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ALTERATION OF THE CD34+Tf-1β CELL LINE PROFILE IN RESPONSE TO LONG-TERM EXPOSURE TO IL-15
ALTERATION OF THE CD34+ Tf-1b CELL LINE PROFILE IN RESPONSE TO LONG-TERM EXPOSURE TO IL-15 Nancy L. Farner,1 Jacek Gan,1 Jill L. O. de Jong,1 Thomas P. Leary,2 Timothy S. Fenske,3 Patrick Buckley,5 Sabrina Dunlap,1 Paul M. Sondel1,4,6 Interleukin 15 (IL-15) is a cytokine with many functional characteristics that are similar to IL-2. Most of the functional activities that IL-2 and IL-15 support have been evaluated in short-term assays. It was our intention, then, to determine the long-term effects of IL-15 in comparison to IL-2. These studies were performed using the growth factor-dependent myelomonocytic cell line, Tf-1, which has been well characterized with regard to morphology, CD marker expression, responses to certain growth factors and cytokines (GM-CSF, IL-4, erythropoietin), and can differentiate through the myeloid and erythroid lineages. In order to study IL-2 and IL-15 responses, Tf-1 cells were retrovirally infected with the IL-2Rb chain gene as a means to confer IL-2 responsiveness to this cell type. The results of this study demonstrate that retroviral infection of Tf-1 successfully generated a stable IL-2 responsive cell line, Tf-1b, without interfering with the original characteristics of the Tf-1 cell. Tf-1b cells respond functionally to both IL-2 and IL-15. When Tf-1b cells are grown for 8 weeks in IL-2 (Tf-1b2), rather than GM-CSF, the original morphology, CD marker expression, esterase activity and proliferative response is unaltered in comparison to that of the original Tf-1b line maintained in GM-CSF. However, long-term growth of Tf-1b in IL-15 (Tf-1b15) results in morphological alterations, downregulation of CD33, CD38, and HLA-DR, and a decreased response to IL-15 in comparison to Tf-1b2. These studies support the concept that retroviral infection, even when it confers new functions upon a cell, does not necessarily alter all other functions, as assessed by evaluation of its phenotypic profile. Furthermore, the production of the Tf-1b2 and Tf-1b15 sublines demonstrates that IL-2 and IL-15 can support long-term cell growth. However, this long-term growth in IL-15 leads to subtle alterations in the cell profile that are not seen with IL-2, suggesting that distinctions in IL-2 and IL-15 function do exist. Further study of the Tf-1b15 cell line will be useful to clarify these functional distinctions between IL-2 and IL-15. 7 1997 Academic Press Limited
Interleukin 15 (IL-15) was initially isolated from the supernatant of the simian kidney epithelial cell line, CV-1/EBNA as it supported proliferation of the IL-2 dependent cell line, CTLL-2.1 It is expressed in virtually all tissues, including bone marrow-derived cells and placenta.1 Subsequent cloning documented that IL-15 did not have any appreciable homology to other proteins, but that it is structurally similar to IL-2.
