Differentiation of human dendritic cells from monocytes ill vitro using granulocyte-- macrophage colony stimulating factor and low concentration of interleukin-4

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC, such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast, adherent cells cultivated with GM-CSF alone were predominantly macrophages, as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.

Several laboratories have developed culture systems that allow the generation of large numbers of tillman dendritic cells (DC) f rom monocytes using granulo cytemacrophage colony stimulating [a ctor (GM•CSF).and imerle ukin-a (lL-4) .In this work we provided evidence that GM-CSF (/OO nglm/) in combination with a low concentration of lL-4 (5 ng/m/) was efficient in the generation of immature.non-adherent, monocyte-derived DC as the same concentration of GM-CSF.and ten times higher concentra tion of IL-4 (50 ng/ml).This conclusion was based on the similar phenotype profile of DC. such as the expression of CD /a.CD80.CD86.and HLA-DR.downregulation of CD/ 4. and the absence of CDB3.as well as on their similar allonimu!atoryactivity fo r T cells.A higher number of cells remained adherent in cultures with lower concentra tions oj IL-4 than in cultures with higher eoncemrations of the cytokine.Howe ver, most of these adherent cells down-regulated CD I4 and stimulated the proliferation of alloreactive T cells.In contrast, adherent cells cultivated with GM.CSF alone were predominantly macrophages, as judged by the expression of CDI4 ami the ineffi ciency to stimulate allo reactive T cells.DC generated in the presence of lower concentrations of IL-4 had higher proapoprotic potential fo r the Jur kut cell line than DC differentiated with higher concentrations of IL-4.suggesting their stronger cytotoxic.anti-tumor effec t.

Int rod uct ion
Dendritic cells (DC) are a family of bone marrowder ived professional antigen presenting ce lls (APC) with sparse, but wide (issue distri bution ( I , 2).Thei r capacity for migration, co nstitutive expression of major histocompatibility complex (MIlC) class 1 and 11 molecules, costi rnulawry and adhesion molecules, make them ideal for the acuvution of naive T cells (3,4) .It has been shown that DC are heterogeneous by their origin, maturati on stage, and func-tion (5-8).DC reside in tissues in an immature form , where they are adapted for capturing and acc umulating antigens.A variety of danger signals, including microo rganisms.dying ce lls, or proinflammatory cytokiues, ind uce terminal differentiation, also known as maturation of DC.Mature DC migrate to lymph nodes, acquire potent antigen-presenting capacity, and stimulate T cell responses vigoro usly.Moreover, the maturation of DC is strengthened during interactions with T ce lls by signals provided by T cells themselves  Cuhure co nditions for the generation of myeloid DC from blood.bone marrow , or C D34+ stern cells.have been deve loped by seve ral laboratories (I (}-13) .In recent yea rs, there has been a growing interest in using human DC prepared ex vivo (or immunotherapy.For such purposes.optimization of culture conditions was needed for the preparation of large numbers of functionally potent DC so they could be transferred in "h'o.Immunotherapy with mature DC is required for rhe imJuclion of ami-tumor immunity (14. 15).In contrast.immature DC possess natural killer (NK) activity against various tumor ce ll lines in vitro (16,17) .On the other hand , tolerant DC co uld be used for the treatment of autoimmune diseases.and for the prevention of transplant rejec tion (7. 18-20).
Therefore, the aim of this study was 10 check whether a combination of G ~1-CSF ( 100 ng/ml) with lower conce ntrations of IL-4 (5 nglml) was as efficient in the generation of human monocyte-derived DC as GM-CSF together with a ten times higher concentration of IL-4 (50 ng/m l).
washed in phosphate buffered saline (PBS) with 0, I% sod ium azide and 2% FCS (PBS/FCS).and adjus ted at concentrations of I x 10' cells/tube.Cells were incubated with the apprcpriate dilutions of (he following monoc lonal antibodies (mAbs ) to: HLA-DR (Becton-Dickinson), COl a, CDSO, CDS3, and CD86 (Serorec), and CDI4 (ICN) , for 45 Olin at +I"C.After washing in PBS/FCS anti-mouse (Fab-2), Ig-FITC antibody (Scrotec) with 5% normal human serum was added and the cells were further incubated for 30 min at +4 Cl c. Controls consisted of samples with an irrelevant mouse mAb.After washing.the cells were analyzed on EPICS XL-~ICL now cytometer, (Coulter, Krefeld, Germany).lmmunocytochetnistry Cytospins prepared from adherent cells were fixed with co ld acetone.and then incubated with anti -Cp !a or 3mi-C D 14 mAbs for 60 min at room temperature.After washing in Tris-buffered saline (TBS), slides wac incubated with anti-mouse Ig (Duke) for 30 min.and then with a mouse alkaline phosphatase-anti-alkaline pshosphatasc (APAAPj complex (DAKO) for 30 min .Enzyme reaction was visualized using naphtol AS-~1X phosphate dissolved in di met hyl formarnide and fast red as a subs trate.Lcvamisole was added to the e nzyme co mplex to block endogenous alkaline phosphatase.Cytospins were analyzed hy light microscopy.The perce ntage o f positive ce lls was determined on the basis of 500 ce lls calculated per sample.

