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Monoclonal antibody assessments of T cell interactions in erythropoietin studies
Int J Immunopharma~
Vol 3 No a pp 2"~-24"
1981
0192-0561/81/030233-1450200/0 Pergamon Pre,,,, I td
Printed In the U ~ A
M O N O C L O N A L ANTIBODY ASSESSMENTS OF T CELL INTERACTIONS IN E R Y T H R O P O I E T I N STUDIES DAVID G. NATHAN* Department of Pedmtncs, Harvard Medical School, Boston, Massachusetts, U.S.A (Recetved f o r pubhcatwn 12 May 1981)
Abstract--The contemporary apphcatlon of clonal assay techntques has greatly expanded our knowledge of the regulation of hematopolesls. Our efforts have been directed toward the mvestlgatmn of non-erythropo~etm-medmted regulation of human erythropolesls m the form of cell-to-cell interaction between mature T cells and erythrold progemtors. Our data indicate that three such progemtors, the earll~marrow erythrocyte precursor BFU-E, the more mature marrow erythrocyte precursor, CFU-E and the penpheral blood BFU-E, each exhlblt totally different reqmrements for their colony expressmn in culture, with respect to the absence of erythropoletm and the presence of mature T ceils or their products The capacity of erythrold progemtors to w~thstand mcubatwn m the absence of erythropo~etm appears to be a charactensnc of ~mmature rather than mature erythrold progemtors Furthermore, use of OKT3 anubody depletmn techmques shows that peripheral blood-derived BFU-E appear to depend upon mature lymphocytes or T cell-condmoned medmm for erythropoletm-stlmulateddffferentmtmn whde bone marrow BFU-E and CFU-E have no reqmrement for mature T cells to produce erythropoletm-dependent maturauon Our results, plus a vast array of data provided by other mvemgators m the field, are integrated into a proposed framework for further mvest|gatlon of T cell mductmn of erythropo~etm-dependent erythrmd dffferentmtmn tamed at more specifically ldentff)qng the reducer cell subset(s) m that system.
The development o f m vtvo and m vttro d o n a l assays for both pluripotent and committed hematopoietic progenitor cells has greatly expanded our understanding of the regulation o f hematopoiesis. Thts subject has already been extensively reviewed (Cline & Golde, 1979; Quesenberry & Levitt, 1979; Lipton & Nathan, 1979), and need not be d|scussed m detail here. Figure 1 outlines our contemporary concepts o f hematopoiet|c differentmtmn based upon assays conducted m both routine and human systems. The pluripotent stem cell is the most fundamental hematopoietic progenitor. Its offspnng ultimately populate the entire marrow with recogmzable nucleated red cell, white cell, and platelet precursors. Its nnmediate offspring are the committed progenitors of lymphocytes (CFU-L) and the tnpotent myeloid precursor (CFU-S). CFU-S cells have the potential to give rise to any of three committed progenitors. Each o f the three possesses a unique hmlogic program of differentiation and rephcatlon that provide the recognizable precursors o f the erythrocyte, granulocyte, and megakaryocyte populauons. These cells are known as the BFU-E, CFU-C, and CFU-M cells, respect|rely. They are all present in small numbers m
hone marrow. Their precise morphology is unknown, but they are most likely mononuclear cells that resemble lymphocytes. In addition to these cellular components of the system, there are other cells and humors that influence the pathway of hematopotetic differentiation described above. Among these regulators are: erythropoietin, colony-stimulating activity, thromhopoietin, and possibly lymphopoietin. Much o f the process, we believe, is also under the influence o f thymus-derived (T) lymphocytes since some o f these inducer substances may be secreted locally by the marrow stromal cells or T lymphocytes that may provlde the microenviroument of the developing hematopoietic cells. The erythroid progenitor assay system developed by Axelrad and colleagues in plasma clot (Stephenson, Axelrad, McLeod & Shreeve, 1971; McLeod, Shreeve & Axelrad, 1974; Axelrad, McLeod, Shreeve & Heath, 1974) and adapted to methylceUulose (Gregory, 1976; Messner & Fauser, 1978) offers a method for the in mtro investigation of erythroid regulatton. Eaves & Eaves (1978) have demonstrated three erythroid progemtors. Two of these consist of specific subgroups of the erythroid burst-forrmng
* Address all correspondence to: Davld G Nathan, M.D Chlef, Dwislon of Hematology and Oncology, Children's Hospital Medical Center, 300 Longwood Ave., Boston, MA 02115, U.S.A. The materlal presellted in thls article is the expressed vlew of the authors w h o were cooductlOg research on O R T H O C L O N E * monoclonal antibodies These reagents are intended for research use only and are not to be used In diagnostic procedures ("Trademark)
233
234
DAVIDG. NATHAN
----------- T Lymphocyte
,CFU-L
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Proerythroblast--Erythrocyte Hegakaryocyte Platelet
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CFU-E0
~--COMMITTEDSTEMCELLS---~I
Granulocyte Monocyte
(Macrophage)
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BONEMARROW_.__JL__PERIPHERAL___J PRECURSORCELLS~I--BLOODCELLS-----]
Fig. 1. Schematic diagram of hematopolcsls as defined by clonal assays m the human and murme systems and G-6PD lsoenzyme studies m diseases of clonal prohferauon. CFU-S, colony-forming umt--spleen; CFU-L, colony-forming umt--lymphocyte; BFU-E and CFU-E, burst-forming and colony forrmng umt---erythrmd; CFU-M, colony-forming umt--megakaryocyte; CFU-C, colony-forrmng umt m culture; and CFU-E0, colony-forming umt--eosmophd. unit (BFU-E), and are termed the primitive and mature BFU-E, respectivdy. In the presence of erythropoietm, ~ach of these subgroup types develops into large colomes composed of multiple subcolomes. The rate of colony expression from primitive BFU-E is relatively slow, requiring 2 - 3 weeks for compleUon, depending upon culture conditmns. Mature BFU-E colonies develop more rapidly. The third and most mature class of progenitors is called the erythroid colony-forming unit (CFU-E). This cell is derived from BFU-E and forms single erythroid cell colonies m 6 - 7 days in culture. It ~s the immediate precursor of the proerythroblast. Figure 2 shows typical BFU-E and CRU-E derived colomes m plasma clot at high magnificatmn. The large colony comprising 6 multiple subcolonies d~nved from the BFU-E contrasts wlth the single CFU-E derived colony. Clarke and Housman 0977) and Ogawa and co-workers (Ogawa, Grush, O'Dell & Sexton, 1976) have demonstrated that the mononuclear cell fraction of normal human peripheral blood contmns small numbers of BFU-E. They most likely represent immature marrow progemtors which escape the marrow and enter the clrculation under ambient conditions. Most peripheral blood BFU-E are probably analogous to the most prLrmtwe bone marrow BFU-E that are demonstrated m methylcellulose.
Table I summarizes some of the salient differences between bone marrow and peripheral blood progenitors evaluated m plasma clot cultures. CFU-E expresslon occurs earlier m culture than BFU-E colony expression, and small colony-forming, mature BFU-E are expressed earlier than large colony-forming BFUE (Gregory & Eaves, 1977). Peripheral blood BFU-E are presem in much fewer numbers than e~ther of the marrow progenitors. They differ also wlth respect to their sensltiwty to erythropoictin. In the human system, it has been shown that CFU-E are the most sensitive, and pnmmve BFU-E less sensmve, to ¢rythropoletin than most mature BFU-E (Gregory, 1976; Eaves & Eaves, 1978). Marrow BFU-E are less susceptible to tnUatedthymidin¢ suicide than CFU-E, and peripheral blood BFU-E exlublt httle, if any, sensitivity to tntiated thymlchn¢, indicating that a lower fraction of penpheral blood BFU-E are m cycle than either marrow BFU-E or CFU-E (Adamson, 1978). With respect to hemoglobin synthesis, the peripheral blood BFU-E give rise to colomes capable of substanUal hemoglobm F production (Clarke, Nathan, Alter, Forget, Hillman & Housman, 1979). In contrast, marrow progenitor colomes produce much more hemoglobin A than F. In fact, hemoglobin F synthesis is barely detectable m our particular plasma clot culture system for marrow BFU-E. This difference m hemo-
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- "S..a. > Fig 2 Photomicrograph at high magmficat]on of erythrold progemtor derived colomes showing single large benzadme posture colomes for CFU-E (left) contrasted with large single colony with multiple subcolomes from BFU-E (right)
235
Monoclonal Antibody Assessments of T Cell Interactmns m Erythropoletm Studies Table 1
237
Charactensucs of human bone marrow CFU-E, BFU-E and peripheral blood BFU-E
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Peak day m culture Morphology Numbers of colomes/ 105 mononuclear cells plated Epo sensmvity Thymldme smclde Hemoglobin F synthes~s T cell or LMF requirement Sensmvlty to epopnval state
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globin synthesis will be further discussed later m this paper since it serves as an indicator of progenitor maturity. Our most recent studies also show that the three progenitors exhibit totally different requirements for colony expression m culture with respect to the absence of erythropoietin and the presence of mature T cells or their products.
Clarke, Breard, Merler & Housman, 1978). Clottmg is inmated by the addition of 0.1 ml of Grade I bovine thrombm (Slgrna Chemical Company, St. Louis, MO) m 0.1 ml of culture mechum. One-tenth millihter allquots of the clotting mixture are then dispensed into 0.2 ml mlcromer culture wells and incubated for up to 14 days. CFU-E are enumerated on day 7 and BFU-E on day 14.
