Human Dendritic Cells Express Functional Interleukin-7

Immunobiol., vol. 198, pp. 514-526 (1997/98)

Haematoiogyllmmunology Research Group, Christchurch School of Medicine and Christchurch Hospital, Christchurch, New Zealand

Human Dendritic Cells Express Functionallnterleukin-7 RUDIGER V. SORG\ ALEXANDER D. McLELLAN, BARRY D. HOCK, DAVID B. FEARNLEY, and DEREK N. HART

J.

Received August 29, 1997; Accepted in revised form November 24, 1997

Abstract Interleukin-7 (IL-7) supports the proliferation of mature T lymphocytes, however, the cellular source of IL-7 for T lymphocyte activation has not been well established. We therefore investigated whether human peripheral blood dendritic cells (DC) produce IL-7 as a contribution towards T lymphocyte activation. Human CMRF-44+/CD14-/CD19- low density DC, purified after overnight tissue culture, contained IL-7 transcripts, detected by direct cell reverse transcription-polymerase chain reaction. Intracytoplasmic staining confirmed IL-7 protein in at least a subpopulation of cultured low density DC. In contrast, restinglimmature DC, isolated directly by immunodepletion of lineage marker positive cells, contained no IL-7 mRNA_ Thus, the expression of IL-7 by DC follows the pattern described previously for CD80, CD86 and CD40. However, tissue culture of purified resting/ immature DC, in contrast to CD80, CD86 and CD40, failed to induce IL-7 transcripts. The functional importance of DC IL-7 expression was demonstrated in an allogeneic mixed leukocyte reaction (MLR). Neutralising mAb to IL-7 significantly inhibited T lymphocyte proliferation when low DC numbers were used, but at higher stimulator numbers, anti-IL-7 mAb failed to inhibit an allogeneic MLR. This suggests, that when DC are in excess, other co-stimulatory pathways can compensate for the lack of IL-7. Addition of IL-7 to a MLR caused a significant increase in the proliferative response stimulated by monocytes and B lymphocytes but not by DC. These data support the concept of an initial phase of antigen uptake by DC followed by the optimisation of DC co-stimulatory potential. The co-stimulatory repertoire expressed, including IL-7, may be regulated by exogenous stimuli, thereby ensuring DC flexibility in mounting a response appropriate to the environmental changes.

Abbreviations: DC = dendritic cell; IL-7 = interleukin-7; MLR = mixed leukocyte reaction; RTPCR = reverse transcription-polymerase chain reaction; Lin = lineage marker ':-Present address: Bone Marrow Donor Center, Heinrich Heine University of Dusseldorf, Moorenstrasse 5, 40225 Dusseldorf, Germany °1998 by Gustav Fischer Verlag