Allogeneic mixed leukocyte reaction (MLR)
Peripheral blood mononuc lear (PB MN) cells were isolarcd from buffy coa ls using Lymphoprc p gradicnt.T cells were purified Irom PBMN using immuuomagnctic so rting with a pan-T ce ll isolation kit by ~lA C S technology (Myltenyi Biotec.Bcrgish Gladbach.Germany), following instructions of the manufac turer.The purity of T cells recovered in the negative fraction was higher than 95 %, as checked by ami-CD3 FITC mAb (Sc rotcc) and now cytometry.

Results
A human T cell leukemic line, Jurkat.was previously obtained from the ATCC collection (Wa shington DC.USA).Jurkat cells were cultivated alone or with DC at a ratio 10: I in complete RPMI medium for 24 or 48 hrs.After that, cells were stained with TUrk reagent.as we originally described (26).and analyzed hy lighl microscopy.Apopt osis was detected using c1 assical morph ological criteria (co ndensation of chromatin 3n(VOr tragmenration of nuclei).The percentage of apoptotic Jurkat cells.that were easily recognized from DC. was determined after the calculation of at least 500 cells per sample.

Statistical analysis
Differences ill parameters between various groups were evaluated usin g the Studem 's t-tcst.
/L --I is a necessary cytok ine for fire deve lopme nt of DC fro m monocvtes lIuman monocytcs.mostly CDl a -.ClJ I4 • (Fig. I).were cultivated with GM-CSF ( 100 ng/ml) alone.GM-CSF.and low concentrations of IL-4 (5 nglml ). or G ~I-C S F. and high concentrations of IL-4 (50 nglml ) for 7 days.Then. the numbers of non-adherent and adherent cells were calculated.As presented in Fig. 2 and Fig. 3. in cultures with GM-CSF and IL-4, the cells rapidly became non -adherent and displayed veiled and dendritic morphology to a different extent.The percent age of non-adherent DC was higher using GM-CSF and high co ncentrations of IL-4 than the percentage of non-adherent cells generated in the presence of lower concentrations of IL-4 (89.I ± 6.2 vs 72.

SS lOG File FITe
Fi~. 1-Expression ofC D l a and CDI4 hy human monocytes.
Monocyt es were iso lated as adh erent the ce lls from PRMN ce lls.After the detach cm ent with co ld RPM I-serum free medium co n- Note thai most ce lls displayed veiled and dendrit ic morph ology.May Grtinwnld-Giemsa staining of a cy tospi n: magni ficat ion : x 840.
cells were non-adherent cells.whereas the rest of them were adherent, macrophage-like cells.

DC generated in the presence of GM•CSF and IL•4 are phenotypi ca lly immatu re
We next studied phenotypic characteristics of DC cultivated with GM•CSF and IL-4.TIlC results given in Table I. and Fig. 4 showed that most non-adherent cells had typical characteristics of immature DC. such as ex pression of COla, CD80, CD86 and HLA-DR, down-regulation of CD I4, and the absence of CD83.No significant differences in the expression of any marker were detected between DC cultured with low and high concentrations of IL-4.

