(CANCER RESEARCH 49, 1125-1129, March I, 1989]

Modulation of Lymphocyte Proliferation and Immunoglobulin Production byTransferrin-Gallium1

Christopher R. Chitambar,2 Margaret C. Seigneuret, William G. Matthaeus, and Lawrence G. LumDivision o]'Hematology/Oncology, Department of Medicine, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ABSTRACT

Gallium resembles iron with respect to transferrin (Tf) binding andcellular uptake via Tf receptors. We have previously shown that trans-ferrin-gallium (Tf-Ga) complexes interfere with the cellular incorporationof iron and inhibit the proliferation of III 6(1 cells. Since mitogen-stimulated peripheral blood lymphocytes express Tf receptors, we examined the effect of Tf-Ga on lymphocyte proliferation and on immuno-globulin synthesis by B-lymphocytes. Tf-Ga inhibited phytohemaggluti-nin, pokeweed mitogen, and tetanus toxoid-stimulated lymphocyte proliferation by >SO%, an effect which appeared to be cytostatic rather thancytotoxic. In cocultures of T-lymphocytes or CD4+ T-lymphocytes andB-lymphocytes, Tf-Ga also inhibited pokeweed mitogen-stimulated im-munoglobulin production by 84 to 100%. Tf-Ga inhibited both T-inde-pendent Epstein Barr virus-stimulated B-lymphocyte proliferation andimmunoglobulin production; however, these effects appeared to be independent of each other, since immunoglobulin production was inhibited by75% by a concentration of Tf-Ga which did not uniformly inhibit proliferation. Tf-Ga is capable of targeting Tf receptor-bearing T- and B-lymphocytes and interfering with their proliferation and function. Sucheffects may be of relevance to patients being treated with this metal. Thepotential immunosuppressive activity of gallium warrants further investigation.

iron deficiency induced by continuous exposure of these cellsto gallium (11). Like erythroid cells, mitogen-activated peripheral blood lymphocytes also synthesize and express large numbers of Tf receptors (12,13), thus making them potential targetsfor Tf-Ga.

To date, there have been no studies examining the interactionof gallium with lymphocytes. Information from such studieswould be of relevance to patients being treated with this metal.We have therefore examined the interaction of Tf-Ga complexes with the immune system in vitro at three levels: (a)effects on mitogen-induced proliferation of peripheral bloodlymphocytes; (b) effects on T-lymphocyte-dependent immunoglobulin synthesis; and (c) effects on T-lymphocyte-independentimmunoglobulin synthesis by B-lymphocytes. We report thatTf-Ga suppresses the mitogen-induced proliferation of peripheral blood lymphocytes and inhibits both T-dependent and T-independent immunoglobulin production by B-lymphocytes.However, the inhibitory effects on immunoglobulin synthesisappear to be independent of the antiproliferative effects of Tf-Ga, suggesting that, in lymphocytes, this metal may affectintracellular mechanisms independent of DNA synthesis.

INTRODUCTION

Recent clinical studies have shown gallium nitrate (NSC15200) to be an effective agent in the treatment of hypercal-u:mia (1) and certain malignancies (2). Although informationregarding the interaction of gallium with various cell systemsis incomplete, studies have shown that gallium binds avidly invitro (3) and in vivo (4) to the iron transport protein Tf3 andthat the cellular uptake of 67Ga is enhanced by Tf (5). In recentstudies, we have shown that the uptake of 67Ga by human

leukemic HL60 cells is mediated through the Tf receptor (6).Furthermore, we have shown that stable Tf-Ga complexesinhibit the cellular uptake of iron by HL60 cells and block theproliferation of these cells (7). The inhibition of DNA synthesisby Tf-Ga appears to be the result of a diminution in the activityof the iron-containing M2 subunit of ribonucleotide reducíase,the enzyme responsible for the synthesis of deoxyribonucleo-tides from ribonucleotides (8).

Although gallium nitrate administration appears to be reasonably well tolerated, a number of patients receiving this agentby continuous i.v. administration have developed microcytichypochromic anemia (9). Since erythroid precursors express Tfreceptors which can be targeted by Tf-Ga complexes (10), theanemia is most likely the result of a state of an intracellular

Received 9/1/88; revised 11/28/88; accepted 12/1/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by USPHS Grant CA41740 awarded by the

National Cancer Institute to C. R. C. and by an American Cancer SocietyInstitutional Grant to the Medical College of Wisconsin.