EXPERIMENTAL PROCEDURES
Anttbody treatment Orthoclone OKT3 is a monoclonal cytotoxic IgG antibody prepared by hybrldoma technology (Kohler & Milstein, 1975). It is reacuve with a cell surface antigen common to all human circulating T cells (Kung, Goldstem, Reinherz & Schlossman, 1979, Reinherz, Kung, Pesando, Ritz, Goldstem & Schlossman, 1979). Its activity was established by serial dituuon of the murine asciuc fired m which It was secreted. It was used m all of our studies at a dilution of 1:500 m mmmmm essential medmm (S-MEM; Grand Island BIological Company, Grand Island, NY) wath 10 mM Hepes buffer plus 507o fetal calf serum (FCS) (Flow Laboratories, Rockville, MD). A similarly diluted m u n n e ascmc flmd lacking monoclonal antibody activity was used as a control. Evaluation of mononuclear cell elements was accomphshed by the use of a fluorescence acuvated cell sorter (FACS I, Becton Dickinson, FACS Systems, Mountain View, CA) (Benna, Hullett, Swett & Herzenberg, 1972) using OKT3 and a fluorscem conjugated anu-mouse IgG Treatment of cells w~th OKT3 or control ascltes flmd was accomphshed by mcubation of 20 to 40× 106 mononuclear cells in
Clonal assays Clonal assay methods for assessment of BFU-E and CFU-E in human bone marrow and BFU-E in blood have been described elsewhere (Clarke & Housman, 1977; Nathan, Clark, Hillman, Alter & Housman, 1979). Briefly, mononuclear cells are prepared by Flcoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N J) centrifugaUon. The appropriate cell numbers are added in 0.1 ml of alpha minus medmm (Grand Island Biological Company, Grand Island, NY) to 0.8 ml of the erythropoietmdependent plasma clot system of McLeod and coworkers (1974), modified by Clarke & Housman (1977). Erythropoietm (kindly provided by Drs. Anne Ball and Peter Dukes, NIH, and Connaught, Step III, Connaught Laboratories, Willowdale, Ontario, Canada) is employed at a concentration of 2 1.u. ml-~. In some experiments, 0.1 of medium m which tetanus toxold-stimulated mononuclear cells had proliferated is substituted for 0.1 ml of culture medmm This medmm contains lymphocyte mttogenie factor (LMF) (Nathan, Chess, Hillman,
238
DAVID G. NATHAN
I ml of the medium to whlch e~ther anUbody-contaznmg or control ascit~c fluid had been added for 30 min at 4°C. Then 0.15 ml of fresh frozen rabblt serum (Lot C192108, Grand Island Biologlcal C o m pany, Grand Island, NY) was added as a source of complement. The incubation was contained at 37°C for 1 h w~th gentle shaking. Following this the cells were thrice washed m a-medium plus 5% FCS. RESULTS AND DISCUSSION
The epopr~vai state As mentioned above, a dlstingmshmg characterlstic of progenitor classes is their response to the absence of erythropoietm m the culture medmm. Iscove (1978) first noted that murine marrow BFU-E and CFU-E differ wRh respect to their ability to proliferate m culture m the absence of erythropoletm. If erythropoietin was w~thheld from the culture for even a few hours, only a small percentage of the CFU-E were able to produce colonies, whde BFU-E were able to proliferate for days. Tsang & Aye (1979) reported a significant fraction of human marrow BFU-E were also capable o f colony production after prolonged erythropoietin deprivauon. Thus, the capacity of erythroid progemtors to withstand incubation in semi-solid media in the absence of erythropoietm appears to be a characteristlc of immature rather than mature erythroid progenitors. Table 2 shows the influence of the delay of erythropoletin addition on bone marrow BFU-E and CFU-E and peripheral blood BFU-E colony formation. Maximal colony growth follows addiuon of erythropoietin at control day zero. This is compared Table 2.
to the percentage of maxzmal growth achieved by each type after an established number of epopnval days. The results show that the expression of penpheral blood BFU-E is preserved for greater than three days m such experiments. The expression of marrow BFU-E is preserved to a lesser degree than that of peripheral blood BFU-E. In sharp contrast, a rapid decline m CFU-E colomes occurs when erythropoletin is withheld from the culture medium. We postulate that the more ~mmature the progemtor the greater its potenual to divide m response to an reducer that can be derived from T ceils. In culture, the immature cell replicates under the influence of reducer(s) and acqmres erythropoletm receptors. At this point, ff erythropoletm is not added, the progemtor will no longer differenuate and colony growth is lost. The more immature the progenitor, the more div~slons can occur prior to the accrual of erythropotetin receptors and the longer the time that the ¢poprival state can be tolerated. T cell regulatton o f erythropotests The inducer role of T lymphocytes in hematopolesis has been extensively studied in m vttro and in wvo systems. A decrease in the splemc engraftment of granulopoletlc and erythropoletlc colomes in lethally irradiated lsogemc recipient mice from anti-0 treated bone marrow has been described (Goodman, Bosfond & Shlmpock, 1978). Further, there is a failure of engraftment and correction of the anemm m the congenitally anemic w/w ~ mouse when 0 positwe cells are removed from the reconstituting marrow graft (Wiktor-Jedrzejczak, Sharkis, Ahmed & Sell, 1977). Recent work has implicated thymus-derived cells as regulators of erythropoletlC engraftment in
Influence of delay of erythropoletm addmon on bone marrow (BM) BFU-E and CFU-E and peripheral blood (PB) BFU-E colony formation
Percentage of mayamum colomes compared to epo added at day 0 control Day of erythropoletm addR1on
PB BFU-E
B M BFU-E
B M CFU-E
0
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100 32 10 0 0 0
1
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Monoclonat A n t i b o d y Assessments o f T Cell Interactions m Erythropoietin Studies
239
EFFECTOF T AND B LYMPH(X~Y'TESON BFU-EIN NULLCELLS
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F~g. 3 Three examples o f the effects o f varying n u m b e r s o f isogemc T ( • ) and B ( O ) cells on the f o r m a t m n o f BFUE colonies T h e T or B cells were added to 10~ null cells per ml o f plasma clot m the presence o f 2 Lu. o f erythropo~etm at the cell c o n c e n t r a t m n s s h o w n m the abscissa. The ordinate represents the total n u m b e r o f B F U - E colomes o f all s~zes observed after 14 days o f culture o f the cell mxxtures. In a d d m o n to the numerical Increase m BFU-E colomes observed when T ceils were added to null cells, there was also an increase m the average sxze o f mdlwdual colomes. Previously published m J exp Med. 147, 324--339, 1978. Q 6O u.J o.
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Fig. 5. Influence o f T cell-condmoned m e d m m and intact T cell on 3 - 4 + BFU-E colony growth m null cells The null cells were incubated at 106 cells per ml o f clot m the presence of 2 I.U. erythropoletm The different a d d m v e s were tetanus toxo~d, at a final c o n c e n t r a u o n o f 4 #g per ml o f clot, T cell-condmoned m e d m m , prepared as described m the text, at 10% v / v or 2 . 5 x 106 T cells per ml of clot. The mtxtures were cultured for 14 days and the 3 - - 4 + BFU-E colontes were enumerated. Prevmusly pubhshed m J. exp. M e d. 147, 324-339, 1978
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Fig 4. Influence o f various T ceil preparations and the effects o f ~rradIatmn on 3--4 + B F U - E colony growth m null cells The fresh or Irradiated null cells were incubated at l06 cells m l - 1 and l07 various treated isogenelc or allogeneic T cells were added to them The cell mixtures were incubated for 15 days m the presence of 2 t u of erythropoletin. Only 3 - - 4 + BFU-E colomes were included in the colony scores Previously p u b h s h e d in J exp M e d 147, 324--339, 1978
240
DAVID G. NATHAN
these w/w v rmce (Shark;s, Spwak, Ahmed, Mism, Stuart, Wlktor-Jedrzejczak, Sell & Sensenbrenner, 1980). The role of thymus-derwed cells ts further supported by expenments demonstrating the defective restoratwe capacity of bone marrow from congemtally athymic (Zipon & Tramm, 1973) and neonatally thymectomized (Risitzky, Z~pon & Trainin, 1971; Zipori & Trainm, 1975) rmce. The cellular products of dividing T cells or T cell leukemic lines (Golde, Bersch, Quan & Lusls, 1980) have multiple effects on hematopoletlc cells, regulating synthes~s of antibody (Geha, Schneeberger, Rosen & Merler, 1973; Strelkauskas, Wilson, Callery, Chess & Schlossman, 1977) and influencing them to divide and differentmte into eosinophllic (Basten & Beeson, 1970), granulocytlc (Cline & Golde, 1974; Ruscetti & Chervemck, 1975) and megakaryocytlC (Hara & Ogawa, 1978) as well as erythroid colonies (Iscove, 1978). It has been shown that the regulation of tn v i t r o as well as m m v o erythroid production and maturation is strongly controlled by erythropoletm. Recently, however, a number o f investigators have demonstrated that there is sigmficant erythropoiesls which requires other factors in addition to the hormone. It ~s hkely that these nonerythropo~etin factors mfiuence primitive erythroid differentmuon to a greater extent than the differentiation of the more mature progenitor, as shown by the relatwe resistance to the epoprwal state of the immature progemtor compared to the more mature progenitor. Udupa & Re~sman (1979) have demonstrated m the munne system that immature BFU-E are probably derived from CFU-S and that the repllcauon of mature BFU-E is less sensitive to erythropoietin than CFU-E. In addition, Iscove's work (1978) demonstrated a role for a substance called burst-promoting activity (BPA) derived from P H A and Con A stimulated mouse spleen ceils. Iscove's experiments have been interpreted to show that BPA may somehow increase the number of erythropoletin receptors on BFU-E. Investigations by Johnson & Metcalf (1977) have demonstrated that erythroid colonies could be derived from cultures of fetal liver stimulated by a PHA-treated mouse spleen cell conditioned medium w~thout the addition of erythropoletm. Their studies, however, did not rule out the presence of small amounts of endogenous erythropoietin in the culture media. Eaves & Eaves (1978) have shown that there is an adherent population o f marrow cells necessary for erythropoietln-mduced m vitro expression of the immature bone marrow BFU-E. This adherent cell requirement is also fulfilled by leukocyte-conditioned medmm. Studies reported previously from our laboratory
have shown the tn vitro inducer effect of T lymphocytes on peripheral blood BFU-E expression m humans. In those studies, peripheral blood null cells from normal mdwlduals were separated Into null, T, and B cell fractions by lmmunoaffimty chromatography and E rosettmg (Chess & Schlossman, 1976) BFU-E colony expression was found in unfracuonated and null cell populations, but not m the T or B cell fractions Minunal growth of small, poorly hemoglobmtzed colomes was evident in the null cell fractions. However, when T cells were added back to null cells, large numbers of typical BFU-E colomes were formed. Figure 3 shows previously published data in which this T cell effect is noted. Increasing numbers of T ceils were added to a constant number of null cells. As the number of T cells added was increased, the number of large BFU-E colomes formed m culture also increased. Figure 4 shows that when irradiated or allogenelc T cells were added, there was no effect (Nathan et al., 1978). These data demonstrate that the peripheral blood BFU-E is in fact found In the null cell fracuon of peripheral blood and that T ceils can serve an inducer funcUon for erythropoietm-stmaulated proliferation and differentiation of this progemtor. As shown m Fig. 5, intact T cells can be replaced m th~s inducer function by a tetanus toxo~d stimulated T cell conditioned medium, a medium that is known to contmn lymphocyte m~togemc factor, or LMF. Th~s medium was mixed in plasma clot culture with null cells at 10% v/v and m most studies, stzmulated BFU-E colony expression to the same extent as approxamately 2 x 106 T cells. The failure of patients deficient m T ceils to exhibit anerma represents an important clinical contradiction to the concept that T ceils are reqmred for the induction of erythropoietln-dependent erythrotd progemtor differentiation. An experimental approach was designed (Lipton, Remherz, Kudisch, Jackson, Schlossman & Nathan, 1980) in an attempt to resolve this issue, using highly specific monoclonal hybrldoma antibodies (Kung et al., 1979; Remherz et al., 1979). These antibodies permit further separation and analysis of the cell-cell interactions which operate m vitro in plasma clot. As reported elsewhere, we worked with an anUbody which is umquely reactive vath the entire class of peripheral blood T lymphocytes (Kung et al., 1979). Peripheral blood or bone marrow was collected m preservatwe-free heparm. The mononuclear cells were separated by Ficoll-Hypaque centnfugatlon. The peripheral blood mononuclear cells were subjected to nylon wool adherence to remove 80% or more of the contaminating monocytes and B lymphocytes. The bone marrow mononuclear cells were not exposed to nylon wool. The cells were then lncu-
Monoclonal Antibody Assessments of T Cell Interactmns m Erythropoletm Studies
241
NEGATIVE CONTROL NEGATWE CONTROL
NTI-T CELL ANTIBODY LABELED
LABELEO
~FLUORESCENCE
I N T E N S I T Y -
-
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INTENSITy
Fig 6. (a) FACS analysis of mature T cell content of Flcoll--Hypaque separated nonadherent peripheral blood mononuclear ceils. Analysis was done using OKT3 with a fluorescem conjugated ant~-mouse IgG The area under the curve shows that 77070 of the cells were mature T lymphocytes (b) A simdar FACS analysis of mature T cell content of Flcoll--Hypaque separated nonadherent peripheral blood mononuclear cells after treatment vnth OKT3 and rabbit complement. There are virtually no T cells remmnmg m this separation. Prewously pubhshed m J exp. Med. 152, 350, 1980.