Monocyte-derived DC better stimu late the proliferotion ofallogeneic T cells than the manoe)'les
Stimu lation of alloge neic T ce lls is a typical characteristic of DC.The refore, we compa red the extent of proliferation of alloreactive T cells in vitro, using either monocytes or DC generated from the monocyres with GM-CSF and IL-4.The results presented in Fig. 5 showed that monocytes were much poorer stimulato rs of alloge nic T cells.especia lly at higher monocy te/T cell ratio.than monocytederived DC.However.DC genera ted with lower co ncentrations of IL-4 were as efficient in allostimulatory activity as DC gene rated with higher conce ntrations of IL-4.Purified al logeneic T ce lls (2x lo-~/well) were cultiva ted with differen t numbers of monccytcs (Mo).or DC differentiated from monocyres in the presence of G M-CSF, and low ('j or high concentrations (hi) of IL.4.as described in Methods .Va lues (mean c pm ± S D of triplicates.from o ne represe ntative expe riment) are given.All the d ifferences in the proli feration of T ce lls be twee n monocy res and mo nocy te-derived DC. used as stimulato rs. were p < 0.005 .No statistica lly significant difference s were observed in T ce ll prolifera tion be twee n cult ures using DC as stimulators .Co ntrol = cp m (T cells, alone) + cpm (DC, alone)

Adherent cells generated in cultures with GM-CSF and tL-4 art pllfnotypica lly and f unctiona lly diffe rent from the adherent cells cultivated with GM-CS F alone
Hascd on previous results that in cultures with OM-CSF and lower co ncentrations of IL-4 a higher number of ce lls remained adherent than in the cultures with a higher co nccntrarton of IL-4 , we framed a question whether these adherent cells differrcd from a predomin ant population of adherent ce lls developed from rnonocytes in cultures with OM-CSt' alone.Result s presented in Fig. 6 showed that both types of adherent ce lls were CDl a-, hUI the percentage of C D I4+ cells was significa ntly higher in cultures with GM-CSF alone.In addition, while adherent cells from GM-CSFnL-4 cultures showed a moderat e allostimul arory activity, adherent ce lls from GM -CSF cultures were inefficient in the MLR assay.

DC differentiated in the presence of lower concentra• lions of IL-4 had stronge r proapoptotic activityfor the Jurkat cell tine
Finally.we exa mined the ability of monoc yte-derived DC 10 induce apoptosis of the Jurkat ce ll line.based on rece nt results, showi ng that immature DC possesse d NK-Jike activity ( 16,17).As shown in Fig. 7 apop tosis of Jurkat ce lls was higher in cultures with DC than their spo ntaneous apoptosis.The percentage of apoptotic cells (especially after 48 hrs) was higher using DC generated with OM-CSF and low concentrations of IL-4.co mpared to the apoptosis induced with DC generated in the prese nce of higher concentrations of the cytokinc.
60% of DC (23) .These facts and our findings that the expression of CD Ia and down-regulation of CD 14 varied between donors, sugges ted that preactivation state of monocyres and/or qua lily o f F S could be responsible for the observed phenomenon .
We showed that immature DC were efficient activators of allorcactive T cells , in spite of relatively low expression of CDSO and CD86.These costimulatory molecules "vere of crucia l importance in generati ng signal :2 hy bind ing 10 C D28 o n T cells.and together with sig nal I tran smitted through TCR and coreccptor molecul es.enabled the prolifera tion of T ce lls (9.18. 27) .These con tradic tory findi ngs could be exp lained hy the matura tion of DC co-cu ltured with T cells.Among numerous signals provided by T cells , the interaction between CD4O-ligand expressed by activated T cells and CD40.expressed by DC. was most important for the final maturation of DC (2,12).Mature DC up-regu lated costirnclatory molecules and produce IL-12, a key cytokinc that induced polarization of the T cell response towards Th I (2. 3. 9. 12).Ad herent ce lls developed in monocyte cultures arc mostly considered bei ng macrophages hy their phenotype (CD I a -CD 14 "), expression of the M•CSF receptor and in- However.under such co nditions a proportion of cells still remai ned adherent ( I I. 28).Althoug h <he dynamics of phenotypic and functional changes o f such cultivated macrophages had not been fully charac terized, our result s supported the concept that monocyte -or macrophage-derived DC were adherent cells at the ear ly stage of their diffcrcntiation.
One importa nt finding prese nted in this work is the ability of imma ture DC to induce apoprosis of the Jurkat leukemic T ce ll line.The prop:n y of immatur e. but not mature, human monoc yte-der ived DC lO induce apop tosis of different tumo r cel l lines in vitro (16,17) made these cells functionally similar to NK cells.1l1C~characteristics of DC shou ld be considered, when DC arc used for anti -tumor therapy.However, we are at the beginning of understanding the mechanisms involved in ce ll apoptosis induced by DC, and defin ing optimal cond itio ns for the generation of DC with strong cy totoxic pote ntial.Our finding that the proapo ptotic ef fect of DC was higher when these cells were generated using lower co nce ntrations of IL-4, a phcnomc-Flg.7 -Apoptosis of l urkat cells induced by monocy tederived DC.Apoptosis was determined usmg morphological criteria after staining of cell with Tu rck reagent.as described in Methods.V31Ũ C'S (mean :t SD; n= 6) are givcn as percentagesof apopl()(;c cells.• = P < 0.05: •• = p < 0.01 compared 10 values in cultures using DC generatedin the presence' of higher concentrationsof IL-4.
DC are recog nized as the most efficie nt professional A Pe for the: induction of primary immune responses ( 1, 2.

