in normal volunteers led to a large fraction of multiwells with EBV transformants secreting antibodies reactive with the indicator cell lines. When the volunteers were healthy the fraction of positive wells was significantly lower and equal to levels seen in other healthy volunteers.

volunteers and patients were evaluated using the CML lymphoid leukemic cell line NALMl and the monocytic cell line U937 as screening targets. As shown in Table 1, a fraction of transformed PBM culture from normal volunteers and patients were found to secrete antibodies that reacted with cell surface antigens on the leukemic cell lines. A large fraction of PBM cultures from normal volunteers with acute infections obtained in the immediate symptomatic period of infection produced antibodies. Figure 1 demonstrates that in normal volunteers, after recovery from these self limited infectious events, the fraction of transformable antibody producers decreases in whole PBM cultures and approaches that seen in healthy, normal volunteers (Table 1). During the stress of certain acute infections the fraction of

cultures positive for antibody production (83% k lo%, mean f standard error) was significantly higher than during the recovery phase or after a period of established good health in these volunteers (15% k 3%, for the paired samples, p < 0.05, for the difference). In contrast to these acute viral infections, patients with a chronic viral infection, HIV, when not acutely ill, or during remission from leukemia, showed negligible differences compared to normal healthy volunteers. In healthy appearing volunteers, as shown in Tables 1 and 2, EBV transformants secreting cell surface reactive antibodies could be obtained from whole PBM, but the fraction of cultures secreting these antibodies is generally low. There is a great deal of variation in the fraction of positive cultures between volunteers. We interpret this to be a result of the natural variability between individuals, including unrecognized illness; slight variations in culture conditions, such as increasing or decreasing the number of cells in each culture; and, more importantly, dynamic and subtle changes in the health status and immune system that are a function of uncontrolled environments. The majority of antibodies produced by EBV transformants were of the IgM class, although occasional patients and healthy volunteers produced IgG antibodies, Unselected PBM from healthy volunteers yielded approximately 17% of cultures producing IgM antibodies and 0.5% producing IgG antibodies (data not shown), a ratio of 1gM:IgG antibodies of 36: 1. Similarly, the majority of antibodies produced by EBV transformants from patients with active acute and chronic infections are of the IgM class. Examples of the cytofluorographic results of screening against NALMl and U937 are shown in Figures 2 and 3 respectively. Figure 2 demonstrates that the antibodies from three different multiwells, (Figures 2D, 2E, and 2F) containing EBV trans-

Table 2 The in vitro growth of cell surface reactive antibody secreting EBV transformants is regulated by CD8 + cells. Norniul voluntcw\

12 12 4


Trun.sJormution conditions

we11.s .seeded

well.^ producing untihody (76 SE)

Unmanipulated cells CD8 Positive cells depleted Depleted CD8 positive cells added hack U n manipulated cells with CDX antibody added


17.4 & 5.2

I x0









25.0 & 5.0

'These results are significantly greater than paired unmanipulatcd cells ( p < 0.05). These voluntars are not included in Table 1

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Figure 2 Flow cytofluoriinetry testing for antibody reactivity of EBV transformant supernatants against NALM I demonstrates differences in reactivity patterns. The figures are from one experiment and are histograms of cell number (Y-axis) versus log green fluorescence (X-axis). This experiment used fluoresceinated goat anti-human IgM antibodies as the second antibody as described in the materials and methods, a: Negative Control, F5C9 IgM anti-tetanus antibody (9.2% positive), b: Positive Control, HL4.1 IgM antibody (98.6%), c: Negative EBV-#37 (9.7%), d: Positive EBV-#28 (16.20/0). e: EBV-18 (24.9%), f : EBV-#23 (53.1%).

formants from a single volunteer, appear to have different patterns of reactivity with the lymphoid cell line, NALMI. The positive control (Figure 2B) is HL4.1, a HuMoAb obtained from an EBV transformant culture of an ALL patient that reacts with all leukemic cell lines and normal nucleated peripheral blood cells testedzx. In Figure 3 several cultures, negative for reactivity with NALM 1, are shown to react with U937. Cultures from a patient with HIV infection and a

normal volunteer, selected for secretion of antibody reacting with cell surface antigens on the leukemic cells, were also tested for reactivity with autologous or allogeneic PBM. As can be seen in Figures 4 and 5 respectively, cultures containing IgM antibodies reacted with allogeneic and/or autologous PBM. Thus, many of the IgM antibodies detected in this system also react with antigens expressed on normal autologous and allogeneic lymphocytes and/or monocy tes.