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Fig. 7 FACS analysis of mature T cell content of Flcoll--Hypaque separated bone marrow mononuclear cells There are virtually no T cells in th~s preparatmn Prevmusly published m J exp M e d 152, 350, 1980
bated with the monoclonal anUbody and complement, then washed and plated m plasma clot vnth an erythropoietm concentrauon of 2 Lu. m l . - ) In each experiment, a portion of the cells was analyzed by FACS before and after antibody treatment. In experiments in which mature T cells were reqmred, they were prepared from the same samples by standard E rosetting techmques. Figure 6a shows a representative FACS analysis of Ficoll-Hypaque-separated, non-adherent peripheral blood mononuclear cells. In this study, 77°7o of the mononuclear cells were identified as T cells by indirect lmmunofluorescence of OKT3 coated cells. Figure 6b shows the FACS analysis of this population after treatment with OKT3 and complement. It can be reachly seen that within the ILmltS of detection of the assay, the mature T cells that comprise the majority of the penpheral blood lymphocytes were eliminated by this treatment. FACS analysis of the bone marrow mononuclear cells was qmte different. Figure 7 shows a representative analysls. Again, within the hmlts of detecUon (<5%), there were virtually no detectable mature T cells m a Ficoll-Hypaque-separated sample of marrow mononuclear cells. It should be emphaslzed, however, that slmple calculations based on total cellularlty of blood and marrow would reveal that the absolute number of T cells m marrow would
242
DAVID G. NATHAN 60
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F~g 8. Influence of OKT3 and complement (OKT3 + C') treatment on nonadherent (NA) peripheral blood mononuclear cell BFU-E colony expresslon. In two separate experiments, peripheral blood mononuclear cells were separated by F1coll--Hypaque (FH) cemnfugatlon and NA cells prepared as prevmusly described. The cells were treated voth elther OKT3 or with a nommmune murme ascstes control and cultured m plasma clot at a concentratmn of 106 cells per ml m the presence of 2 l.U. erythropoletm. The size of the colomes was graded from l + to 4 + as prewously described. Panel A shows the BFU-E growth of control and antlbody-treated cells after additmn of varying amounts of NCTC containing LMF acuvlty Panel B shows the same results after addmon of T cells or I0% LMF to the cultures. Cultures of T cells alone reveal nearly absent colony growth. Previously pubhshed m J'. exp Med 152, 350, 1980. equal those in peripheral blood if T cells comprised about 4% of marrow cells. The effect of depletion of T cells by the hybridoma antibody on peripheral blood BFU-E colony formaUon was studied in two different types of experiments. In the experiment shown in Fig. 8a, the control cells (treated w~th nonspecific mouse ascttes and complement) were cultured alone and in the presence of varying proportions o f LMF m the plasma clot system. BFU-E colony formatton was not affected morphologically or numerically by this treatment, even in the presence of added LMF. In contrast, treatment of the cells with orthoclone
OKT3 and complement greatly reduced colony formauon, both numerically and wlth respect to colony sine, unless the cultures contmned as little as 1% of LMF. The experiment shown m Fig. 8b was performed differently. Control cells treated with nonspectfic mouse ascltes antibody fired and complement, developed BFU-E colomes in culture, and growth was enhanced in this experiment by added T cells or LMF, suggesting that thls sample differed somewhat from the one obtmned from the expenmerits shown m Fig. 9, in that the inducer substance released by T cells are present in this culture at less than optimal levels. Treatment of the cells with
Monoclonal Antibody Assessments of T Cell Interactions m Erythropoletm Studies OKT3 and complement greatly reduced BFU-E colony formation, both numerically and morphologically, and growth was restored by added T cells and by added L M F containing medmm when it was present at 10070 of the plasma clot. T cell preparations themselves exhibited virtually no BFU-E colony expression, indicating minimal contamination of T lymphocytes with BFU-E. The results with OKT3 treated bone marrow were qmte different, as shown m a representatwe study depicted m Fig. 9. In th~s study, It is evident that treatment with OKT3 and complement does not reduce marrow CFU-E or BFU-E colony growth. The colony morphology was not affected, nor was there any effect of the addition o f T cells of 10070 L M F after antibody and complement treatment. It appears, therefore, that immature peripheral blood derived BFU-E depend upon mature lymphocytes or on T cell-conditioned medium (LMF) for erythropoxetin-sumulated differentmtion m plasma clot, and bone marrow BFU-E have no requirement for mature T cells for erythropoietin-dependent colony expression. These findings, however, do not preclude the presence in bone marrow of mature T ceils undetectable by these methods or the presence of immature T cells or other cells whach may serve as reducers of erythropo~etin-dependent dlfferentaatlon. However, mature T cell independence of mature marrow BFU-E may explain the lack of anemia m patients deficient in these cells. There are still many unanswered questions. These studies of marrow BFU-E colony formation provide insight only into differentmtlon of these BFU-E Into erythro~d colomes and not into their rephcat~on. The r61e of erythropoietm in the development of the erythrold progenitors has been explored as menhoned by Udupa & Reisman (1979). They clearly demonstrate that the rephcation of mature BFU-E and CFU-E is highly erythropoletm-responslve, and the development of immature BFU-E and CFU-E ~s also highly erythropoietm-responsive whereas the development of ~mmature BFU-E from CFU-S and their replication is less, ff at all, influenced by the hormone. Amphficatlon of erythropolesis occurs as BFU-S mature and rephcate and respond to ambient erythropoletm. Other celt-cell mteracttons
The hematopo~euc cell-cell interactions described above may be comphcated somewhat by the high proportion o f monocytes that contaminate cell fracuons used m typical expdrlments. Monoeytes comprise 25-50070 of the cells m a Flcoll-t-Iypaque blood cell separation (Zucker-Frankhn, 1974) and m-
243
hlbmon of BFU-E expression by monocytes has been reported (Rinehart, Zanjani, Nomdedeu, Gormus & Kaplan, 1978). Despite the possibihty of nonspeclfic inhibition produced m vitro by extruded products of stimulated monocytes (Unanue, Belier, Calderon, Klely & Stadecker, 1976), we have been unable to demonstrate an inhibitory role of these cells. In recent studies (Lipton, Link, Breard, Jackson, Clarke & Nathan, 1980), we have prepared peripheral blood cell fracuons by physical separation techmques continning up to 9507o monocytes which still expressed BFU-E colomes ff a source of L M F was added to the culture. In addition, null cell preparations m which no attempt was made to remove monocytes by adherence contained up to 8407o monocytes. These preparations required T ceils or L M F for optunal BFUE expression. The results of an experiment demonstrating a T cell or L M F dependence for optimal BFU-E colony expression using this populaUon is demonstrated m Table 3. Null cell expression of BFU-E is abundant despite this monocyte contamination ff T cells or L M F are added. Recent work by Kurland (Kurland, Meyers & Moore, 1980) has shown m the murme system that. macrophages added to their culture system sttmulated both CFU-E and BFU-E prohferation. Cells related to macrophages may comprise a sigmficant portion of the marrow microenwronment and their rSle m hematopo~euc regulation needs further exploration. M o d e l o f erythropotests