From the 1Department of Human Oncology, 3Cellular and Molecular Biology, 4Pediatrics, 5Pathology and Lab Medicine, and 6 Genetics, University of Wisconsin and Wisconsin Comprehensive Cancer Center, Madison, WI 53792, USA; 2Experimental Biology Research, Virus Discovery Group, Abbott Laboratories, North Chicago, IL 60064, USA Correspondence to: Paul M. Sondel, K4/448 University of Wisconsin Comprehensive Cancer Center, 600 Highland Avenue, Madison, WI 53792 Received 14 May 1996; revised and accepted for publication on 16 November 1996 7 1997 Academic Press Limited 1043–4666/97/050316 + 12 $25.00/0/ck960171 KEY WORDS: CD34+/IL-15/Tf-1b cell line/retrovirus 316
Both IL-2 and IL-15 belong to the four alpha helix bundle cytokine family.1,2 IL-2 and IL-15 also share many functional similarities.1,3–8 Both induce proliferation of PHA-activated T cells from peripheral blood lymphocytes (PBL), including activation of both CD4+ and CD8+ subsets.1,3,4 Generation of cytolytic activity by PBL can be induced by IL-2 and IL-15 [the lymphokine activated killer (LAK) effect].1,3,5 Natural killer cells of both the CD56bright and CD56dim populations (expressing high-affinity abgc IL-2 receptors and intermediateaffinity bgc IL-2 receptors, respectively) proliferate in response to either IL-2 or IL-15.5 The CD56dim population can also be induced by both IL-2 and IL-15 to elicit a cytolytic response against NK-resistant target cells. B cells can undergo proliferation or differentiation in the presence of IL-2 or IL-15, depending on the culturing conditions.6 Other specific activities that IL-2 and IL-15 support have been documented, including their chemoattractant potential on lymphocytes,7 ability to expand and support the in vitro culture growth of tumour-derived activated cells (TDAC or CYTOKINE, Vol. 9, No. 5 (May), 1997: pp 316–327
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TIL),8 and induce anti-tumour activities in vivo in a tumour bearing mouse model system.9 The ability of IL-2 and IL-15 to elicit functional responses is mediated by the IL-2 and IL-15 receptor complexes. The IL-2 receptor is expressed in three forms, according to their affinity for IL-2.10–15 The high-affinity IL-2 receptor (IL-2R) is composed of the a, b, and gc subunits, and has a Kd of 2–50 pM. The intermediate-affinity IL-2R is composed of the b and gc subunits, and has a Kd of 0.5–2 nM. The a chain of the IL-2R binds IL-2 with low affinity (Kd = 2–20 nM), but is incapable of eliciting a signal of its own. Both the high- and intermediate-affinity IL-2Rs are functional as they contain the b and gc subunits.16–18 The b and gc subunits are important components of the IL-15R as well; signalling and subsequent functional responses to IL-15 cannot occur without these subunits in the IL-15R complex.1,19 The IL-15Ra chain can bind IL-15 with high-affinity alone, but cannot signal on its own.19–21 It is unclear at this time what purpose high-affinity binding of IL-15 by IL-15Ra plays in the immune system. Most of the functional characterizations of IL-15 to date have involved short-term treatment with IL-15. Responses of lymphoid cells transient IL-15 exposure (usually less than 7 days) are usually parallel to those of IL-2. Our hypothesis was that long-term growth stimulated by IL-2 vs IL-15 may reveal a differential effect on cell differentiation or function. Here we describe responses and characteristics of an immature
myeloid precursor (Tf-1b)22 to long-term IL-15 growth in comparison to long-term in vitro growth stimulated by IL-2. The parental line from which Tf-1b is derived (Tf-1), has been used extensively as a model for stem cells, expresses many haematopoietic markers, responds functionally to many haematopoietic growth factors, and can be induced to differentiate through the erythroid and myeloid lineages.23–25 Tf-1b does not express the IL-2Ra chain, but does express both IL-2Rb and gc chains, each involved in the response to IL-2 and IL-15. Thus, TF-1b is an appropriate model to compare the long-term effects of IL-2 and IL-15 exposure. These studies show that following 8 weeks of exposure to IL-15, Tf-1b cells are, in some aspects (expression of CD33, CD38, HLA-DR, morphology, and proliferation to IL-15), distinct from those cells exposed to IL-2 during the same period and indicate that although short-term effects of IL-2 and IL-15 are similar, some long-term effects of those cytokines are distinct.
RESULTS Tf-1 cells express IL-2Rb following retroviral infection The retroviral vector containing the gene for IL-2Rb (Fig. 1) was generated as described in Materials and Methods, and used to infect Tf-1 cells. Those Tf-1 cells infected with the vector containing the gene for IL-2Rb and expanded in the presence of G418 were
IL-2Rβ Chain (1.7 kb)
pA
ψ LXSN
LTR
SV
NEO
LTR
Multiple cloning site Figure 1.
Schematic of the IL-2Rb gene-containing retroviral vector.