1To whom requests for reprints should be addressed, at Division of Hematol-ogy/Oncology, Medical College of Wisconsin, 8700 W. Wisconsin Ave, Milwaukee, WI 53226.

3The abbreviations used are: Tf, transferrin; Tf-Ga, transferrin-gallium; PHA,phytohemagglutinin; PWM, pokeweed mitogen; EBV, Epstein-Barr virus;ELISA, enzyme-linked immunosorbent assay; Tf-Fe, transferrin-iron.

MATERIALS AND METHODS

Materials. Human Tf was obtained from Sigma Chemical Co. (St.Louis, MO). Gallium nitrate was purchased from Alfa Products (Dan-vers, MA). PWM and PHA were obtained from GIBCO (Grand Island,NY) and DIFCO Laboratories (Detroit, MI), respectively. Tetanustoxoid was obtained from the Commonwealth of Massachusetts PublicHealth, (Boston, MA).

Preparation of Tf-Ga. Tf-Ga (stock solution of 50 mg of protein/ml)was prepared as previously described (7). Briefly, 3 mol of gallium (asgallium nitrate) were added to each mol of Tf dissolved in 20 mM aceticacid, 150 mM NaCl (pH 3.5) buffer, and the pH of this solution wasraised in gradual increments to 7.4 with l M NaHCOj. Saturation ofTf by gallium (1 mol of Tf binding to 2 mol of gallium) was confirmedusing a Beckman DU 40 spectrophotometer and by measuring thechange in absorbance (which occurs with saturation of both metalbinding sites of TO at a wavelength 242 nm (3).

Lymphocyte Subset Purification. Peripheral blood lymphocytes wereisolated by Ficoll-Hypaque density gradient centrifugation of heparin-ized blood from normal volunteers (14). T (E-rosette positive)- andnon-T (E-rosette negative)-cells were separated by sheep erythrocyteresetting (14). T-lymphocyte subsets were purified by negative selectionusing monoclonal antibodies directed at CD4 and CDS determinantsby antibody treatment and complement lysis (15). B-Lymphocyte-enriched fractions were isolated from the non-T (E-rosette negative)-lymphocyte populations after incubation of the non-T-lymphocytes onplastic to remove adherent monocytes.

Lymphocyte Proliferation Assay. Proliferation assays were performedin a standard fashion in flat-bottomed microculture wells containing1.5 x 10s cells. All cultures were performed in RPMI 1640 mediumsupplemented with penicillin-streptomycin, 4 mM glutamine, and 10%heat-inactivated fetal calf serum (Hyclone, Logan, UT). Heat-inactivated fetal calf serum was prescreened for its ability to support proliferation or immunoglobulin production. The various reagents added tothe assays were diluted in RPMI medium. PWM (final dilution,1:1600), PHA (final dilution, 1:400), and tetanus toxoid (final concentration, 25 ng/ml) were used to stimulate lymphocyte proliferation (14).

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TRANSFERRIN-GALLIUM AND LYMPHOCYTES

Tf-Ga (final concentration, 0 to 5000 ^g/rnl) was added to the culturemedium at the start of the incubation. Peripheral blood lymphocytesincubated with PHA. PWM, or tetanus toxoid for 3 or 6 days werepulsed with ['H|thymidine (20 n\ of a 6.7 ¿iCi/mMspecific activity

solution or 2 ¿iCiper culture) during the last 4.5 h of incubation,harvested, and counted in a beta counter. In parallel experiments, cellsexposed to the various mitogens plus Tf-Ga were harvested after 3 to6 days of incubation and assayed for viability using trypan blue exclusion.