Dendritic cell IL-7 expression . 515

Introduction Interleukin-7 (IL-7) was first identified as a bone marrow stroma - derived cytokine, which promoted the growth and differentiation of early B-lineage committed progenitor cells (1-3). Subsequently, IL-7 production from spleen, liver, kidney (2, 3), thymus (2-4), keratinocytes (5), B lymphocytes (6) and intestinal epithelial cells (7) was described. However, T lymphocytes do not produce IL-7(8, 9). In addition to its non redundant role in B lymphopoiesis (10), IL-7 also promotes the growth and differentiation of thymocytes (11), particularly the development of r/8 T lymphocytes (12), generates lymphokine - activated killer cells (13) and induces cytokine expression and tumoricidal activity of monocytes (14). IL-7 also acts as a co-stimulus for the phytohemagglutinin, phorbol ester, concanavalin A, CD3 and CD2/CD28 induced proliferation of mature CD4+ and CD8+ T lymphocytes, predominantly in an IL-2 independent fashion (15-20). The generation of CD8+ cytotoxic T lymphocytes (21) is augmented by IL-7 and IL-7 promotes their conversion from memory to effector phenotype (22). Furthermore, IL-7 enhances T lymphocyte cytokine expression (16, 20, 23, 24) and has been suggested to playa role in the preferential development of Thl/Th2 T lymphocyte effector phenotypes (25). Although intestinallymphocytes and epidermal r/8 T lymphocytes may be stimulated by intestinal epithelial cell and keratinocyte derived IL-7 respectively (7, 26), the cellular source of any IL-7 involved in primary T lymphocyte activation has not been established. Dendritic cells (DC) are the predominant cellular initiators of primary T lymphocyte responses (27, 28). The majority of DC in human peripheral blood are resting or immature and can be isolated as a HLA-class II+ myeloid cell population, which lacks surface markers typical for other cell lineages (29-31). Immature DC have well developed antigen uptake capacity (32) yet lack, or only weakly express, the co-stimulatory molecules CD80 (B7.1), CD86 (B7.2) and CD40, but these and the DC associated CD83 and CMRF-44 antigens are upregulated during tissue culture (29, 30, 33-35) as the cells develop typical DC morphology. More activated or mature low density blood DC, isolated following short term tissue culture (36, 37), have lesser antigen uptake capability (32) but express both the CD83 and CMRF-44 antigens and the co-stimulatory molecules CD80, CD86 and CD40 (29, 33-38). Thus, these two types of blood DC preparations probably represent successive DC populations in a different state of activation. Nevertheless, both are potent inducers of T lymphocyte responses and utilise the co-stimulatory molecules CD80, CD86 and CD40 for activation of T lymphocytes (33, 34, 38). Until recently, DC were thought to produce few cytokines and their functional contribution to T lymphocyte activation was unclear. Human DC have now been shown to produce IL-12 (39,40) and a recent cytokine reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD83+ DC (41) suggests that they express a unique repertoire of cytokines. Our own RT-PCR screening of DC encouraged more detailed studies and we now provide data indicating that activated human peripheral blood DC are a source of IL-7 which contributes to DC co-stimulation of T lymphocytes.

516 . R. V.

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Materials and Methods Monoclonal antibodies (mAbj and cytokines

The mAb CMRF-31 (CD14; IgG2a), CMRF-44 (IgM; (42)) and IgM isotype control CMRF-50 were produced and characterized in this laboratory. OKT3 (CD3; IgG2a) and OKM1 (CDllb; IgG2b) were obtained from the American Type Culture Collection (Rockville, MD, USA). HuNK-2 (CD16; IgG2a) was kindly provided by Prof. I. F. C. McKENZIE (Austin Research Institute, Heidelberg, Australia); FMC63 (CD19; IgG2a) and isotype control mAb Sal-4 (lgG2b), Sal-5 (IgG2a) and X63 (IgG1) by Prof. H. 20LA (Child Health Research Institute, Adelaide, Australia); HB15a (CD83; IgG2b) by Prof. T. F. TEDDER (Duke University, Durham, NC, USA). Phycoerythrin (PE)-conjugated anti-HLA-DR (IgG2a), CD14 (IgG2a), CD19 (IgG2a), isotype control mAb and streptavidin were purchased from Becton Dickinson (Mountain View, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse immunoglobulin (FITC-SAM-Ig) was obtained from Silenus Laboratories (Hawthorne, Australia). Biotin-conjugated rat anti-human IL-7 (IgG2a) and isotype control mAb were obtained from Pharmingen (San Diego, CA, USA). The neutralizing mouse anti-human IL-7 mAb M25 (IgG2b) and human recombinant IL-7 were kindly provided by Dr. M. B. WIDMER (Immunex Corporation, Seattle, WA, USA). Cell preparations

Peripheral blood monocytes and B lymphocytes were sorted on a FACS Vantage (Becton Dickinson) from PBMC, following depletion of T lymphocytes by rosetting with neuraminidasetreated sheep erythrocytes, as CD14+ and CD19+ populations, respectively. Sorting was performed either directly or after 16 h tissue culture in RPMI-1640 supplemented with 10% heatinactivated fetal calf serum (Irvine Scientific, Santa Ana, CA, USA), 2mM L-glutamine, 100U/ml penicillin and 100 pg/ml streptomycin. Peripheral blood DC were isolated from T lymphocyte depleted PBMC either (1) directly by immunomagnetic depletion (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) of lineage marker (lin; CD3, CDllb, CD14, CD16 and CD19) positive cells, followed by FACS sorting of lin-/HLA-DR+ cells(31,33,34) or (2) following 16 h tissue culture and enrichment of low density cells via Nycodenz (1.068 g/cm 3; Nycomed Pharma, Oslo, Norway) gradient centrifugation followed by FACS sorting of the CMRF-44+/CD14-/CD19- population (33-35, 37). Purity of sorted populations was greater than 95%. T lymphocytes were isolated from PBMC by rosetting with neuraminidase-treated sheep erythrocytes (90 min on ice), followed by Ficoll-Hypaque gradient separation and hypotonic lysis of erythrocytes with double-distilled water. Immunostaining