The cDNA encoding IL-2Rb was subcloned into the multiple cloning site of pLXSN (see Materials and Methods for further details). LTR, retroviral long terminal repeat; C, packaging signal; SV, SV40 early promoter; NEO, neomycin phosphotransferase; pA, polyadenylation signal. Arrows indicate transcriptional start sites and direction of transcription.
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Tf-1β
THP-1
Tf-1L
120 000
Tf-1
PHA blasts
CYTOKINE, Vol. 9, No. 5 (May, 1997: 316–327)
100 000
Counts
80 000 1850 bp
β-actin
60 000 40 000 20 000 0
4500 bp
Tf-1
Tf-1β
Figure 3. Response of Tf-1 and Tf-1b to GM-CSF Q, IL-2 ; and IL-15 <.
IL-2Rβ Tf-1 and Tf-1b were tested for their ability to proliferate in response to IL-2 and IL-15 in a standard 3-day proliferative assay as outlined in Materials and Methods. Total counts of incorporated [3H]thymidine are indicated. All cytokines were used in the assay at a concentration of 160 ng/ml. As demonstrated in this figure, Tf-1 cells respond by proliferation only to GM-CSF, while Tf-1b cells respond equally well to GM-CSF, IL-2 and IL-15. (q), control.
1400 bp
γc Figure 2.
Northern blot analyses of IL-2Rb and gc mRNA expression.
The blot was prepared as described in Materials and Methods. Was probed for b-actin, for IL-2Rb, and for gc . PHA blasts were used as a positive control for both IL-2Rb and gc expression, while THP-1 was used as a negative control for both IL-2Rb and gc expression. Molecular weight markers are indicated to the left of each panel.
designated Tf-1b, while those Tf-1 cells infected with the vector alone and expanded in the presence of G418 were designated Tf-1L. Expression of the gene at the mRNA level was determined by both RT-PCR22 and Northern blot (Fig. 2). As indicated in this figure, only the Tf-1b cells, and not the Tf-1L cells express the gene for IL-2Rb. PHA blasts are shown here as a positive control for IL-2Rb expression, while THP-1 cells, a monocytic cell line, are shown here as a negative control for IL-2Rb expression. The expression of gc was also evaluated here as IL-2 responsiveness cannot occur in the absence of this receptor component. As indicated in the bottom panel of Figure 2, Tf-1, Tf-1L, Tf-1b cells and PHA blasts express gc mRNA. THP-1 cells do not, which is consistent with previously published work.22,26,27
Tf-1b, but not Tf-1 or Tf-1L cells are responsive to IL-2 and IL-15 The functional expression of IL-2Rb in the Tf-1b cell line had been documented in an earlier study by determining that Tf-1b could respond by proliferation to IL-2.22 Here, we expand those studies to document IL-15 induced proliferation of Tf-1 cells. As indicated
in Figure 3, both Tf-1 and Tf-1b respond by proliferation to GM-CSF. In contrast, only Tf-1b cells respond by proliferation to IL-2 and IL-15. These results indicate that the transfection process resulted in expression of IL-2Rb which is reflected in the ability of the Tf-1b cells to proliferate to IL-2 and IL-15, and that the response of Tf-1 cells to IL-15 requires IL-2Rb expression as neither the Tf-1 parental line (nor the Tf-1L cell line, not shown) proliferate in response to IL-15.
IL-2 and IL-15 sustain long-term Tf-1b cultures and do not alter IL-2Rb expression One aspect of IL-15 characterizations that has not yet been studied is whether this new cytokine is capable of sustaining cell growth in long-term culture beyond the initial stimulus, and whether this ability parallels that of IL-2. In order to determine this, Tf-1b cells were grown in either IL-2 or IL-15 without GM-CSF since IL-2 or IL-15 alone should stimulate growth of these cells. Furthermore, growth stimulated by IL-2 or IL-15 requires expression of the IL-2Rb chain, therefore IL-2 and IL-15 should each function as ‘‘selecting’’ agents favouring growth of the IL-2Rb expressing transfectants. Thus, no G418 was added to these cultures either. After 8 weeks (20 passages) of culturing either in IL-2 or IL-15, the cells were still viable and replicating in vitro. No ‘‘crisis period’’ in cell growth was noted during this time. The resultant cells were renamed Tf-1b2, for those grown long term in IL-2, and Tf-1b15, for those grown long term in IL-15.