B-lymphocyte proliferation assays were performed in a similar fashion by incubating purified B-lymphocytes (in the absence of T-lympho-cytes) with 50 ¡tiof EBV-containing supernatant from the marmosetB958 cell line (16, 17). Cells were pulsed with [3H]thymidine after 3 or

6 days of incubation and treated as described above.PWM-stimulated (T-Lymphocyte-depcndent) Immunoglobulin Pro

duction Assay. Cocultures for PWM-stimulated immunoglobulin synthesis were performed in round-bottomed microculture wells containing5.0 x 10" T-lymphocytes or T-lymphocyte subsets (CD4 and CDS) and5.0 x 10" B-lymphocytes. PWM was omitted in the negative controlwells. Tf-Ga or Tf was added to triplicate wells (final concentration,1000 jig/ml) at the start of the incubation. After 12 days of culture,supernatants were tested for polyclonal IgG and IgM production byELISA (18, 19), and the amount of immunoglobulin produced wasdetermined from a standard curve which was generated using knownconcentrations of commercially available immunoglobulin standards.

EBV-stimulated (T-Lymphocyte-independent) Immunoglobulin Production Assay. B-Lymphocytes (5 x IO4) were incubated in 96-wellround-bottomed microculture plates with 50 n\ of EBV-containingsupernatant from the marmoset B958 cell line. EBV was omitted inthe negative control wells. Tf-Ga (final concentration, 50 or 1000 ^g/ml) was added to wells either on Day 0 of initial plating or on Day 4of culture (after EBV infection of B-lymphocytes had been established).The supernatant from each well was harvested on Day 12 and assayedfor IgG and IgM by ELISA.

290^

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^ 50**> IJ 40

30

20

10

100%-o 9,385 cpn.•¿�98,798 cp«,

42,146 CPU!

1 10 50 100 500 1000 5000

TRANSFERRIN-GALLIUM CONCENTRATION (Mg/ml)

Fig. 1. Effect of Tf-Ga on the proliferation of PHA-stimulated peripheralblood lymphocytes. Tf-Ga was added at the start of the incubation. Cells werepulsed with 2 ¿iCi/wellof ['Hjthymidine for 4.5 h after 3 days of culture. [3H]-Thymidine uptake in the absence of Tf-Ga was considered to be 100% (control),and cpm incorporated in the presence of different concentrations of Tf-Ga werenormalized to this value and expressed as the percentage of control. Data fromthree normal subjects are shown.

RESULTS

Effects of Tf-Ga on Mitogen Activation of Peripheral BloodLymphocytes. Peripheral blood lymphocytes were stimulatedby exposure to PHA, PWM, or tetanus toxoid in the presenceof 0 to 5000 Mg/ml of Tf-Ga. Fig. 1 shows the effect of Tf-Gaon PHA-stimulated lymphocyte proliferation. At a concentration of 1 Me/ml, Tf-Ga stimulated ['H]thymidine incorporation

above that of the control; however, at concentrations above 500Mg/ml, Tf-Ga markedly inhibited lymphocyte proliferation toless than 50% of control. A similar but somewhat variableresponse to Tf-Ga was seen when lymphocytes were stimulatedwith PWM (Fig. 2): peripheral blood lymphocytes from onesubject showed an 80% inhibition of [3H]thymidine incorporation with 500 Mg/ml of Tf-Ga; whereas lymphocytes from theother two subjects displayed a 40 to 50% inhibition of [3H]-thymidine incorporation with 1000 Mg/ml of Tf-Ga. The response of tetanus toxoid-stimulated peripheral blood lymphocytes to Tf-Ga was more uniform, and 1000 Mg/ml of Tf-Gaproduced a >60% inhibition of [3H]thymidine incorporation(Fig. 3). In the assays shown, lower concentrations of Tf-Garesulted in a variable enhancement of lymphocyte proliferation;however, this enhancement of lymphocyte proliferation was notuniform and was not seen in the absence of mitogens (data notshown).

To determine whether the inhibition of lymphocyte proliferation by Tf-Ga was the result of a direct toxic action onlymphocytes, lymphocytes plated in the presence of differentconcentrations of Tf-Ga were harvested, and cell viability wasdetermined by trypan blue exclusion. Although higher concentrations of Tf-Ga inhibited the proliferation of mitogen-acti-vated lymphocytes, these cells maintained a viability equal to

100% <o 48,177 cpm

•¿�35,514 cpm

A 67,063 cpm

1 10 50 100 500 1000 5000

TRANSFERRIN-GALLIUM CONCENTRATION (Mg/ml)