Based on our studies and those of others (Eaves & Eaves, 1978; Iscove, 1978; Papayannopoulou & S t a m a t o y a n n o p o u l o s , 1979; P a p a y a n n o p o u l o u , Chen, Maniat~s & Stamatoyannopoulos, 1980; Iscove, 1977) we proposed a hypothetical model to provide a framework for the investigation of human erythropoiesis. As shown m Fig. 10, the plunpotent stem cell, CFU-S, which has the capacity for selfrenewal, becomes committed to erythrold differentiation, and stochastically dffferentmtes into a prtmmve class o f BFU-E, the BFU-EFA. This primitive erythro~d progenitor can dtrectly gwe rise to CFU-E and on to erythroblasts, capable o f synthesizing fetal hemoglobin. They are sttmulated m vtvo to undergo increased funcUonal dffferentmtion to F cells during anemia under the influence of high levels of erythropoletin. Those F erythroeytes, so called because they contain fetal hemoglobin as well as hemoglobin A, represent 1-5070 of erythrocytes under normal condluons and greatly increase m number during stress erythropoiesls (Dover, Boyer, Zmkham, 1979). In addition to erythropoietln, the
DAVID G
244
-I--
100
-500
1
1T
-400
~
"300
NATHAN
90
,'!l,o
I
+ ÷ I
-200
h -100
Cell
Treatment Control Addlhves
. Ab+C LMF 1obT/ml
Control LMF 106T/m)
• Ab+C LMF 106T/ml
LMF 106T/ml
F~g. 9 Influence of OKT3 and complement ( O K T 3 + C ' ) treatment of Flcoll--Hypaque separated bone marrow mononuclear cells on CFU-E and BFU-E colony expression The different addltwes were LMF at 10°70 v/v and 106 T cells per ml plasma clot culture to control and O K T 3 + C ' treated cells Prewously pubhshed m J exp Med. 152, 350, 1980. Table 3 *
BFU-E colomes derwed from null cellst m the presence of T lymphocytes or LMF
Populauon
Number of cells plated m l - i
BFU-E/ 105 cells plated~/
2 x 105 8 × 104 106 2 x l0 s 106 8 x 104 I06 2 × l0 s 8 x 104
41 5-)-7 5 41 3")'15 3-*-1 7
Null cells Null cells T lymphocytes Null cells and T lymphocytes Null cells and T lymphocytes Null cells and LMF Null cells and LMF
170~10 229+_11 229-~9 188±15
* Previously pubhshed m J chn Invest 65, 219, 1980 t The "null cells" contmn 84070 monocytes and approximately 5% T cells as determined by fluorescence-activated cell sorting For null cell" T lymphocyte mixtures this ts expressed as BFU-E/105 null cells plated. f u n c t i o n a l d i f f e r e n t i a t i o n o f this p r o g e n i t o r to erythrocytes reqmres a T cell p r o d u c t . This c a n b e supplied m the b l o o d b y m a t u r e T cells. In the m a r r o w , the inducer cell is m the a d h e r e n t cell fraction. T h e hneage o f t h a t cell h a s n o t been determ i n e d . T h e assay for F cell p r o g e m t o r d l f f e r e n t l a u o n ts t h e expression m p l a s m a clot o f BFU-EFA colomes
f r o m peripheral b l o o d null cells in the presence o f e r y t h r o p o l e t m a n d m a t u r e T ceUs. T h e existence o f the CFU-EFA is referred, b u t these are n o t detected m the b l o o d a n d few are n o t e d in the b o n e m a r r o w ( S t a m a t o y a n n o p o u l o s & P a p a y a n n o p o u l o u , 1979). T h e vast m a j o r i t y o f BFU-EFA do not chfferentlate functlonally, as s h o w n vertically m the model, but
Monoclonal Antibody Assessments of T Cell Interactions m ErythropoieUn Studies undergo maturat]on-dtfferentiauon, as shown horizontally, to BFU-E A. They then, under the influence of erythropoietin, contmue to divide and amplify the system, filling the marrow with many BFU-E A. As they mature, they acqmre increased erythropoietin sensitivity and lose the requirement for T cell factors to induce functional differentmtion to the red cells. Finally, they differentiate to CFU-E A, continuing to amplify the system, and give rise to " A cells" which under nonstress conditions represent 95% of the total red cells. Smce BFU-E A are highly responsive to ambient erythropoletin, BFU-F~A dffferenttate to F cells to a relatively greater extent than BFU-E^ do to A cells in response to the high erythropoietin levels in anemia. Hence, the accumulation of F cells m peripheral blood in response to anemia. Although we are beginning to understand the nature o f T cell induction o f erythropotetindependent BFU-F~A differentiation, we do not have a grasp o f the probable cell-cell mteract)ons in I
COMMITMENT
245
BFU-EF^ repl]catmn and maturatmn. As well, understanding of the molecular biology of T cell ]nductton of erythropoietm-dependent erythroid differentiatton awmts purification of sufficient quantities of labeled functmnal erythropoietin and lsolatmn of the progenitor cell ttself so that the interaction of the hormone with its target can be studied in a reasonably homogeneous system. Recently, a subset of T cells defined by a monoclonal hybndoma antibody, OKT4, has been found to be responsible for L M F production (Reinherz, Kung, Breard, Goldstein & Schlossman, 1980). This same subset of cells ts required for B cell proliferation and differentiatmn, secretion of immunoglobulins, as well as generation of cytotoxlc T cells (Evans, Lazarus, Penta & Schlossman, 1978; Reinherz, Kung, Goldstein & Schlossman, 1979a; 1979b). Further investigation into the T cell subsets responsible for induction of erythropoietm-dependent erythroid differentiation will more specifically identify the inducer cell m that system.
] MATURATION - DIFFERENTIATION'[
Granutocyte f f Megakaryocyte /I
AMPLIFICATION H,gh Epo + T cells or T cell product
W er W
11l )-
0 Z
u.
Fig 10. Model of crythropolesls descrlblng maturation of erythrold progenitors from CFU-S and tcrmlnal differentiation of immature and mature BFU-E to F cells and A cells,respectively For description, see text
REFERENCES
ADAMSON, J W., m dlscusslon with NATHAN, D O. & HILLMAN, D (1978) Studies of erythropoles~s in culture Blood Cells 4, 228. AX~LRAD,A., McLEOD, D L., SHREEVE,M M. & HEATH, D. A. (1974) In Hemopmes~s m Culture (cd. Robinson, W. A.) p 26. DHEW Pubhcatlon NIH74-205 BASTEN,A. & BEESON,P. B. (1970). Mechanism of ¢osmophlha II. Role of the lymphocyte. J exp Med. 131, 1288--1305
246
DAVID G. NATHAN
BENNA, W A., HULLETT, H R., SWETT, R. G. & HERZENBERG, L A. (1972) Fluorescence-activated cell sorting Rev Sct Inatrum. 43, 404-409. CHESS, L & SCHLOSSMAN, S F (1976) Methods for preparatlon of unique human lymphocyte populations In Manual o f Chmcal Immunology, (eds. RosE, N R. and FRIEDMAN, H ) p. 77 Washington, D C , American Society for Microbiology CLARKE, B. J & HOUSMAN, D (1977). Characterization of an erythrold precursor cell of high proliferative capacity in normal human peripheral blood. Proc. natn. Aead. Scl. USA 74, 1105-t 109 CLARKE, B. J , NATHAN, D G , ALTER B. P , FORGET, B G , HILLMAN, D. G. ,~" HOUSMAN, D E. (1979) Hemoglobin synthesis in human BFU-E and CFU-E derived erythroid colonies. Blood 54, 805-817 CLXNE M J & GOLDE D W (1974) Production of colony-stimulating activity by human lymphocytes Nature 248, 703--704 CLINE, M. J. & GOLDE, D W. (1979). Cellular interactions m haematopoiesis Nature 277, 177--181 DOVER, G. J., BORER, S. H & ZINKHAM, W H (1979) Production of erythrocytes that contain fetal hemoglobin in anemia: transient m wvo changes. J chn Invest 63, 173-176. EAVES, C. J & EAVES, A C. (1978) Erythropoietm (EP) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood $2, 1196-1210. EVANS, R. L., LAZARUS, H., PENTA A. C. & SCHLOSSMAN, S. F (1978) Two functionally distinct subpopulatlons of human T cells that collaborate In the generation of cytotoxic cells responsible for cell mediated lympholysls J I m m u n . 120, 1423-1428 GEHA, R. S , SCHNEEaERGER, E., ROSEN, F S & MERLER, E. (1973). Interaction of human thymus-derived and nonthymus-derived lymphocytes m vitro Induction of prohferation and antibody synthesis m B lymphocytes by a soluble factor released from antigen-stimulated T lymphocytes. J. exp M e d 138, 1230--1247 GOLDE, D. W., BERSCH, N , QUAN, S G. & LUSIS, A J (1980). Production of erythrold-potentiatmg activity by a human T-lymphoblast cell line. Proc. natn Acad. Scl U S.A., 77, 593-596. GOODMAN, J W , BOSFOND, N L. & SmMPOCK, S. G (1978). On the role of thymus in hemopoletic differentiation Blood Cells 4, 53--64 GREGORY, C J (1976) Erythropoietln sensitivity as a differentiation marker m the hemopoletic system: studies of three erythropoietic colony responses in culture. J. Cell Physiol. 