Cells were incubated with saturating concentrations of mAb for 15 min on ice, prior to labeling with FITC-SAM-Ig and then analyzed on a FACS Vantage using Cellquest software. For double labeling, cells which had undergone primary staining, were incubated in 10% mouse serum for 5 min and then labeled with PE-conjugated mAb. Intracytoplasmic detection of IL-7 was performed as described (43), using the Caltag fix/perm kit (Caltag, South San Francisco, CA, USA). Briefly, T lymphocyte depleted, cultured low density PBMC were incubated for 4 h in medium containing 10 pg/ml Brefeldin A (Epicenter Technologies, Madison, WI, USA), washed twice with 0.5% bovine serum albumin (BSA)/phosphate buffered saline (PBS) and stained with the DC-restricted CD83 mAb HB15a (38), CMRF-44 (42) or isotype controls, followed by FITCSAM-Ig. After two washes, cells were incubated for 5 min with 10% mouse serum and 10% rat serum, washed and fixed for 15 min at room temperature in solution 1 (Caltag). Next, cells were washed twice and incubated with 10 pg biotin-conjugated rat-anti human IL-7 or isotype control mAb in solution 2 (Caltag) in the presence of 10% mouse serum and 10% rat serum, prior to detection with PE-conjugated streptavidin and analysis on a FACS Vantage.

Dendritic cell IL-7 expression' 517 Allogeneic mixed leukocyte reaction (MLR)

Mitomycin C-treated cultured low density CMRF-44+/CD14-/CD19- DC, cultured CD14+ monocytes or cultured CD19+ B lymphocytes (10 2-10 4 cells/well) were co-cultured with 105 allogeneic T lymphocytes in a final volume of 200 pI in round-bottom 96-well plates (Falcon, Franklin Lakes, N], USA). After 5 days of culture in medium as described above at 37°C and 5% CO 2, cultures were pulsed with 0.5 pCi (3H) thymidine (Amersham Int., Arlington Heights, IL, USA) per well for the final 16-18 h, before harvesting and scintillation counting. For neutralization experiments, mouse anti-human IL-7 or isotype control mAb Sal-4 were added at the onset of MLR at the concentrations indicated. For stimulation experiments, IL-7 was added at 10ng/ml from the beginning of co-culture. IL-7 alone induced a dose-dependent proliferation of T lymphocytes in a 5-day assay, with maximum proliferation (1635 ± 735% (mean ± SEM) of T lymphocyte background; range 500-3000%; n = 3) at 10 ng/ml (data not shown). IL-7 did not induce tritiated thymidine uptake of DC, monocytes or B lymphocytes. All assays were performed in triplicate. The two-tailed Student's t-test was used to determine statistical significance of data. Direct cell reverse transcription-polymerase chain reaction (RT-PCR)