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Tf-1
Tf-1β
90
90 A
0 100
B
1
10
102
103
104
0 100
1
10
Tf-1β2
103
104
103
104
Tf-1β15
90
90 C
0 100
102
D
1
10
102
103
104
0 100
1
10
102
Figure 4. Flow cytometric analysis of IL-2Rb expression on Tf-1 (A), Tf-1b (B), Tf-1b2 (C), and Tf-1b15 (D) cells. Tf-1, Tf-1b, Tf-1b2 (grown in IL-2 alone) and Tf-1b15 (grown in IL-15 alone) were stained for expression of IL-2Rb using the anti-IL-2Rb antibody, 561, as outlined in Materials and Methods. The shaded areas represent staining with the isotype control for mAb 561, UPC10. The unshaded areas represent staining with mAb 561. As demonstrated by this figure, Tf-1 cells do not express IL-2Rb, as shown previously.10 Tf-1b, Tf-1b2, and Tf-1b15 all express IL-2Rb at roughly the same levels, indicating that selection of Tf-1b cells in IL-2 or IL-15 is sufficient to maintain IL-2Rb expression.
To confirm that culturing in IL-2 or IL-15 selected for IL-2Rb expression, Tf-1, Tf-1b, Tf-1b2, and Tf-1b15 cells were analysed by flow cytometry for their expression of IL-2Rb. As indicated in Figure 4, expression levels of IL-2Rb in Tf-1b, Tf-1b2, and Tf-1b15 are comparable, and the parental line Tf-1, does not express IL-2Rb. Hence, selection by IL-2 or IL-15 was adequate to maintain expression of IL-2Rb.
Long-term culture in IL-15 alters morphology, but not esterase activity Since IL-15 mRNA is expressed in virtually all human tissues (including those derived from bone marrow), it becomes of interest to determine if one of the functions of IL-15 is to support cellular differentiation which may in part be reflected in morphological changes. Samples from Tf-1, Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 were used to determine whether any changes in size, shape or morphological features could be observed, especially in the cells that had been grown in IL-2 or IL-15. This was also
an opportunity to determine if the retroviral infection itself had caused any alterations, and so Tf-1L was included in these studies. No gross alterations in morphology were detected in Tf-1L, Tf-1b, or Tf-1b2 in comparison to Tf-1, or to each other. This indicated that neither the retroviral infection process, nor growth in IL-2 rather than in GM-CSF, had altered the morphology of these cell lines. However, the Tf-1b15 cells were somewhat smaller in size, had enlarged cytoplasm, and smaller nuclei in comparison to Tf-1, Tf-1L, Tf-1b, and Tf-1b2. Figure 5 illustrates the morphological staining of Tf-1b and Tf-1b15 for comparison. These features are consistent with, but do not prove, some maturation or differentiation of the Tf-1b15 cells had occurred. Neither long-term selection in IL-2, nor the retroviral infection process itself, alters the morphology of these cells, although IL-15 seems to have a differentiating effect according to this morphological analysis. The parental line, Tf-1 was originally shown to be highly positive for esterase activity, a characteristic indicative of cells derived from the myeloid lineage.23
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TABLE 1.
Figure 5.
Morphological and cytochemical staining of the cells.
Tf-1, Tf-1L, Tf-1b, Tf-1b2, and Tf-1b15 were used to determine whether any morphological changes in size, shape or morphologic features could be noted, especially in the cells that had been grown in IL-2 or IL-15. Samples from each cell line were Giemsa stained on the same day as described in Materials and Methods. (A) depicts the Giemsa staining on Tf-1b, and (B) depicts the Giemsa staining on Tf-1b15. No gross alterations in morphology were detected in Tf-1L, Tf-1b, or Tf-1b2 in comparison to Tf-1, or to each other. However, the Tf-1b15 cells (lower panel) were somewhat smaller in size and had enlarged cytoplasm, and smaller nuclei in comparison to Tf-1, Tf-1L, Tf-1b, and Tf-1b2.