89, 289-302. GREGORY, C J & EAVES, A. C (1977) Human marrow cells capable of erythropoIetlc differentiation m wtro definition of three erythroid colony responses B l o o d 49, 855--864 HARA, H & OGAWAM (1978). Munne hemopoietic colonies in culture containing normoblasts, macrophages, and megakaryocytes A m . J. H e m a t o l 4, 23--34 ISCOVE, N N. (1977) The role of erythropoletm in regulation of population size and cell cycling of early and late erythrold precursors in mouse bone marrow. Cell Tissue Kmet. 10, 323--334. ISCOVE, N. N. (1978). Erythropoletm-independent stimulation of early erythropoIesis in adult marrow cultures by conditioned media from lectln-stlmulated mouse spleen cells. ICN-UCLA Symposium on HematopoIetlc Cell Differentlation in Molecular and Cell Biology, Academic Press, New York, Vol. 10, pp. 37-52 JOHNSON, G. R. & METCALF, D (1977) Pure and mtxed erythrold colony formation m wtro stimulated by spleen condltloned medmm with no detectable erythropoietln. Proc. natn A c a d Sct. U S A . 74, 3879-3882. KOHLEIt, G. & MILSTEIN, C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity Nature 256, 495--497 KUNG, P C., GOLDSTEIN, G., REINHERZ E. L. & SCHLOSSMAN, S F (1979) Monoclonal antibodies defining distractive human T cell surface antigens. Science 205, 347-349 KURLAND, J I , MEYRES, P. A. & MOORE, M. A S (1980) Synthesis and release of erythrold colony- and burstpotentiating activities by purified populations of murine peritoneal macrophages J exp. Med. 151,839-852 LIPTON, J. M., LtNK, N A., BP,JEARD, J., JACKSON, P L., CLARK~, B. J & NATHAN, D G. (1980) Monocytes do not Inhibit peripheral blood erythroid burst-forming umt colony formation. J. chn. Invest 65, 219-226. LIPTON, J M & NATHAN, D. G , T cells and erythropoteslS. Proc. of Conf on Aplastic Anemia, sponsored by NHLBI and NIAMDD, San Francisco, CA, 17-19 June 1979 (in press). LIPTON, J M , REINHERZ, E L., KUDISCH, M., JACKSON, P L., SCHLOSSMAN,S F & NATHAN, D G (1980). Mature bone marrow erythroId burst-forming units do not require T cells for reduction of erythropoIetin-dependent differentlation. J. exp. M e d 152, 350--360 McLEoD, D L., SnREEVE, M M. & AXELRAD, A A (1974). Improved plasma culture system for production of erythroCytlc colonies m vitro: quantitative assay method for CFU-E. B l o o d 44, 517-534. MESS~,q-.'R, H. A & FAUSER, A A. (1978). Distribution of fetal hemoglobin in individual colonies by RIA: a lead toward human stem cells. In Cellular and Molecular Regulation o f Hemoglobin Swttchmg, (eds. STAMATOVANNOPOULOS,G and NEINHUIS, A.), Grune and Stratton, New York. p 39 NATHAn, D. G., CHESS, L., HILLMA~, D. G., CLARKE, B., BREARD, J., MERLER, E. & HOUSMAN, D E. (1978). Human erythroid burst-forming unit: T-cell requirement for proliferation m vitro J. exp. Med. 147, 324--339 NATHAN, D G , CLARKE, B. J., HILLMAN, D G , ALTER, B. P & HOUSMEN, D E. (1979) Erythroid precursors in congenital hypoplastlc (Diamond--Blackfan) anemia J c h n , Invest 61, 489-498
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247
OGAWA, M., GROSH, O C., O'DELL, R. F. & SEXTON, J (1976) C~reulatmg erythropoletic precursors in patients with hemogloblnopathles' absence of correlation with retlculocyte radices. Exp HaematoL 4, 41 PAPAYANNOPOULOU, T., CHEN, P., MANtATIS, A. & STAMATOYANNOPOULOS, G (1980). Simultaneous assessment of l-antigen expression and fetal hemoglobin in single red cells by tmmunofluorescence. Blood 55, 221-232 PAPAYA~NOPOULOU, T. & STAMATOYANNOPOULOS,G. (1979) On the onbqn of F cells in the adult: clues from studies in clonal hemopathles. In Cellular and Molecular RegulaUon o f Hemoglobin Swztchmg (eds. STAMATOYANNOPOULOS, G and NEINHUIS, A.), Grune and Stratton, New York. pp 87--195. QUESENnERRY, P. & LEVITT, L. (1979). Hematopmetlc stem cells. New Engl J. M e d 301, 755-760, 819-823,868-872. REI~HERZ, E L., KONG, P C., BR~ARD, J M., GOLDS'r~IN, G. & SCHLOSSMAN, S F. (1980). T cell reqmrements for generation of helper factor(s) In man" analysis of the subsets involved. J. Immun 124, 1883--1887. I~INHEnZ, E. L., KUNG, P C., GOLDSTEIN, G & SCHLOSSMAN, S F (1979a). Separauon of functional subsets of human T cells by a monoclonal antibody Proc natn Acad Scz., U.S A. 76, 4061-4065 REINHERZ, E L , KUNG, P C., GOLDSTEIN, G & SCHLOSSMAN, S F (1979b) Further characterization of the human Inducer T cell subset defined by monoclonal antibody. J lmmun 123, 2894-2896. REIN~RZ, E L., KUNG, P C., PESANDO, M. J , RITZ, J., GOLDSTEXNG & SCHLOSSMAN, S. F (1979). la determinants on human T-cell subsets defined by monoclonal antibody. Activation stlmuh required for expression. J exp M e d 150, 1472-1482. RINEHART, J. J., ZANJANI, E D., NOMDEDEU, B , GORMUS, B. J & KAPLAN, M. E (1978). Cell-cell interaction in erythropolesls Role of human monocytes. J chn Invest 62, 979-986 RISITZKY, P., ZWORh D. & TRAININ, N. (1971) Effect of neonatal thymectomy on hemopoletlc tissue m mice Blood 37, 634--646. RUSCETTI, F. S & CHERVENICK, P A (1975) Release of colony-sumulatmg acUvlty from thymus-derwed lymphocytes J chn Invest. 55, 520--527 SHARKIS, S J , SPIVAK, J L., AHMED A., MISITI, J., STUART, R. K. WIKTOR-JEDRZEJCZAK, W., SELL, K. W & SENSENi)RENNEN, L L (1980) Regulatmn of hematopolesls" Helper and suppressor influences of the thymus Blood 55, 524--535 STAMATOYANNOPOULOS,G & PAPAYANNOPOULOU,T. (1979). Fetal hemoglobin and the erythro~d stem cell dffferentiatmn process In Cellular and Molecular Regulatmn o f Hemoglobin Swstchmg (eds STAMATOYANNOPOULOS,G and NE1NHUIS, A ) p 323. Grune and Stratton, New York. STEPHENSON, J. R , AXELRAD, A A., MCLEOD, D & SHREEVE, M M (1971). Induction of colonies of hemoglobinsynthesizing cells by erythropoletm m mtro Proc natn Acad Sc~., U S.A 68, 1542--1546 STRELKAUSKAS,A J., WILSON, B S., CALLERY, R. T CHESS, L & SCHLOSSMAN, S F (1977) T cell regulatmn of human peripheral blood B cell responsweness J exp M e d 146, 1765--1772 TSANG, R W & AYE, M T. (1979) Evidence for prohferatlon of erythrold progenitor cells m the absence of added erythrope~etm. Exp. Hematol 7, 383-388 UDLIPA, K B. & REISSMAN, K R (1979) In mvo erythropoletm requirements of regenerating erythrold progenitors (BFU-E, CFU-E) m bone marrow of mice Blood 53, 1164-1171 UNANUE, E R., BELLER, D I , CALDERON, J., KmLY, M J x, STADECKER, M O. (1976) Regulatmn of lmmumty and inflammation of mediators from macrophages A m d Pathol 85, 465-478 WIKTOR-JEDREZEJCZAK, W., SHARKIS, S , AHMED, A. & SELL, K W (1977) Theta-sensltwe cell erythrOpolesls" Identtficauon of a defect in w / w v anemic mace. Sctence 196, 313-315. ZIPORl, D. & TRAININ, N (1973) Defectwe capacity of bone marrow for nude mace to restore lethally irradiated reagents Blood 42, 671-678 ZIPORI, D & TRAINIU, N (1975) Impaired radioprotectlve capacity and reduced proliferative rate of bone marrow from neonatally thymectormzed mice Exp Hematol 3, 1--11 ZUCKER-FRANKLIN, D (1974). The percentage of monocytes among mononuclear cell fractmns obtmned from normal blood J Immun 112, 234--240
Vol 3 No a pp 2"~-24"
1981
0192-0561/81/030233-1450200/0 Pergamon Pre,,,, I td
Printed In the U ~ A
M O N O C L O N A L ANTIBODY ASSESSMENTS OF T CELL INTERACTIONS IN E R Y T H R O P O I E T I N STUDIES DAVID G. NATHAN* Department of Pedmtncs, Harvard Medical School, Boston, Massachusetts, U.S.A (Recetved f o r pubhcatwn 12 May 1981)
Abstract--The contemporary apphcatlon of clonal assay techntques has greatly expanded our knowledge of the regulation of hematopolesls. Our efforts have been directed toward the mvestlgatmn of non-erythropo~etm-medmted regulation of human erythropolesls m the form of cell-to-cell interaction between mature T cells and erythrold progemtors. Our data indicate that three such progemtors, the earll~marrow erythrocyte precursor BFU-E, the more mature marrow erythrocyte precursor, CFU-E and the penpheral blood BFU-E, each exhlblt totally different reqmrements for their colony expressmn in culture, with respect to the absence of erythropoletm and the presence of mature T ceils or their products The capacity of erythrold progemtors to w~thstand mcubatwn m the absence of erythropo~etm appears to be a charactensnc of ~mmature rather than mature erythrold progemtors Furthermore, use of OKT3 anubody depletmn techmques shows that peripheral blood-derived BFU-E appear to depend upon mature lymphocytes or T cell-condmoned medmm for erythropoletm-stlmulateddffferentmtmn whde bone marrow BFU-E and CFU-E have no reqmrement for mature T cells to produce erythropoletm-dependent maturauon Our results, plus a vast array of data provided by other mvemgators m the field, are integrated into a proposed framework for further mvest|gatlon of T cell mductmn of erythropo~etm-dependent erythrmd dffferentmtmn tamed at more specifically ldentff)qng the reducer cell subset(s) m that system.