For RT, sorted cells were resuspended in PBS and 32-500 cells in a volume of 5 pI added to 15 pI pre-prepared RT reactions (100 mM Tris-HCI pH 8.3, 50 mM KCl, 10mM MgCI 2, 0.1 % Triton X-100, 10 mM dithiothreitol, 1 mM dNTP each, 1 U/pl RNasin (Promega, Madison, WI, USA), 1 U AMV per reaction reverse transcriptase (Promega) and 50 nM specific primer final concentrations). The specific anti-sense primers were: 1) IL-7: 5'-CAGTATTGTTGTGCCTTCTG-3' (nt. 707-726) (3) or 2) ~-actin: 5'-CAGGTCCAGACGCAGGATGG-3' (nt. 562-581) (44, 45). Samples were placed immediately in a 55°C waterbath and incubated for 1 h. Samples without reverse transcriptase but containing cells and all other components of RT, served as negative controls. For PCR, 20 pI of RT products were added to 80 pI pre-prepared PCR reactions (10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCI 2, 0.1% Triton X-100, 0.2 mM dNTP each, 1 U per reaction Taq DNA polymerase (Boehringer-Mannheim, Mannheim, Germany) and 0.5 pM primers) and overlaid with 100 pI mineral oil. The sense primers were: 1) IL-7: 5'-TAGGTATATCTTTGGACTTCCT-3' (nt. 402-423) (3) and 2) ~-actin: 5'-CTACAATGAGCTGCGTGTGG-3' (nt. 311-330) (44,45). The anti-sense primers for IL-7 and ~-actin are shown above. Samples were placed in a 94°C pre-heated DNA thermal cycler (Perkin-Elmer-Cetus, Norwalk, CT, USA), incubated for 1 min and then subjected to 35 cycles of PCR (1 mini 94 °C-1 min/55 °C-1 min/72 0q, with a final incubation at 72 °C for 5 min. The primers employed allow the amplification of 325 bp and 271 bp fragments of IL-7 and ~-actin eDNA, respectively. For detection, 20 pI of RT-PCR products were separated on a 2% agarose gel, alkaline transferred (0.5 M NaOH/1.5 M NaCI) to Hybond N+ membrane (Amersham Int.) and hybridized with internal oligonucleotides: 1) IL-7: 5'-AACTTGCGAGCAGCACGGA-3' (nt. 629-647) (3) and 2) ~-actin: 5'-TAGCACAGCCTGGATAGCAAC-3' (nt. 441-461) (45) using the digoxigenin 3'-endlabeling and detection system according to the instructions of the manufacturer (Boehringer-Mannheim). Stringency washes were performed twice at 60°C (lL-7) or 64 °C (~-actin) in 6xSSC/0.1 % SDS for 15 min.

Results IL-7 production by human peripheral blood DC

Resting/immature human peripheral blood DC were isolated directly from T lymphocyte depleted PBMC by immunomagnetic depletion of lin+ (CD3, CDllb, CD14, CD16, CD19) cells, followed by FACS sorting of lin-/HLA-DR+

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Figure 3. Intracytoplasmic staining for IL-7 of human peripheral blood DC. Cultured low density, T lymphocyte-depleted PBMC were incubated for 4 h in the presence of 10 pg/ml Brefeldin A and then labeled with CDS3 (B, C) or IgG2b isotype control mAb (A) and FITC-SAM-Ig. After fixation, cells were permeabilized and stained with biotin-conjugated IgG2a isotype control (A, B) or anti-IL-7 (C) mAb and PE-conjugated streptavidin. Percent positive cells are indicated.

cells (Fig. lA). The lin-/HLA-DR+ DC comprised 63.3 ± 20.5% (mean ± SD; 40-95%; n = 6) of the immunodepleted preparations (75-97% lin-). Activated/more mature DC were obtained by FACS sorting CMRF44+/CD14-/CD19- DC (Fig. lB), after 16 h tissue culture of T lymphocyte depleted PBMC and subsequent enrichment of low density cells. CMRF44+/CD14-/CD19- DC comprised 8.1 ± 3.7% (mean ± SD; range = 3.7-16.2%; n = 11) of low density cells. To determine IL-7 expression, 32-500 sorted cells from each type of DC preparation were subjected to direct cell RT-PCR analysis. In addition, both directly sorted and cultured CD14+ monocytes and CDI9+ B lymphocytes were studied. Representative results (n = 3) are shown in Figure 2. Monocytes and directly isolated lin-/HLA-DR+ DC did not express readily detectable levels of IL-7 mRNA and only a very weak band was observed for 500 cells after prolonged exposure. In contrast, IL-7 transcripts were readily detected in B lymphocytes and CMRF-44+/CD14-/CDI9- DC, employing as few as 200 cells and 80 cells, respectively. Culture alone of directly isolated DC did not induce IL-7. In three experiments, directly isolated lin- DC were cultured for 16 h, sorted for lin-/HLA-DR+ and then subjected to RT-PCR, however no IL-7 mRNA was detected (data not shown). The presence of IL-7 protein within cultured low density DC was established by intracytoplasmic staining of Brefeldin A treated low density cells with antiIL-7 mAb, using CMRF-44 or CD83 mAb to confirm DC staining. CD83 stained all CMRF-44+ DC, without labeling of CD14+ or CD19+ cells (data not shown). In two separate experiments, 11 % and 33% of CD8Y DC showed weak expression of IL-7 protein. The dot plots of one of the two experiments are shown in Fig. 3.