Samples from each of the cell lines were also tested to determine if they had maintained this myeloid characteristic. All of the cells, including those grown long term in IL-2 or IL-15, maintained their positivity for esterase staining, indicating that long term growth in IL-2 or IL-15, as well as retroviral infection, has not altered this particular characteristic (data not shown).
Long-term culture of Tf-1b cells in IL-15 alters CD marker expression of CD33 , CD38 and HLA-DR Defined steps of differentiation are also characterized by the expression of certain CD markers.28–30 Flow cytometry was used then to determine if long-term culture in IL-2 or IL-15 had caused alterations in CD marker expression on the cells in comparison to the parental line, Tf-1. Descriptions of the CD markers tested here are summarized in Table 1. Since Tf-1 cells have many of the same markers as haematopoietic
Antibodies used in CD marker expression analysis
mAb
Isotype
CD3 CD5 CD7 CD10 CD11b CD14 CD16 CD19 CD33 CD34 CD38 CD45 HLA-DR
IgG1 IgG2a IgG2a IgG2a IgG1 IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
Antigenic specificity T cells T cells/thymocytes T cells/thymocytes/NK cells CALLA/lymphoid progenitors/granulocytes Granulocytes/precursor monocytes/NK cells Monocytes/granulocytes/macrophages NK cells/FcgRIII Pan B cells Monocytes/myeloid precursors/granulocytes Hematopoietic precursors/endothelial cells Plasma cells/thymocytes/activated T cells Leukocyte common antigen (LCA) MHC Class II
stem cells, it was possible that some step towards differentiation may have occurred that could not be detected by the gross cytochemical studies, but that may be reflected in CD marker expression. Each of the cells was subjected to a series of flow cytometric stainings in parallel, and the results of several separate experiments summarized in Table 2. For each cell line and each marker, data are presented as a percentage of positive cells, MFI (mean fluorescence intensity), and the level of expression measured by the fold increase of the MFI of the test antibody (i.e. CD11b, CD33, etc) over the MFI of the isotype control antibody on the same cells. As indicated in this table, the expression of CD11b, CD33, CD34, CD38 and HLA-DR on Tf-1, Tf-1L, Tf-1b, and Tf-1b2 were quite similar. This indicated that the retroviral transfection, selection in G418 and GM-CSF or selection in IL-2 had little effect on the expression of these markers as compared to expression by the parental line, Tf-1. This further supports that retroviral infection, acquisition of new protein expression, and long-term growth in IL-2 does not appreciably alter the original characteristics of the cell, other than the documented functional expression of the IL-2Rb molecule. However, this was not the case with Tf-1b15 cells, which were grown long-term in IL-15. The majority of the CD marker expression profile on Tf-1b15 cells is similar to that on the other cell lines, with the exception of CD33, CD38, and HLA-DR expression which are all decreased on Tf-1b15 cells. CD33, a marker expressed commonly on myeloid precursors and monocytes, but not on mature myelocytes,31,32 is less than half as intense on Tf-1b15 cells as on the other cell lines (MFI = 52 in comparison to MFI = 114 on the parental Tf-1 cell line). CD38, a marker expressed most commonly on lymphoid and myeloid precursor cells and 80 percent of monocytes,33,34 is less than 1/7 as intense on Tf-1b15 cells than it is on the other cell lines (MFI = 13 in comparison to MFI = 96 on Tf-1L). The MHC Class
IL-2 and IL-15 Effects on the Tf-1b cell line / 321
TABLE 2.