The development o f m vtvo and m vttro d o n a l assays for both pluripotent and committed hematopoietic progenitor cells has greatly expanded our understanding of the regulation o f hematopoiesis. Thts subject has already been extensively reviewed (Cline & Golde, 1979; Quesenberry & Levitt, 1979; Lipton & Nathan, 1979), and need not be d|scussed m detail here. Figure 1 outlines our contemporary concepts o f hematopoiet|c differentmtmn based upon assays conducted m both routine and human systems. The pluripotent stem cell is the most fundamental hematopoietic progenitor. Its offspnng ultimately populate the entire marrow with recogmzable nucleated red cell, white cell, and platelet precursors. Its nnmediate offspring are the committed progenitors of lymphocytes (CFU-L) and the tnpotent myeloid precursor (CFU-S). CFU-S cells have the potential to give rise to any of three committed progenitors. Each o f the three possesses a unique hmlogic program of differentiation and rephcatlon that provide the recognizable precursors o f the erythrocyte, granulocyte, and megakaryocyte populauons. These cells are known as the BFU-E, CFU-C, and CFU-M cells, respect|rely. They are all present in small numbers m
hone marrow. Their precise morphology is unknown, but they are most likely mononuclear cells that resemble lymphocytes. In addition to these cellular components of the system, there are other cells and humors that influence the pathway of hematopotetic differentiation described above. Among these regulators are: erythropoietin, colony-stimulating activity, thromhopoietin, and possibly lymphopoietin. Much o f the process, we believe, is also under the influence o f thymus-derived (T) lymphocytes since some o f these inducer substances may be secreted locally by the marrow stromal cells or T lymphocytes that may provlde the microenviroument of the developing hematopoietic cells. The erythroid progenitor assay system developed by Axelrad and colleagues in plasma clot (Stephenson, Axelrad, McLeod & Shreeve, 1971; McLeod, Shreeve & Axelrad, 1974; Axelrad, McLeod, Shreeve & Heath, 1974) and adapted to methylceUulose (Gregory, 1976; Messner & Fauser, 1978) offers a method for the in mtro investigation of erythroid regulatton. Eaves & Eaves (1978) have demonstrated three erythroid progemtors. Two of these consist of specific subgroups of the erythroid burst-forrmng
* Address all correspondence to: Davld G Nathan, M.D Chlef, Dwislon of Hematology and Oncology, Children's Hospital Medical Center, 300 Longwood Ave., Boston, MA 02115, U.S.A. The materlal presellted in thls article is the expressed vlew of the authors w h o were cooductlOg research on O R T H O C L O N E * monoclonal antibodies These reagents are intended for research use only and are not to be used In diagnostic procedures ("Trademark)
233
234
DAVIDG. NATHAN
----------- T Lymphocyte
,CFU-L
BLymphocyte
Plurlpotent/ StemCell ~
/
/BFU-E
CFU-E--
Proerythroblast--Erythrocyte Hegakaryocyte Platelet
CFU-S~ ' ~
CFU-C
~Myeloblast ~-
Monoblast
Eoslnophlllc' Myeloblast
CFU-E0
~--COMMITTEDSTEMCELLS---~I
Granulocyte Monocyte
(Macrophage)
Eoslnoph|]
BONEMARROW_.__JL__PERIPHERAL___J PRECURSORCELLS~I--BLOODCELLS-----]
Fig. 1. Schematic diagram of hematopolcsls as defined by clonal assays m the human and murme systems and G-6PD lsoenzyme studies m diseases of clonal prohferauon. CFU-S, colony-forming umt--spleen; CFU-L, colony-forming umt--lymphocyte; BFU-E and CFU-E, burst-forming and colony forrmng umt---erythrmd; CFU-M, colony-forming umt--megakaryocyte; CFU-C, colony-forrmng umt m culture; and CFU-E0, colony-forming umt--eosmophd. unit (BFU-E), and are termed the primitive and mature BFU-E, respectivdy. In the presence of erythropoietm, ~ach of these subgroup types develops into large colomes composed of multiple subcolomes. The rate of colony expression from primitive BFU-E is relatively slow, requiring 2 - 3 weeks for compleUon, depending upon culture conditmns. Mature BFU-E colonies develop more rapidly. The third and most mature class of progenitors is called the erythroid colony-forming unit (CFU-E). This cell is derived from BFU-E and forms single erythroid cell colonies m 6 - 7 days in culture. It ~s the immediate precursor of the proerythroblast. Figure 2 shows typical BFU-E and CRU-E derived colomes m plasma clot at high magnificatmn. The large colony comprising 6 multiple subcolonies d~nved from the BFU-E contrasts wlth the single CFU-E derived colony. Clarke and Housman 0977) and Ogawa and co-workers (Ogawa, Grush, O'Dell & Sexton, 1976) have demonstrated that the mononuclear cell fraction of normal human peripheral blood contmns small numbers of BFU-E. They most likely represent immature marrow progemtors which escape the marrow and enter the clrculation under ambient conditions. Most peripheral blood BFU-E are probably analogous to the most prLrmtwe bone marrow BFU-E that are demonstrated m methylcellulose.
Table I summarizes some of the salient differences between bone marrow and peripheral blood progenitors evaluated m plasma clot cultures. CFU-E expresslon occurs earlier m culture than BFU-E colony expression, and small colony-forming, mature BFU-E are expressed earlier than large colony-forming BFUE (Gregory & Eaves, 1977). Peripheral blood BFU-E are presem in much fewer numbers than e~ther of the marrow progenitors. They differ also wlth respect to their sensltiwty to erythropoictin. In the human system, it has been shown that CFU-E are the most sensitive, and pnmmve BFU-E less sensmve, to ¢rythropoletin than most mature BFU-E (Gregory, 1976; Eaves & Eaves, 1978). Marrow BFU-E are less susceptible to tnUatedthymidin¢ suicide than CFU-E, and peripheral blood BFU-E exlublt httle, if any, sensitivity to tntiated thymlchn¢, indicating that a lower fraction of penpheral blood BFU-E are m cycle than either marrow BFU-E or CFU-E (Adamson, 1978). With respect to hemoglobin synthesis, the peripheral blood BFU-E give rise to colomes capable of substanUal hemoglobm F production (Clarke, Nathan, Alter, Forget, Hillman & Housman, 1979). In contrast, marrow progenitor colomes produce much more hemoglobin A than F. In fact, hemoglobin F synthesis is barely detectable m our particular plasma clot culture system for marrow BFU-E. This difference m hemo-
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- "S..a. > Fig 2 Photomicrograph at high magmficat]on of erythrold progemtor derived colomes showing single large benzadme posture colomes for CFU-E (left) contrasted with large single colony with multiple subcolomes from BFU-E (right)
235
Monoclonal Antibody Assessments of T Cell Interactmns m Erythropoletm Studies Table 1
237
Charactensucs of human bone marrow CFU-E, BFU-E and peripheral blood BFU-E
Perlpheral blood
Bone marrow
Peak day m culture Morphology Numbers of colomes/ 105 mononuclear cells plated Epo sensmvity Thymldme smclde Hemoglobin F synthes~s T cell or LMF requirement Sensmvlty to epopnval state
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globin synthesis will be further discussed later m this paper since it serves as an indicator of progenitor maturity. Our most recent studies also show that the three progenitors exhibit totally different requirements for colony expression m culture with respect to the absence of erythropoietin and the presence of mature T cells or their products.
Clarke, Breard, Merler & Housman, 1978). Clottmg is inmated by the addition of 0.1 ml of Grade I bovine thrombm (Slgrna Chemical Company, St. Louis, MO) m 0.1 ml of culture mechum. One-tenth millihter allquots of the clotting mixture are then dispensed into 0.2 ml mlcromer culture wells and incubated for up to 14 days. CFU-E are enumerated on day 7 and BFU-E on day 14.
EXPERIMENTAL PROCEDURES
Anttbody treatment Orthoclone OKT3 is a monoclonal cytotoxic IgG antibody prepared by hybrldoma technology (Kohler & Milstein, 1975). It is reacuve with a cell surface antigen common to all human circulating T cells (Kung, Goldstem, Reinherz & Schlossman, 1979, Reinherz, Kung, Pesando, Ritz, Goldstem & Schlossman, 1979). Its activity was established by serial dituuon of the murine asciuc fired m which It was secreted. It was used m all of our studies at a dilution of 1:500 m mmmmm essential medmm (S-MEM; Grand Island BIological Company, Grand Island, NY) wath 10 mM Hepes buffer plus 507o fetal calf serum (FCS) (Flow Laboratories, Rockville, MD). A similarly diluted m u n n e ascmc flmd lacking monoclonal antibody activity was used as a control. Evaluation of mononuclear cell elements was accomphshed by the use of a fluorescence acuvated cell sorter (FACS I, Becton Dickinson, FACS Systems, Mountain View, CA) (Benna, Hullett, Swett & Herzenberg, 1972) using OKT3 and a fluorscem conjugated anu-mouse IgG Treatment of cells w~th OKT3 or control ascltes flmd was accomphshed by mcubation of 20 to 40× 106 mononuclear cells in
Clonal assays Clonal assay methods for assessment of BFU-E and CFU-E in human bone marrow and BFU-E in blood have been described elsewhere (Clarke & Housman, 1977; Nathan, Clark, Hillman, Alter & Housman, 1979). Briefly, mononuclear cells are prepared by Flcoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N J) centrifugaUon. The appropriate cell numbers are added in 0.1 ml of alpha minus medmm (Grand Island Biological Company, Grand Island, NY) to 0.8 ml of the erythropoietmdependent plasma clot system of McLeod and coworkers (1974), modified by Clarke & Housman (1977). Erythropoietm (kindly provided by Drs. Anne Ball and Peter Dukes, NIH, and Connaught, Step III, Connaught Laboratories, Willowdale, Ontario, Canada) is employed at a concentration of 2 1.u. ml-~. In some experiments, 0.1 of medium m which tetanus toxold-stimulated mononuclear cells had proliferated is substituted for 0.1 ml of culture medmm This medmm contains lymphocyte mttogenie factor (LMF) (Nathan, Chess, Hillman,
238
DAVID G. NATHAN
I ml of the medium to whlch e~ther anUbody-contaznmg or control ascit~c fluid had been added for 30 min at 4°C. Then 0.15 ml of fresh frozen rabblt serum (Lot C192108, Grand Island Biologlcal C o m pany, Grand Island, NY) was added as a source of complement. The incubation was contained at 37°C for 1 h w~th gentle shaking. Following this the cells were thrice washed m a-medium plus 5% FCS. RESULTS AND DISCUSSION
The epopr~vai state As mentioned above, a dlstingmshmg characterlstic of progenitor classes is their response to the absence of erythropoietm m the culture medmm. Iscove (1978) first noted that murine marrow BFU-E and CFU-E differ wRh respect to their ability to proliferate m culture m the absence of erythropoletm. If erythropoietin was w~thheld from the culture for even a few hours, only a small percentage of the CFU-E were able to produce colonies, whde BFU-E were able to proliferate for days. Tsang & Aye (1979) reported a significant fraction of human marrow BFU-E were also capable o f colony production after prolonged erythropoietin deprivauon. Thus, the capacity of erythroid progemtors to withstand incubation in semi-solid media in the absence of erythropoietm appears to be a characteristlc of immature rather than mature erythroid progenitors. Table 2 shows the influence of the delay of erythropoletin addition on bone marrow BFU-E and CFU-E and peripheral blood BFU-E colony formation. Maximal colony growth follows addiuon of erythropoietin at control day zero. This is compared Table 2.
to the percentage of maxzmal growth achieved by each type after an established number of epopnval days. The results show that the expression of penpheral blood BFU-E is preserved for greater than three days m such experiments. The expression of marrow BFU-E is preserved to a lesser degree than that of peripheral blood BFU-E. In sharp contrast, a rapid decline m CFU-E colomes occurs when erythropoletin is withheld from the culture medium. We postulate that the more ~mmature the progemtor the greater its potenual to divide m response to an reducer that can be derived from T ceils. In culture, the immature cell replicates under the influence of reducer(s) and acqmres erythropoletm receptors. At this point, ff erythropoletm is not added, the progemtor will no longer differenuate and colony growth is lost. The more immature the progenitor, the more div~slons can occur prior to the accrual of erythropotetin receptors and the longer the time that the ¢poprival state can be tolerated. T cell regulatton o f erythropotests The inducer role of T lymphocytes in hematopolesis has been extensively studied in m vttro and in wvo systems. A decrease in the splemc engraftment of granulopoletlc and erythropoletlc colomes in lethally irradiated lsogemc recipient mice from anti-0 treated bone marrow has been described (Goodman, Bosfond & Shlmpock, 1978). Further, there is a failure of engraftment and correction of the anemm m the congenitally anemic w/w ~ mouse when 0 positwe cells are removed from the reconstituting marrow graft (Wiktor-Jedrzejczak, Sharkis, Ahmed & Sell, 1977). Recent work has implicated thymus-derived cells as regulators of erythropoletlC engraftment in
Influence of delay of erythropoletm addmon on bone marrow (BM) BFU-E and CFU-E and peripheral blood (PB) BFU-E colony formation
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Monoclonat A n t i b o d y Assessments o f T Cell Interactions m Erythropoietin Studies
239
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F~g. 3 Three examples o f the effects o f varying n u m b e r s o f isogemc T ( • ) and B ( O ) cells on the f o r m a t m n o f BFUE colonies T h e T or B cells were added to 10~ null cells per ml o f plasma clot m the presence o f 2 Lu. o f erythropo~etm at the cell c o n c e n t r a t m n s s h o w n m the abscissa. The ordinate represents the total n u m b e r o f B F U - E colomes o f all s~zes observed after 14 days o f culture o f the cell mxxtures. In a d d m o n to the numerical Increase m BFU-E colomes observed when T ceils were added to null cells, there was also an increase m the average sxze o f mdlwdual colomes. Previously published m J exp Med. 147, 324--339, 1978. Q 6O u.J o.