Figure 3 Flow cytofuorimetry for antibody reactivity of EBV transformant supernatants unreactive against NALM 1 and reacting with IJ937 demonstrating dilferences in reactivity patterns with this cell line. The figures are from one experiment and are histograms of cell number (Y-axis) versus log green fluorescence (X-ax],). This experiment used fluoresceinated goat anti-human IgG and IgM antibodies as the second antibody as dcscribcd in the niatcrials and methods. a: Ncgative Control. F5C9 1gM anti-tetanus antibody (7.2% positive), b. Positive Control. H1.4.I IgM antibody (81.7%). c: EBV-# 12 (31.4%1), d: EBV-#3 (55.6%).

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Figure 4 Flow cytofluorimetry tcsting for antibody reactivity with normal PBM of EBV transformant supernatants from a patient with HIV infection (HIV27) already known to be positive on NALMl or U937. The histograms have been generated by gating on the lymphocyte area of a scattergram of forward vs 90 degree light scatter of ficoll-hypaque prepared PBM. The figures are from one experiment and are histograms of cell number (Y-axis) versus log green fluorescence (X-axis). This experiment used fluoresceinated goat anti-human IgM antibodies as the second antibody as described in the materials and methods, a: Negative Control, D8 an IgM immunoglobulin (8.0% positive), b: Positive Control, HL4.1 IgM antibody (87.1%). c: EBV-A3 (53.7%), d: EBV-A4 (76.0%), e: EBV-AS (44.3%), f : EBV-B8 (72.7%).

CD8 + Cells Regulate the Transformation of Antibody Producing B Cells

We investigated the effect of CD8 + cells on the EBV transformation of B cells producing antibodies reactive with the cell surface antigens of our indicator cell lines. Complement lysis, panning, or immunomagnetic beads were used to remove CD8 + cells from PBM. Both the depleted and whole PBM cultures were transformed with EBV. As can be seen in Tables 2, CD8 depletion consistently and significantly increased the fraction of multiwells producing cell surface reactive antibodies ( p < 0.005). In healthy individuals, 17.4% & 5.2% of undepleted cultures

produced antibodies, while 47.5% & 8.9% of simultaneously cultured, matched CD8 depleted multiwell cultures produced antibodies, a greater than 2 fold increase in antibody producing cultures after CD8 depletion. The proportion of IgG secretors did not change (data not shown). To investigate whether the increased transformation of antibody producing B cells was an effect of the OKT8 antibody used to deplete the cells and whether magnetically depleted cells could restore inhibition of EBV transformation of these cells, PBM from normal volunteers were transformed before and after exposure to CD8 antibody and after the readdition of magnetically depleted CD8 cells to depleted PBM. As shown in Table 2 , CD8 antibody


Figure 5 Flow cytofluorimetry testing for antibody reactivity with autologous PBM of EBV transformant supernatants from a normal volunteer already known to be positive on NALM 1- or U937. The histograms have been generated by gating on the lymphocyte area of a scattergram of forward vs 90 degree light scatter of ficoll-hypaque prepared PBM. The figures are from one experiment and are histograms of cell number (Y-axis) versus log green fluorescence (X-axis). This experiment used fluoresceinated goat anti-human IgM antibodies as the second antibody as described in the materials and methods, a: Negative Control, D8 an IgM immunoglobulin (7.9% positive), b: Positive Control, HL4.1 IgM antibody (90.3%), c: EBV-A6 (18.9%), d: EBV-T8A2 (74.796). e: EBV-A4 (31.50/0), f: EBV-T8A9 (30.2%).



CD8 Cells specifically effect the EBV transformation of peripheral blood B cells producing cell surface reactive antibodies. Culture manipulotion (density)


Transformation eficiency ( T E )

Antibody producing nells

Antibody producing B cell rransformation ejiciency*



2 3

(150) (15)

1/7,100 112,500

Unmanipulated CD8 depleted Unmanipulated CD8 depleted CD8 cells added back

(150) (20) (100) (150) (150)

112400 1/ 13,400 1126,700 1133,700

Unmanipulated ( I 50) CD8 depleted (120) CD8 cells added back (120) Unmanipulated ( I 50) CD8 depleted ( I 50) CD8 cells added back (150)


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Unmanipulated CD8 depleted





113,600 116,700

116700 114300



> 11210 1/24

20 20 45 40 20

11125 1/41 1/33 1/13 1/21

40 80 40

1/84 1/42 1/44

20 80 40

111 1 I


1/43 1/67


'Density indicates the number olcells/well x 10 dctailcd in the Matcrials and Methods