CD11b CD33 CD34 CD38 HLA-DR
Flow cytometric analysis of Tf-1-derived cell lines
%Positive MFI Fold Increase % Positive MFI Fold Increase % Positive MFI Fold Increase % Positive MFI Fold Increase % Positive MFI Fold Increase
Tf-1
Tf-1L
Tf-1b
Tf-1b2
Tf-1b15
26 7.98 2.7 100 113.96 31 94 32.23 11 96 124.29 33.8 100 243.85 70
53 13.0 3.7 100 119.73 29.3 97 36.35 12.7 99 96.34 23.6 100 240.55 64.8
40 9.82 3.4 100 148.45 49.9 98 35.64 12.5 99 108.79 36.5 100 282.41 93.2
42 10.32 2.7 100 164.42 35 95 37.89 9.9 99 100.74 21.5 100 248.79 53.5
27 3.87 1 99 52.48 13.7 86 41.16 10.7 78 13.33 3.5 97 125.46 32.7
CD5, CD10, CD14 and CD19 all Q1% Positive. CD3, CD7, CD16 and CD45 all Q5% Positive. Tf-1, Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 cells were stained with antibodies specific for the CD marker listed and described in Table 1. The values listed are given as ‘‘% Positive’’, which is the percentage of live cells in the population that stained positive with each respective antibody, ‘‘MFI’’, which is the Mean Fluorescence Intensity values for each respective antibody, and ‘‘Fold Induction’’ which is the MFI of the antibody staining divided by the MFI of the isotype control staining for that particular antibody.
II molecule, HLA-DR, is also decreased on Tf-1b15 cells in comparison to the other cell lines (MFI = 125 in comparison to MFI = 243 on Tf-1). Although expression of these three markers is co-regulated to some extent, dim or no expression of these markers generally indicates an immature state of the cell.34–41 It is also possible that the general decrease in surface expression of the CD markers on Tf-1b15 is a result of a decline in the robustness of this cell line. Indeed, analysing the percentage of dead cells as determined by uptake of propidium iodide in the flow cytometry analysis reveals that the Tf-1b15 cells are slightly less viable than Tf-1, Tf-1b, or Tf-1b2 (85% viable in comparison to 93–94%). This decrease in viability of Tf-1b15 may also account for its altered morphology.
Elevated concentrations of IL-15 are required to stimulate Tf-1b15 cells in comparison to Tf-1b2 Although it was unclear whether Tf-1b15 had truly undergone differentiation, it was clear that some of its morphological and molecular features were altered in comparison to the original Tf-1 cell line. Tf-1b15 cells show larger cytoplasm, smaller nuclei, smaller overall size, and decreased expression of CD33, CD38, and HLA-DR. Tf-1b2, however, do resemble the original cell line, Tf-1, with the exception that Tf-1b2 cells can proliferate in response to IL-2 (or IL-15), whereas Tf-1 cannot. In the Tf-1 cell line, treatment with DMSO results in partial inhibition of proliferation, although this does not cause differentiation to the next defined step of the process.23 It was possible then that long-term growth in
IL-15 may have altered the ability of Tf-1b15 cells to proliferate in response to cytokines, in comparison to the Tf-1b2 cell line. Tf-1, Tf-1b2 and Tf-1b15 cells were each incubated with a range of concentrations of GM-CSF, IL-2, F42K (an IL-2 variant that does not bind the IL-2Ra)42–44 and IL-15 concentrations in a standard proliferative assay. One representative graph of this data is shown in Figure 6. The concentration of each cytokine required to reach half-maximal proliferation (EC50 values) for these responses are shown in Table 3 and the statistical comparisons are shown in Table 4. Tf-1, Tf-1b2 and Tf-1b15 cells respond to GM-CSF in a dose–response manner, with EC50 values of 1.2, 7 and 2.3 pM, respectively (Table 3). Statistical analysis demonstrates that these EC50 values reflecting the response to GM-CSF by these cell lines are similar. Tf-1 cells (Fig. 6A) do not respond to F42K, IL-2, or IL-15 as they do not express the IL-2Rb subunit, consistent with previously published results.1,45 The remaining