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Fig. 5. Influence o f T cell-condmoned m e d m m and intact T cell on 3 - 4 + BFU-E colony growth m null cells The null cells were incubated at 106 cells per ml o f clot m the presence of 2 I.U. erythropoletm The different a d d m v e s were tetanus toxo~d, at a final c o n c e n t r a u o n o f 4 #g per ml o f clot, T cell-condmoned m e d m m , prepared as described m the text, at 10% v / v or 2 . 5 x 106 T cells per ml of clot. The mtxtures were cultured for 14 days and the 3 - - 4 + BFU-E colontes were enumerated. Prevmusly pubhshed m J. exp. M e d. 147, 324-339, 1978
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Fig 4. Influence o f various T ceil preparations and the effects o f ~rradIatmn on 3--4 + B F U - E colony growth m null cells The fresh or Irradiated null cells were incubated at l06 cells m l - 1 and l07 various treated isogenelc or allogeneic T cells were added to them The cell mixtures were incubated for 15 days m the presence of 2 t u of erythropoletin. Only 3 - - 4 + BFU-E colomes were included in the colony scores Previously p u b h s h e d in J exp M e d 147, 324--339, 1978
240
DAVID G. NATHAN
these w/w v rmce (Shark;s, Spwak, Ahmed, Mism, Stuart, Wlktor-Jedrzejczak, Sell & Sensenbrenner, 1980). The role of thymus-derwed cells ts further supported by expenments demonstrating the defective restoratwe capacity of bone marrow from congemtally athymic (Zipon & Tramm, 1973) and neonatally thymectomized (Risitzky, Z~pon & Trainin, 1971; Zipori & Trainm, 1975) rmce. The cellular products of dividing T cells or T cell leukemic lines (Golde, Bersch, Quan & Lusls, 1980) have multiple effects on hematopoletlc cells, regulating synthes~s of antibody (Geha, Schneeberger, Rosen & Merler, 1973; Strelkauskas, Wilson, Callery, Chess & Schlossman, 1977) and influencing them to divide and differentmte into eosinophllic (Basten & Beeson, 1970), granulocytlc (Cline & Golde, 1974; Ruscetti & Chervemck, 1975) and megakaryocytlC (Hara & Ogawa, 1978) as well as erythroid colonies (Iscove, 1978). It has been shown that the regulation of tn v i t r o as well as m m v o erythroid production and maturation is strongly controlled by erythropoletm. Recently, however, a number o f investigators have demonstrated that there is sigmficant erythropoiesls which requires other factors in addition to the hormone. It ~s hkely that these nonerythropo~etin factors mfiuence primitive erythroid differentmuon to a greater extent than the differentiation of the more mature progenitor, as shown by the relatwe resistance to the epoprwal state of the immature progemtor compared to the more mature progenitor. Udupa & Re~sman (1979) have demonstrated m the munne system that immature BFU-E are probably derived from CFU-S and that the repllcauon of mature BFU-E is less sensitive to erythropoietin than CFU-E. In addition, Iscove's work (1978) demonstrated a role for a substance called burst-promoting activity (BPA) derived from P H A and Con A stimulated mouse spleen ceils. Iscove's experiments have been interpreted to show that BPA may somehow increase the number of erythropoletin receptors on BFU-E. Investigations by Johnson & Metcalf (1977) have demonstrated that erythroid colonies could be derived from cultures of fetal liver stimulated by a PHA-treated mouse spleen cell conditioned medium w~thout the addition of erythropoletm. Their studies, however, did not rule out the presence of small amounts of endogenous erythropoietin in the culture media. Eaves & Eaves (1978) have shown that there is an adherent population o f marrow cells necessary for erythropoietln-mduced m vitro expression of the immature bone marrow BFU-E. This adherent cell requirement is also fulfilled by leukocyte-conditioned medmm. Studies reported previously from our laboratory
have shown the tn vitro inducer effect of T lymphocytes on peripheral blood BFU-E expression m humans. In those studies, peripheral blood null cells from normal mdwlduals were separated Into null, T, and B cell fractions by lmmunoaffimty chromatography and E rosettmg (Chess & Schlossman, 1976) BFU-E colony expression was found in unfracuonated and null cell populations, but not m the T or B cell fractions Minunal growth of small, poorly hemoglobmtzed colomes was evident in the null cell fractions. However, when T cells were added back to null cells, large numbers of typical BFU-E colomes were formed. Figure 3 shows previously published data in which this T cell effect is noted. Increasing numbers of T ceils were added to a constant number of null cells. As the number of T cells added was increased, the number of large BFU-E colomes formed m culture also increased. Figure 4 shows that when irradiated or allogenelc T cells were added, there was no effect (Nathan et al., 1978). These data demonstrate that the peripheral blood BFU-E is in fact found In the null cell fracuon of peripheral blood and that T ceils can serve an inducer funcUon for erythropoietm-stmaulated proliferation and differentiation of this progemtor. As shown m Fig. 5, intact T cells can be replaced m th~s inducer function by a tetanus toxo~d stimulated T cell conditioned medium, a medium that is known to contmn lymphocyte m~togemc factor, or LMF. Th~s medium was mixed in plasma clot culture with null cells at 10% v/v and m most studies, stzmulated BFU-E colony expression to the same extent as approxamately 2 x 106 T cells. The failure of patients deficient m T ceils to exhibit anerma represents an important clinical contradiction to the concept that T ceils are reqmred for the induction of erythropoietln-dependent erythrotd progemtor differentiation. An experimental approach was designed (Lipton, Remherz, Kudisch, Jackson, Schlossman & Nathan, 1980) in an attempt to resolve this issue, using highly specific monoclonal hybrldoma antibodies (Kung et al., 1979; Remherz et al., 1979). These antibodies permit further separation and analysis of the cell-cell interactions which operate m vitro in plasma clot. As reported elsewhere, we worked with an anUbody which is umquely reactive vath the entire class of peripheral blood T lymphocytes (Kung et al., 1979). Peripheral blood or bone marrow was collected m preservatwe-free heparm. The mononuclear cells were separated by Ficoll-Hypaque centnfugatlon. The peripheral blood mononuclear cells were subjected to nylon wool adherence to remove 80% or more of the contaminating monocytes and B lymphocytes. The bone marrow mononuclear cells were not exposed to nylon wool. The cells were then lncu-
Monoclonal Antibody Assessments of T Cell Interactmns m Erythropoletm Studies
241
NEGATIVE CONTROL NEGATWE CONTROL
NTI-T CELL ANTIBODY LABELED
LABELEO
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Fig 6. (a) FACS analysis of mature T cell content of Flcoll--Hypaque separated nonadherent peripheral blood mononuclear ceils. Analysis was done using OKT3 with a fluorescem conjugated ant~-mouse IgG The area under the curve shows that 77070 of the cells were mature T lymphocytes (b) A simdar FACS analysis of mature T cell content of Flcoll--Hypaque separated nonadherent peripheral blood mononuclear cells after treatment vnth OKT3 and rabbit complement. There are virtually no T cells remmnmg m this separation. Prewously pubhshed m J exp. Med. 152, 350, 1980.