27)
. Seve ral previous stud ies showe d that human DC might have developed from CD I4• blood monocy tes cultivated with relatively high co ncentratio ns of GM-CS F (80 ng/ml -100 ng/ml), and IL-4 (20 ng/ml -100 ng/rnl) (10-14. 23).\Vc showed in this wo rk that IL-4 was a necessary cytokinc for the development of DC. since about 20% of nonadherent cells were generated from monocytes when cult ure medium was supplemented with G~I•CSF only.In addition.we provided evi dence that a much lower concentration o f I L~(5 nglml) was sufficient for the d ifferentiation of nonadherent DC that are phenotypically and functio nally similar 10 DC genera ted in the presence of higher concentrations of IL-4 (50 ng/ml).Decreasing conccnrranons of GM-CSF hc llow 80 ng/ml significantly lowered DC yield (da ta not presented ).
In our culture sys tem, non-adherent DC, generated from monocytcs.had typi cal dendrit ic morph ology.expressed high level s of MIlC class II molecules.low level s of C D80, moderate le vels of C 086, and do wn-regul ated C D I4 .More than a half of them ex pressed C D Ia. whereas CDS3 -a typical mar ke r for mature DC. was not detected .Such phenotypic character istics wen: previously pub lished for immature DC (2. 3. 6. 10).but some d ifferences that were not related to lL-4 co ncentrations were obse rved.In our cu ltures CDla was not expressed by all monocytederived DC. uor did CDI4 co mplete ly disa ppear, as previously published fo r DC ge ne rated in the medi um with FCS (10,12).C Dla was not de tect ed on monocytede rived DC di fferenti ated in medium with autologo us serum , whereas the repl acement of seru m with hepar inized plasma led to the ex press ion o f the molecul e hy abo ut IJOJHOCAllllTETCKl1 rn-arnsn Crpasa 537 non that has not been described yet.was a good starting point for further studies.In conclusion.we described phenotypic and functional properties of immature DC generated from human 1110no-C)'h:S using GM•CSF (100 ng/ml) and lower concentrations of IL-4 (5 ng/ml) Ihal have nOI been published so far .AI-lowing cost reduction experiments, this method has some advantages in the generation of more cytotoxic DC against tumor cells over the protocol using len times more IL-4.\Ve are currently testing factors that stimulate maturation of DC in vitro no functional potential of such prepared mature DC.

FITC
Fig. .. -Phenotypic characteristics of DC generated from human monocytcs in the presence of GM-CSFand lower concentrations of IL.4 .Cells wen: stained insuspension with mAbs and analyzed by now cytorncuy.asdescribed in Merhods.and l-ig. 1. Representative histograms from one c ulture arc presented.x -axts represents fluorescence intcsuy (log .scale) ; Y-axis represents Ihe number of cells.
ability 10 stimulate naive T cells (I I. 22) .\Ve demonstrated that adherent ce lls in cultures with GM•CSF. in the absence of IL-4, met these criteria.but unexpectedl y, adherent ce lls in cultures with GM-CS F and IL-4.although phenotypicaly dif ferent from non-adherent immatur e DC. stimulated the proli feration o f alloge neic T cells in MLR .By the characteristics, these ce lls probably represented a subset of the immature DC.The plasticity of monocy te differentiation pathway in vitro was well documented.Monocytcs cultivated in the presence of G~I•CSF differentiated into adheren t macrophages.In contras t. the withdrawal o f M-CSF and the addition o f GM-CSF and IL-4 led to the diffcrcmiation of most macropbages into non-adherent.imma ture DC.

Table I
Phenotypic characteristics of monocyte-derived DC cultivated with