The calculation lor and meaning of the terms "transformallon efficiency" and "antibody producing transformation efficiency" are

alone had little or no effect on 2 volunteers. The readdition of CD8 positive cells to depleted PBM had a minimal but consistent effect in decreasing the yield of antibody producing cells. This minimal decrease in the fraction of positive cultures did not abrogate the effects of CD8 depletion. Since the goat anti-mouse magnetic beads functionally act to immobilize antibody on the cell surface, exposure of OKT8 cell surface coated cells to magnetic beads could be expected to effect a functional inactivation of the CD8 cellsj5. CD8 depletion might also act by merely increasing the overall number of B cells transformed and thus the fraction of positive cultures. Therefore, we determined the transformation efficiency of cells before and after depletion and with readdition of CD8 positive cells in limiting dilution cultures. Limiting dilution cultures allowed us to determine the overall B cell transformation efficiency and thus the relative transformation efficiency of B cells producing reactive antibody. As can be seen in Table 3, it is apparent that depletion of CD8 positive cells specifically increases the EBV transformation of cell surface antibody producing B cells when compared to the overall B cell transformation efficiency. An average of 1/ 1 13 transformable B cells in undepleted cultures is producing a cell surface reactive antibody as compared to 1/33 transformable B cells in CD8 depleted cultures (p < 0.05, for the paired samples). Moreover, the readdition of CD8 cells, which have been partially blocked in activation through the use of goat anti-mouse magnetic beads and OKT8 antibody, leads to a limited reconstitution of the specific suppression while still suppressing overall EBV transformation efficiency.



DISCUSSION In this study, we have demonstrated that there are significant numbers of EBV transformable B cells among the peripheral blood mononuclear cells of healthy volunteers that can produce antibodies that react with cell surface antigens present on leukemic cell lines. Many of these antibodies are also reactive with autologous and allogeneic normal peripheral blood mononuclear cells. We have shown that the relative number of these B cells increases significantly during certain infections. Furthermore, peripheral blood CD8 positive cells specifically inhibit the transformation of these antibody producing B cells. Antibody reactivity with the lymphoid CML cell line NALM 1 and/or the monocytic cell line U937 was used to enumerate antibody producing cultures. These cell lines were chosen arbitrarily, first, to provide uniform screening targets representing different hematopoietic lineages and second, because of our previous success in producing IgG and IgM HuMoAb reactive with human leukemias16.28.In addition to anti-leukemia cell reactivity, the observed reactivity with autologous and allogeneic PBM demonstrated by a high proportion of the antibodies suggest that many may be derived from one of several functionally defined types of antibodies: a) autoantibodies, as they occur in autoimmune diseases or are induced by some infectious event^^-^; b) cold reactive antilymphocyte (auto)antibodies seen in similar situations of immune stress: c) "promiscuous", multi-antigen reactive antibodies; or d) rare antibodies directed at neoantigens related to the neoplastic transformation of the test cell ~ i n e s l h , 1 7 , 2 7 . ~ 0 . ~ l . ~ 6 ~ 2