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Fig. 7 FACS analysis of mature T cell content of Flcoll--Hypaque separated bone marrow mononuclear cells There are virtually no T cells in th~s preparatmn Prevmusly published m J exp M e d 152, 350, 1980
bated with the monoclonal anUbody and complement, then washed and plated m plasma clot vnth an erythropoietm concentrauon of 2 Lu. m l . - ) In each experiment, a portion of the cells was analyzed by FACS before and after antibody treatment. In experiments in which mature T cells were reqmred, they were prepared from the same samples by standard E rosetting techmques. Figure 6a shows a representative FACS analysis of Ficoll-Hypaque-separated, non-adherent peripheral blood mononuclear cells. In this study, 77°7o of the mononuclear cells were identified as T cells by indirect lmmunofluorescence of OKT3 coated cells. Figure 6b shows the FACS analysis of this population after treatment with OKT3 and complement. It can be reachly seen that within the ILmltS of detection of the assay, the mature T cells that comprise the majority of the penpheral blood lymphocytes were eliminated by this treatment. FACS analysis of the bone marrow mononuclear cells was qmte different. Figure 7 shows a representative analysls. Again, within the hmlts of detecUon (<5%), there were virtually no detectable mature T cells m a Ficoll-Hypaque-separated sample of marrow mononuclear cells. It should be emphaslzed, however, that slmple calculations based on total cellularlty of blood and marrow would reveal that the absolute number of T cells m marrow would
242
DAVID G. NATHAN 60
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F~g 8. Influence of OKT3 and complement (OKT3 + C') treatment on nonadherent (NA) peripheral blood mononuclear cell BFU-E colony expresslon. In two separate experiments, peripheral blood mononuclear cells were separated by F1coll--Hypaque (FH) cemnfugatlon and NA cells prepared as prevmusly described. The cells were treated voth elther OKT3 or with a nommmune murme ascstes control and cultured m plasma clot at a concentratmn of 106 cells per ml m the presence of 2 l.U. erythropoletm. The size of the colomes was graded from l + to 4 + as prewously described. Panel A shows the BFU-E growth of control and antlbody-treated cells after additmn of varying amounts of NCTC containing LMF acuvlty Panel B shows the same results after addmon of T cells or I0% LMF to the cultures. Cultures of T cells alone reveal nearly absent colony growth. Previously pubhshed m J'. exp Med 152, 350, 1980. equal those in peripheral blood if T cells comprised about 4% of marrow cells. The effect of depletion of T cells by the hybridoma antibody on peripheral blood BFU-E colony formaUon was studied in two different types of experiments. In the experiment shown in Fig. 8a, the control cells (treated w~th nonspecific mouse ascttes and complement) were cultured alone and in the presence of varying proportions o f LMF m the plasma clot system. BFU-E colony formatton was not affected morphologically or numerically by this treatment, even in the presence of added LMF. In contrast, treatment of the cells with orthoclone
OKT3 and complement greatly reduced colony formauon, both numerically and wlth respect to colony sine, unless the cultures contmned as little as 1% of LMF. The experiment shown m Fig. 8b was performed differently. Control cells treated with nonspectfic mouse ascltes antibody fired and complement, developed BFU-E colomes in culture, and growth was enhanced in this experiment by added T cells or LMF, suggesting that thls sample differed somewhat from the one obtmned from the expenmerits shown m Fig. 9, in that the inducer substance released by T cells are present in this culture at less than optimal levels. Treatment of the cells with
Monoclonal Antibody Assessments of T Cell Interactions m Erythropoletm Studies OKT3 and complement greatly reduced BFU-E colony formation, both numerically and morphologically, and growth was restored by added T cells and by added L M F containing medmm when it was present at 10070 of the plasma clot. T cell preparations themselves exhibited virtually no BFU-E colony expression, indicating minimal contamination of T lymphocytes with BFU-E. The results with OKT3 treated bone marrow were qmte different, as shown m a representatwe study depicted m Fig. 9. In th~s study, It is evident that treatment with OKT3 and complement does not reduce marrow CFU-E or BFU-E colony growth. The colony morphology was not affected, nor was there any effect of the addition o f T cells of 10070 L M F after antibody and complement treatment. It appears, therefore, that immature peripheral blood derived BFU-E depend upon mature lymphocytes or on T cell-conditioned medium (LMF) for erythropoxetin-sumulated differentmtion m plasma clot, and bone marrow BFU-E have no requirement for mature T cells for erythropoietin-dependent colony expression. These findings, however, do not preclude the presence in bone marrow of mature T ceils undetectable by these methods or the presence of immature T cells or other cells whach may serve as reducers of erythropo~etin-dependent dlfferentaatlon. However, mature T cell independence of mature marrow BFU-E may explain the lack of anemia m patients deficient in these cells. There are still many unanswered questions. These studies of marrow BFU-E colony formation provide insight only into differentmtlon of these BFU-E Into erythro~d colomes and not into their rephcat~on. The r61e of erythropoietm in the development of the erythrold progenitors has been explored as menhoned by Udupa & Reisman (1979). They clearly demonstrate that the rephcation of mature BFU-E and CFU-E is highly erythropoletm-responslve, and the development of immature BFU-E and CFU-E ~s also highly erythropoietm-responsive whereas the development of ~mmature BFU-E from CFU-S and their replication is less, ff at all, influenced by the hormone. Amphficatlon of erythropolesis occurs as BFU-S mature and rephcate and respond to ambient erythropoletm. Other celt-cell mteracttons
The hematopo~euc cell-cell interactions described above may be comphcated somewhat by the high proportion o f monocytes that contaminate cell fracuons used m typical expdrlments. Monoeytes comprise 25-50070 of the cells m a Flcoll-t-Iypaque blood cell separation (Zucker-Frankhn, 1974) and m-
243
hlbmon of BFU-E expression by monocytes has been reported (Rinehart, Zanjani, Nomdedeu, Gormus & Kaplan, 1978). Despite the possibihty of nonspeclfic inhibition produced m vitro by extruded products of stimulated monocytes (Unanue, Belier, Calderon, Klely & Stadecker, 1976), we have been unable to demonstrate an inhibitory role of these cells. In recent studies (Lipton, Link, Breard, Jackson, Clarke & Nathan, 1980), we have prepared peripheral blood cell fracuons by physical separation techmques continning up to 9507o monocytes which still expressed BFU-E colomes ff a source of L M F was added to the culture. In addition, null cell preparations m which no attempt was made to remove monocytes by adherence contained up to 8407o monocytes. These preparations required T ceils or L M F for optunal BFUE expression. The results of an experiment demonstrating a T cell or L M F dependence for optimal BFU-E colony expression using this populaUon is demonstrated m Table 3. Null cell expression of BFU-E is abundant despite this monocyte contamination ff T cells or L M F are added. Recent work by Kurland (Kurland, Meyers & Moore, 1980) has shown m the murme system that. macrophages added to their culture system sttmulated both CFU-E and BFU-E prohferation. Cells related to macrophages may comprise a sigmficant portion of the marrow microenwronment and their rSle m hematopo~euc regulation needs further exploration. M o d e l o f erythropotests
Based on our studies and those of others (Eaves & Eaves, 1978; Iscove, 1978; Papayannopoulou & S t a m a t o y a n n o p o u l o s , 1979; P a p a y a n n o p o u l o u , Chen, Maniat~s & Stamatoyannopoulos, 1980; Iscove, 1977) we proposed a hypothetical model to provide a framework for the investigation of human erythropoiesis. As shown m Fig. 10, the plunpotent stem cell, CFU-S, which has the capacity for selfrenewal, becomes committed to erythrold differentiation, and stochastically dffferentmtes into a prtmmve class o f BFU-E, the BFU-EFA. This primitive erythro~d progenitor can dtrectly gwe rise to CFU-E and on to erythroblasts, capable o f synthesizing fetal hemoglobin. They are sttmulated m vtvo to undergo increased funcUonal dffferentmtion to F cells during anemia under the influence of high levels of erythropoletin. Those F erythroeytes, so called because they contain fetal hemoglobin as well as hemoglobin A, represent 1-5070 of erythrocytes under normal condluons and greatly increase m number during stress erythropoiesls (Dover, Boyer, Zmkham, 1979). In addition to erythropoietln, the
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• Ab+C LMF 106T/ml
LMF 106T/ml
F~g. 9 Influence of OKT3 and complement ( O K T 3 + C ' ) treatment of Flcoll--Hypaque separated bone marrow mononuclear cells on CFU-E and BFU-E colony expression The different addltwes were LMF at 10°70 v/v and 106 T cells per ml plasma clot culture to control and O K T 3 + C ' treated cells Prewously pubhshed m J exp Med. 152, 350, 1980. Table 3 *
BFU-E colomes derwed from null cellst m the presence of T lymphocytes or LMF
Populauon
Number of cells plated m l - i
BFU-E/ 105 cells plated~/
2 x 105 8 × 104 106 2 x l0 s 106 8 x 104 I06 2 × l0 s 8 x 104
41 5-)-7 5 41 3")'15 3-*-1 7
Null cells Null cells T lymphocytes Null cells and T lymphocytes Null cells and T lymphocytes Null cells and LMF Null cells and LMF
170~10 229+_11 229-~9 188±15
* Previously pubhshed m J chn Invest 65, 219, 1980 t The "null cells" contmn 84070 monocytes and approximately 5% T cells as determined by fluorescence-activated cell sorting For null cell" T lymphocyte mixtures this ts expressed as BFU-E/105 null cells plated. f u n c t i o n a l d i f f e r e n t i a t i o n o f this p r o g e n i t o r to erythrocytes reqmres a T cell p r o d u c t . This c a n b e supplied m the b l o o d b y m a t u r e T cells. In the m a r r o w , the inducer cell is m the a d h e r e n t cell fraction. T h e hneage o f t h a t cell h a s n o t been determ i n e d . T h e assay for F cell p r o g e m t o r d l f f e r e n t l a u o n ts t h e expression m p l a s m a clot o f BFU-EFA colomes
f r o m peripheral b l o o d null cells in the presence o f e r y t h r o p o l e t m a n d m a t u r e T ceUs. T h e existence o f the CFU-EFA is referred, b u t these are n o t detected m the b l o o d a n d few are n o t e d in the b o n e m a r r o w ( S t a m a t o y a n n o p o u l o s & P a p a y a n n o p o u l o u , 1979). T h e vast m a j o r i t y o f BFU-EFA do not chfferentlate functlonally, as s h o w n vertically m the model, but
Monoclonal Antibody Assessments of T Cell Interactions m ErythropoieUn Studies undergo maturat]on-dtfferentiauon, as shown horizontally, to BFU-E A. They then, under the influence of erythropoietin, contmue to divide and amplify the system, filling the marrow with many BFU-E A. As they mature, they acqmre increased erythropoietin sensitivity and lose the requirement for T cell factors to induce functional differentmtion to the red cells. Finally, they differentiate to CFU-E A, continuing to amplify the system, and give rise to " A cells" which under nonstress conditions represent 95% of the total red cells. Smce BFU-E A are highly responsive to ambient erythropoletin, BFU-F~A dffferenttate to F cells to a relatively greater extent than BFU-E^ do to A cells in response to the high erythropoietin levels in anemia. Hence, the accumulation of F cells m peripheral blood in response to anemia. Although we are beginning to understand the nature o f T cell induction o f erythropotetindependent BFU-F~A differentiation, we do not have a grasp o f the probable cell-cell mteract)ons in I
COMMITMENT
245
BFU-EF^ repl]catmn and maturatmn. As well, understanding of the molecular biology of T cell ]nductton of erythropoietm-dependent erythroid differentiatton awmts purification of sufficient quantities of labeled functmnal erythropoietin and lsolatmn of the progenitor cell ttself so that the interaction of the hormone with its target can be studied in a reasonably homogeneous system. Recently, a subset of T cells defined by a monoclonal hybndoma antibody, OKT4, has been found to be responsible for L M F production (Reinherz, Kung, Breard, Goldstein & Schlossman, 1980). This same subset of cells ts required for B cell proliferation and differentiatmn, secretion of immunoglobulins, as well as generation of cytotoxlc T cells (Evans, Lazarus, Penta & Schlossman, 1978; Reinherz, Kung, Goldstein & Schlossman, 1979a; 1979b). Further investigation into the T cell subsets responsible for induction of erythropoietm-dependent erythroid differentiation will more specifically identify the inducer cell m that system.
] MATURATION - DIFFERENTIATION'[
Granutocyte f f Megakaryocyte /I
AMPLIFICATION H,gh Epo + T cells or T cell product
W er W
11l )-
0 Z
u.
Fig 10. Model of crythropolesls descrlblng maturation of erythrold progenitors from CFU-S and tcrmlnal differentiation of immature and mature BFU-E to F cells and A cells,respectively For description, see text
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