Fig. 2. Effect of Tf-Ga on the proliferation of PWM-stimulated peripheralblood lymphocytes. Studies were performed as in Fig. 1 except that cells werestimulated with PWM and were pulsed with ['H]thymidine after 6 days of culture.

cells plated in the absence of Tf-Ga [86 ±2% (mean ±SD oftriplicate determinations) on Days 1 to 3 for cells exposed to1000 Mg/ml of Tf-Ga, versus 87 ±4% for controls]. Hence, the

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TRANSFERRIN-GALLIUM AND LYMPHOCYTES

100% •¿�o 2985 cpm

•¿�30.710 cpm

A 31.467 cpm

1 10 50 100 500 1000 5000TRANSFERRIN-GALLIUM CONCENTRATION (Mg/ml)

Fig. 3. Effect of Tf-Ga on the proliferation of tetanus toxoid-stimulatedperipheral blood lymphocytes. Studies were performed as in Fig. 1 except thatcells were stimulated with tetanus toxiod and were pulsed with | 'I l]ilu inuline

after 6 days of culture.

marked inhibition of [3H]thymidine incorporation seen withhigher concentrations of Tf-Ga appeared to be the result of anarrest in cellular proliferation rather than a direct cytotoxic orcytolytic effect. These studies therefore show that the proliferation of Tf receptor-bearing, mitogen-activated lymphocytescan be inhibited by Tf-Ga in a dose-dependent manner.

Effect of Tf-Ga on PWM-stimulated (T-Lymphocyte-depen-dent) Immunoglobulin Production. To study the possible immu-nosuppressive activity of Tf-Ga, we compared the effects ofequivalent concentrations of Tf and Tf-Ga on PWM-stimulatedimmunoglobulin production. Immunoglobulin production wasdetermined in assays containing cocultures of B-lymphocyteswith either T-lymphocytes, CD4-enriched T-lymphocytes, orCD8-enriched T-lymphocytes. Cells were incubated understandard conditions (control) or with 1000 Mg/ml of either Tfor Tf-Ga. This concentration of Tf-Ga was used because itconsistently inhibited mitogen-stimulated lymphocyte proliferation. As shown in Table 1, control cells displayed greaterimmunoglobulin production in the CD4-enriched coculturesthan in the CD8-enriched cocultures. These results underscorethe "helper" function of CD4 T-lymphocytes with regard to

immunoglobulin production (when tested in a PWM-stimulatedcoculture system) and are consistent with previously reporteddata (15). The addition of Tf to the assay had a definitestimulatory effect on immunoglobulin production and may havebeen the result of more efficient iron delivery to cells by humanTf. Although Tf was added to cultures in a relatively iron-freeform, it readily binds iron present in the medium to form Tf-Fe. Hence immunoglobulin production can be considered tohave occurred in the presence of Tf-Fe rather than Tf. Incontrast, Tf-Ga inhibited total immunoglobulin (IgG + IgM)production in all three cocultures. In the presence of Tf-Ga, no

Table 1 Effect oftransferrin-gallium on PWM-stimulated immunoglobulinsynthesis

T-lymphocytes or T-lymphocyte subsets (CD4 or CDS) (5 x 10") were incubated with 5x10" B-lymphocytes and 1000 ng/ml of Tf-Ga, or Tf was added to

cultures at the time of initial plating. Immunoglobulin produced (IgG and IgM)is expressed in ng/ml. The percentage of inhibition by Tf-Ga refers to inhibitionof total immunoglobulin (IgG + IgM) production. Data shown represent studieson peripheral blood lymphocytes obtained from three normal individuals.

Immunoglobulin production(IgG/IgM)CellsT

+BCD4

+BCD8

+ BNo.123123123Control1,096/2197,937/662,341/01,818/37310,772/6703,779/265542/575,394/1811,412/0Tf1,748/2357,241/06,986/4215,155/1,02020,000/3,3264,066/285708/685,491/1422,287/0Tf-Ga208/0131/0229/0288/0132/00/0270/0117/00/0%ofinhibitionby

Tf-Ga84989087991005497100

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52 120 y100%

=<yD - 3,071cpm\o - 10,052cpm\•¿�-7,207 cpm

0 100 200 300 400 500

TRANSFERRIN-GALLIUM CONCENTRATION (ug/ml)

Fig. 4. Effect of Tf-Ga on EBV-stimulated proliferation of B-lymphocytes.Tf-Ga was added at the start of the incubation, and cells were pulsed with 2 ><<iwell of [JH]thymidine for 4.5 h after 6 days of culture. Data from three normal

subjects are shown.