520 . R. V. SORG et al. Functional relevance of CMRF-44+/CD 14-/CD19- peripheral blood DC Il-7 expression

The potential contribution of DC-derived IL-7 to T lymphocyte activation was sought using the allogeneic MLR. Sorted CMRF-44+/CD14-/CD19- blood DC had strong allostimulatory activity (Fig. 4A) and as few as 300 DC induced a significant increase (p < 0.001) in allogeneic T lymphocyte (3H) thymidine uptake in as-day MLR. When 2000 DC were co-cultured with allogeneic T lymphocytes in the presence of a neutralizing anti-IL-7 mAb a dose-dependent inhibition of T lymphocyte proliferation (Fig. 4B), with maximum inhibition of 47.5 ± 5.9% (mean ± SEM; range = 37-57.4%; n = 3) at 10 pg/ml anti-IL-7 was observed, compared to the response in the presence of the control mAb. In additional experiments more variable inhibition was observed. Thus, in two experiments, anti-IL-7 mAb inhibited the MLR at low but not at high DC stimulator numbers (Fig. 4C) and in a further two experiments no significant inhibition was observed (Fig.4D).

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Figure 4. Functional relevance of IL-7 expression in a DC stimulated allogeneic MLR. Graded doses of CMRF-44+/CD14-/CD19- DC (A, C, D) or 2000 DC (B) were co-cultured with 105 allogeneic T lymphocytes in the absence (A) or presence of 10 pg/ml (C, D) or graded doses (B) of anti-IL-7 and IgG2b isotype control mAb. After 5 days (3H) thymidine uptake was determined. Results are shown as mean ± SD of triplicates of cpm (3H) thymidine uptake. The background T lymphocyte proliferation was 1040 ± 768 (A, D), 516 ± 306 (B) and 1661 ± 450 (C).

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Figure S. Effect of IL-7 on an allogeneic MLR-stimulated by DC, monocytes and B lymphocytes. 2000 CMRF-44+/CD14-/CD19- DC, cultured CD14+ monocytes and cultured CD19+ B lymphocytes were co-cultured with 105 allogeneic T lymphocytes in the presence or absence of 10 ng/ml IL-7 and after 5 days tritiated thymidine uptake was determined. Results are shown as mean ± SEM of triplicates of T lymphocyte ± IL-7 corrected cpm tritiated thymidine uptake.

Having determined a functional role for DC produced IL-7, the effect of adding IL-7 at 10 ng/ml to an allogeneic MLR stimulated by 2000 CMRF44+/CD14-/CD19- DC, cultured CD14+ monocytes or cultured CD19+ B lymphocytes was compared (Fig. 5). In two separate experiments, additional IL-7 had no significant effect on DC stimulated T lymphocyte (3H) thymidine uptake. Monocytes and B lymphocytes were poor stimulators of the allogeneic MLR, but the addition of IL-7 caused a significant 365 ± 91 % (mean ± SEM, p < 0.05 and p < 0.001) and 455 ± 214% (p < 0.005 and p < 0.001) increase in T lymphocyte (IL-7 T lymphocyte background corrected) stimulation, respectively.

Discussion The molecular basis for the exceptional stimulatory activity of DC is still poorly understood (27, 28). Several adhesion molecules, including LFA-3, ICAM-l and ICAM-3, participate in DC-T lymphocyte interactions and CD40, CD80 and CD86 on DC have been shown to contribute to the activation of T lymphocytes (33,34,38,46). We show here that IL-7 is also part of the stimulatory repertoire available to DC. Activated/mature human CMRF-44+ /CD14-/CD19- peripheral blood DC expressed IL-7 mRNA and at least a subpopulation of these cells pro-

522 . R. V. SORG et al.