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During acute and chronic infections, during active dieS4,15.51 It has been theorized that autoreactive B cells, rather than being clonally eliminated during autoimmune diseases, or after acute vaccination, autoreactive antibodies can frequently be serologic- development, are regulated in the normal host. Thus it is possible that within the natural repertoire of the ally d e t e ~ t e d ~ - ~Some , ~ - ~ ’of , these antibodies react with antigens sharing identical epitopes or having the circulating B cells there are potential high affinity property described as “epitope homology” or autoreactive IgM and IgG antibody producers that “molecular mimicry” leading to high affinity cross may have been among the many antibody producers reactivity with diverse antigens lacking shared detected in these cultures47. Novel neoplasia related antigens can be expressed identical structure^^^^^^^. Occasionally a shared, high on the surfaces of neoplastic cells as a result of affinity reactivity with a viral or bacterial antigen and certain autoantigens occurs, leading to an acute illness neoplasia induced derepression or alteration of such as myocarditis during certain streptococcal normal enzyme . As an example, infections or Chaga’s disease46.More frequently, the naturally occurring IgG antibodies to bacterially shared homology is with cytoskeletal components of associated carbohydrate residues react with malignant the Cold reactive IgM antilymphocyte auto- cells due to the derepression of alpha I-3-galactosylAnti-gal antibodies are frequent in the antibodies are also produced under circumstances of tran~ferase~’,~~. immune stress. These (auto)antibodies are frequently normal population, are predominantly IgG, and have encountered serologically but are of uncertain signifi- been shown to react selectively with malignant ,~~. of the high frequency of autocance in the pathogenesis of the associated diseases or t i s s ~ e s ’ ~Because the regulation of the associated immune response^'^. reactivity observed in these cultures it is likely that Promiscuous antibodies, primarily of the IgM class, antibodies reacting with neoantigens on the malignant have low affinity for antigen, but because they are cells would be observed only rarely in the system pentameric IgM antibodies, a high avidity. This described above, and, if seen, would most likely be of results in multiple cross r e a ~ t i v i t i e s ~ ~These - ~ ~ . an IgG isotype. Elimination of actively functioning T cells from antibodies are detectable in normal sera, and frequently cross react “promiscuously” with intra- PBM prior to EBV transformation leads to a general cellular cytoskeletal components, “hidden antigens” increase in the tr‘ nsformation efficiency of the However, as can be seen in this such as DNA, and circulating soluble antigens, such remaining B cell~’~.~~ff as IgG or thryoglobulin when they are presented in an report, CD8 cells have a specific inhibitory effect on the EBV transformation of B cells producing immobilized format such as an ELISA3.’”,‘2.’”.’7.42.50. The multi-organ and multi-antigen reactive human antibodies reacting with cell surface antigens on our monoclonal antibodies generated by many inves- indicator cell lines. Although possibly a direct effect, tigators studying both autoimmunity and neoplasia CD8+ T cell regulation of the transformation of these B cells in vitro may also be mediated through may fall into this class23’24.’x-40. It has been strongly suggested that these prom- other T cell and effector populations present in the iscuous antibodies cannot react with cell surface cultures. It is also possible that the antibodies antigens because of low antigen density and antigen produced and expressed by these B cells may parmobility on cell surface^^^-^'. Thus, many of IgM ticipate in regulatory interactions with T cells through antibodies captured from humans and those described a direct interaction of cell surface expressed antigens . CD8 T cells in murine systems cannot promiscuously bind antigen or receptors and a n t i b ~ d y ’ ~Although when the antibodies are expressed on the membrane appear to grossly regulate these cells, the fine by the secreting cell49.This is because the advantage of regulation of the EBV transformation and outgrowth high avidity binding seen in the pentameric secreted of B cells producing one or another functionally form is lost with monomeric membrane expression. dissimilar antibody, e.g. promiscuous antibody, auto(auto)antibody, or anti-neoantigen This is also true when antigen is presented in soluble antibody, form or on a fluid cell membrane. Thus, antigen antibody, by various T cell subpopulations are likely driven expression is limited in the case of promiscuous to be different. It is possible that B and T cells may autoreactive antibodies, while expression of high also interact through, and be influenced in this interacaffinity autoantibodies may be down regulated by an tion by, the type or nature of the antibody produced. As a result of these studies, we conclude that EBV undetermined regulatory mechanism that may involve T cells4’. Therefore, in contrast to promiscuous IgM transformable B cells that are capable of secreting autoantibodies, high affinity serum IgG and IgM antibodies reactive with cell surface antigens are autoantibodies reactive with intracellular or soluble present in the circulation and that the CD8+ antigens from patients with autoimmune disease are peripheral blood mononuclear cell population exerts frequently of high affinity, monoreactive and inhibit- regulatory control, in the form of inhibition of transable by soluble antigen ‘ l . Such characteristics may be formation, on these B cells. This regulation is substanexpected among cell surface reactive autoantibo- tially altered during acute infections leading to an



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increased recovery of these B cells in EBV transformed cultures. The antibodies produced by these EBV transformants, although detected by a common reactivity with cell surface antigens on specific leukemic cell lines, may be of several types, representing the products of different subpopulations of B cells. The regulation of B cell transformation in vitro implies an in vivo effect in which large populations of B cells are inhibited from antibody production. Study of the fine regulation of EBV transformation of these B cell populations may help to delineate the in vivo regulation of these cells. Finally, these results imply an effect between the antibody/antigen interaction and the T cell regulation of transformation. Whether this is a function of the B cell population or the antibody is a question for future experiments. Acknowledgements

We would like to thank Rebecca Rosenstein, PhD (Brown University, Providence, RI) who advised us regarding the statistical analysis, David Niedel-Gresh for his photographic assistance, and Herbert Lazarus, PhD (Centocor, Malvern, PA) for reviewing the manuscript and providing helpful advice. Supported in part by grants from the NCI and NIAID: RU1-CA50054, CA13943, and ROI-A1 26926. References 1. Dighiero G , Lymberi P, Guilbert B. Ternynck T, Avrameas S.


3. 4.

S. 6.



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Epstein Barr virus transformation of peripheral blood B cells secreting antibodies reactive with cell surface antigens.

EBV transformable peripheral blood B cells secreting antibodies reactive with cell surface antigens present on two indicator human leukemia cell lines...
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