IgM production was detected, and IgG production was markedly diminished.

Effect of Tf-Ga on EBV-stimulated (T-Lymphocyte-indepen-dent) B-Lymphocyte Proliferation and Immunoglobulin Production. To determine whether the inhibitory effects of Tf-Ga onimmunoglobulin production were due solely to Tf-Ga action onthe proliferation and release of B-lymphocyte-activating factorsby mitogen-activated T-lymphocytes, we examined the effect ofTf-Ga on B-lymphocytes in a T-cell-independent EBV-stimulated system. Fig. 4 shows the effect of increasing concentrations of Tf-Ga on the EBV-stimulated proliferation of B-lymphocytes when assayed after 6 days of incubation. Similarresults were obtained when the assay was performed after 3days of incubation (data not shown). As in the mitogen-stimulated peripheral blood lymphocyte proliferation assay, low concentrations of Tf-Ga stimulated [3H]thymidine incorporationby B-lymphocytes in one subject. However, at concentrations

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TRANSFERRIN-GALLIUM AND LYMPHOCYTES

above 250 /¿g/ml,Tf-Ga uniformly inhibited B-lymphocyteproliferation in all subjects.

Fig. 5 shows the effect of two concentrations of Tf-Ga onEBV-stimulated immunoglobulin production. To better definethe interaction of Tf-Ga with B-lymphocytes, Tf-Ga was addedto the cells at different times: (a) at the time of initial plating,before EBV infection of B-lymphocytes was established (Fig.5/4); or (b) on Day 4 of incubation (Fig. 5B) after EBV infectionof cells had been established (evidenced by an increase in [3H]-

thymidine incorporation; data not shown). Immunoglobulinproduction was inhibited by as little as 50 Mg/ml of Tf-Ga whenTf-Ga was added at Day 0. In contrast, immunoglobulin production was not inhibited when 50 Mg/ml of Tf-Ga were addedon Day 4. With 1000 Mg/ml of Tf-Ga, total immunoglobulinproduction was consistently inhibited regardless of whether thisconcentration of Tf-Ga was added on Day 0 or Day 4. However,when 1000 Mg/ml of Tf-Ga were added on Day 0, only IgGcould be detected in the culture supernatants. When 1000 tig/ml of Tf-Ga were added on Day 4, the majority (>93%) ofimmunoglobulin present in the culture supernatants was IgM.

DISCUSSION

Our study shows that Tf-Ga complexes are capable of suppressing in vitro lymphocyte proliferation and polyclonal immunoglobulin production. At low concentrations, however, Tf-Ga enhanced mitogen-stimulated lymphocyte proliferation to avariable degree. The reason for this increase in cellular [3H]-

thymidine incorporation is not readily apparent. One possibleexplanation is that, at low concentrations, gallium bound to Tfmay exchange with iron present in the incubation mediumresulting in the formation of Tf-Fe complexes. Human Tf-Fe,having a greater affinity for the lymphocyte Tf receptor thanbovine Tf-Fe (present in the medium), could then enhance theproliferative response of these cells to mitogens. It should beappreciated that, in these experiments, mitogen-stimulated lymphocytes plated in medium containing 10% fetal calf serumwere used as controls. Had human Tf (equivalent to the concentration of Tf-Ga) been added to these cells to serve ascontrol, a further increase in the incorporation of [3H]thymidine

would most likely have occurred. The percentage of inhibitionof [3H]thymidine incorporation by Tf-Ga (when compared withthese Tf-containing controls) would have then been even greaterthan that shown in Figs. 1 to 3.

- 6000

P5000*

40000

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20003

1000A

B•ige;CD

IgMr\

É\¿~.rVControlTI-Go TI-Go TI-GoTI-Ga50uo./«l

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Fig. 5. Effect of Tf-Ga on EBV-stimulated immunoglobulin production. Tf-Ga was added on Day 0, at the start of the incubation (A) or on Day 4 (B).Columns, mean; bars. SEM (n = 3).