duced IL-7 protein. In contrast, resting/immature lin-/HLA-DR+ DC did not produce IL-7, nor was IL-7 mRNA induced upon culture. This requirement for a period of in vitro culture in the presence of other cell types to differentiate/activate the DC appropriately to detect IL-7 mRNA may account for the variable results reported in the RT-PCR analysis of CD83+ DC (41). An important difference in the induction of IL-7 compared to other co-stimulatory molecules on DC was noted. CD40, CD80 and CD86 (29, 30, 33, 34) are induced on directly isolated DC during tissue culture, as are the CMRF-44 and CD83 antigens (35). In contrast, no IL-7 mRNA was induced by culturing the purified restinglimmature DC. Thus, other yet to be identified stimuli, which occur during the culture of T lymphocyte-depleted PBMC, prior to the isolation of low density DC, must induce the DC synthesis of IL-7. Although directly isolated and cultured low density DC are assumed to represent successive states of in vitro differentiation/activation, the properties of the latter DC population may differ according to the stimuli present. In vivo, this flexibility could allow DC to interpret the microenvironment during antigen uptake and to respond to certain environmental or danger signals (47) with the expression of a distinct costimulatory repertoire, thereby facilitating stimulation of defined T lymphocyte effector types. IL-7 has been shown to preferentially induce interferon-y (25) and to synergize with IL-12 (24) in the activation of T lymphocytes and the induction of interferon-yo The recently described expression of IL-12 by DC (again dependent on DC activation) (39, 40, 48) and our observation that DC produce IL-7 are indicative of a DC stimulatory repertoire involved in inducing a Thl effector phenotype. Whether the expression of IL-7 within a subpopulation of CD83+ DC reflects the fact that we were detecting the stronger IL-7 producing cells only, incomplete activation of the DC, an example of restricted use of the DC stimulatory repertoire, or even DC subpopulations in human peripheral blood (49), requires further investigation. However, full expression of IL-7 by DC may also be dependent on reciprocal T lymphocyte signaling and thus antigen recognition, as with IL-12 (39). When IL-7 was neutralized in a CMRF-44+/CD14-/CD19- DC stimulated allogeneic MLR, a significant inhibition of T lymphocyte proliferation was observed. It cannot entirely be excluded that T lymphocytes stimulated by DC also express IL-7, however, no evidence for this, even after T lymphocyte activation, exists (8, 9), and we attribute the inhibition to a blockade of DC produced IL-7. The biological relevance of DC produced IL-7 was further supported by the failure of supplementary IL-7 to enhance a DC driven MLR despite significant effects on a monocyte or B lymphocyte driven MLR. For these experiments, a DC stimulator: responder ratio of 1:50 was used. We cannot rule out that exogenously added IL-7 may be able to enhance the MLR when DC numbers are even lower than those described here. The effect of blocking the functional activity of IL-7 was more pronounced at lower DC:T lymphocyte ratios (1:50). This could suggest that at lower ratios a relative lack of co-stimulatory molecules may be compensated for by IL-7 or

Dendritic cell IL-7 expression . 523

that when DC are in excess, other co-stimulatory pathways can compensate for a lack of IL-7. The presence of anti-IL-7 mAb caused no inhibition of a DC stimulated allogeneic MLR, independent of the number of stimulator cells used, in two experiments. This variable contribution of IL-7 to the co-stimulation of an allogeneic T lymphocyte response may reflect the degree of CD40, CD80, CD86 and other co-stimulatory molecule upregulation or that the tissue culture conditions induce variable amounts of DC IL-7 production or IL-7 independent co-stimulatory repertoires. Although the significance is not yet clear, a difference was noted in the low density cell populations prior to DC isolation, which correlated with the ability of the anti-IL-7 antibody to inhibit the subsequent DC stimulated MLR. Preparations yielding DC, which were not inhibited, showed stronger expression of the CMRF-44 antigen on low density monocytes (data not shown). As the CMRF-44 antigen is induced on monocytes by some activating stimuli (42), this may be a surrogate marker for the release of monocyte derived factors during DC preparation, which influenced DC maturation and induction of co-stimulatory molecule expression (29). This apparently variable DC response further emphasizes the importance of the external influences on the DC stimulatory repertoire. In summary, human blood DC prepared after tissue culture and low density isolation are induced, by as yet unknown factors, to produce IL-7 mRNA and IL-7 protein, which contributes to T lymphocyte co-stimulation. The control of DC IL-7 production is different from the membrane co-stimulatory molecules CD40, CD80 and CD86, which implies that the DC co-stimulator repertoire may be defined by environmental signals. Acknowledgments

We are grateful to LISA WHYTE for expert FACS assistance and AMANDA BOYCE for technical assistance. We also thank SUE BANKS and MICHAEL O'DWYER for their patient secretarial assistance. This work was supported by the New Zealand Health Research Council and the Canterbury Health Laboratories.

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