The inhibitory effects of Tf-Ga on immunoglobulin synthesisappeared to be specifically due to the Tf-Ga complex, since nosuch effect was seen with an equimolar concentration of Tf. Todetermine whether Tf-Ga inhibited immunoglobulin productionby action at the T-lymphocyte or B-lymphocyte level (or both),immunoglobulin synthesis was examined in a 1'XVMstimulatedT-lymphocyte-dependent system (containing both T- and B-lymphocytes) and a T-lymphocyte-independent culture containing B-lymphocytes. Since inhibition of immunoglobulin synthesis was seen in both systems, it appears that Tf-Ga acts at twolevels: (a) inhibition of PWM-stimulated helper T-lymphocyteactivation and production of T-lymphocyte replacing factors bythe CD4 subset, including factors which affect B-lymphocytegrowth and differentiation; and (/>}direct inhibition of B-lymphocytes. Others have shown that proliferating T-lymphocytesand proliferating and differentiating B-lymphocytes express Tfreceptors (20-23). Tf-Ga therefore appears to target to both Tfreceptor-bearing cell populations and block their proliferationand function.

Although the growth-inhibitory effects of Tf-Ga may appearto be responsible for the inhibitory effect on immunoglobulinsynthesis, the studies examining EBV-stimulated immunoglobulin synthesis showed that immunoglobulin synthesis was uniformly inhibited by a concentration of Tf-Ga (50 Mg/ml addedon Day 0) which did not consistently inhibit cell growth. Hence,the inhibition of EBV-stimulated immunoglobulin productionby Tf-Ga cannot be explained purely on the basis of inhibitionof B-lymphocyte proliferation. These studies suggest tht gallium also acts on intracellular processes that are independentof DNA synthesis but are necessary for immunoglobulin production.

In our studies, the timing of exposure of B-lymphocytes toTf-Ga was important. Fifty iig/m\ of Tf-Ga added to cultureson Day 0 inhibited immunoglobulin production, whereas thesame concentration added on Day 4 failed to inhibit immunoglobulin production. We hypothesize that, during the first 1 to2 days of incubation, the number of Tf receptors expressed byEBV-activated B-cells is probably low enough to be completelytargeted and inhibited by 50 Mg/ml of Tf-Ga. With furtherproliferation of the EBV-activated cells, the total density of Tfreceptors on B-lymphocytes and the number of Tf receptor-bearing cells exceed that which can be targeted (and inhibited)by 50 Mg/ml of Tf-Ga. Consistent with this hypothesis, a higherconcentration of Tf-Ga (1000 Mg/ml), added on Day 0 or later,uniformly inhibited immunoglobulin production.

The expression of Tf receptors by lymphocytes representscellular requirements for iron. Based on our previous studies inHL60 cells, the Tf-Ga complex could be considered an analogueof Tf-Fe which targets to Tf receptor-bearing cells and blocksiron uptake and iron-dependent processes. Although targetingof the lymphocyte Tf receptor by certain anti-Tf receptor monoclonal antibodies has also been shown to block T- and Blymphocyte activation (22-24), in most cases the mechanismsof action of monoclonal antibodies appear diverse or undefinedand are probably different from that of Tf-Ga.

Since our studies indicate that gallium has immunosuppres-sive activity in vitro, studies of the immune system of patientsbeing treated with this metal appear warranted. Since the concentration of transferrin in vivo ranges from 2 to 4 mg/ml andonly about one-third of this is normally iron saturated, theremaining two-thirds are available to bind gallium. Hence, theinhibitory concentrations of Tf-Ga used in our studies in vitroare theoretically achievable in vivo. The potential use of galliumas an immunosuppressive agent awaits further evaluation in

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TRANSFERRIN-GALLIUM AND LYMPHOCYTES

animal model systems. Gallium may be useful in the treatmentof graft-versHS-host disease or graft rejection where inhibitionof lymphocyte activation is desirable, and it may prove to beless toxic than agents which are currently being used as immu-nomodulators.

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1989;49:1125-1129. Cancer Res   Christopher R. Chitambar, Margaret C. Seigneuret, William G. Matthaeus, et al.   Production by Transferrin-GalliumModulation of Lymphocyte Proliferation and Immunoglobulin

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