proliferation analysis is thus focused on Tf-1b2 and Tf-1b15 cells, which do respond by proliferation to F42K, IL-2, and IL-15. Tf-1b2 cells (Figure 6B) and Tf-1b15 cells (Figure 6C) have similar proliferative responses to IL-2 [EC50 = 66 pM and 128 pM (P = 0.35)] as well as to the IL-2 variant, F42K, which does not bind to the IL-2Ra chain [EC50 = 96 pM and 138 pM (P = 0.072)]. The response of any Tf-1b derivative to IL-2 or F42K is predicted to be similar since Tf-1b cells do not express the IL-2Ra chain.22 Hence, the EC50 values for IL-2 and F42K responses by Tf-1b2 are similar [EC50 = 138 pM and 128 pM (P = 0.88)]. Kit225, which is a human T cell line bearing high-affinity abgc IL-2 receptors, has
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an EC50 value for IL-2 of 2 pM and an EC50 value for F42K of 68 pM (data not shown) which is consistent with the importance of the IL-2Ra chain in the response to IL-2, but not to F42K. These analyses demonstrate that alterations of proliferation by Tf-1b2 and Tf-1b15 to GM-CSF, IL-2, and F42K have not occurred in the process of long-term culture in IL-2 or IL-15 for Tf-1b2 and Tf-1b15 cells. However, the responses of Tf-1b2 and Tf-1b15 to IL-15 are not similar [EC50 = 184 pM vs 336 pM (P = 0.015)]. In fact, the response of Tf-1b15 to IL-15 requires twice as much IL-15 to reach maximal proliferation as is required for Tf-1b2. This result suggests that some subtle alteration of the Tf-1b cell lines’ quantitative ability to proliferate in response to IL-15 has occurred during long-term growth in the presence of IL-15.
DISCUSSION
Figure 6.
Response of Tf-1, Tf-1b2 and Tf-1b15 to cytokines.
These cells were tested for their ability to proliferate in response to GM-CSF (R), IL-2 (Q), F42K (W) (the IL-2 variant that does not bind to IL-2Ra), and IL-15 (T) in a standard 3-day proliferative assay as outlined in Materials and Methods. Total counts of incorporated [3H]thymidine are indicated. As demonstrated here, Tf-1 (A) cells respond by proliferation only to GM-CSF, and not to IL-2, F42K, or IL-15. Tf-1b2 (B) and Tf-1b15 (C) proliferate in response to all four cytokines.
IL-15 is a recently discovered cytokine with many functions similar to that of IL-2.1,3–9 However, many of the comparisons between IL-2 and IL-15 to date have been evaluated in short-term assays. It was our intent to determine if long-term exposure to IL-15 would be similar to long-term exposure of IL-2. We chose to perform these studies on the myelomonocytic Tf-1 cell line which has been characterized extensively.22–24 It has been established that Tf-1 cells express many haematopoietic stem cell markers, respond to haematopoietic stem cell growth factors, and can differentiate through myeloid and erythroid lineages.22–24,26 Because of these characteristics, Tf-1 has been used as a model for stem cell studies, as well as a target for retroviral infection.47 Tf-1b, the cell derivative of Tf-1, was required for these experiments as Tf-1 does not respond to IL-2 or IL-15 in the absence of IL-2Rb expression (Fig. 3).22 Tf-1b cells were generated by infecting Tf-1 cells with a retroviral construct containing the gene for IL-2Rb (Fig. 1) which is successfully transcribed (Fig. 2). Our prior report documents that Tf-1b cells could proliferate in response to IL-2, as they contained both the genes for IL-2Rb and gc which together generate the intermediate-affinity bgc IL-2 receptor.22 However, it was unclear if this single manipulation of Tf-1 cells would also confer IL-15 responsiveness on Tf-1b cells. As indicated in Figure 3, Tf-1b cells can also proliferate in response to IL-15. Since Tf-1b cells could respond by proliferation to IL-2 or IL-15, we then showed that IL-2 and IL-15 could sustain long-term cell growth of Tf-1b cells. Tf-1b2 and Tf-1b15 cell lines were generated from long-term culture in IL-2 or IL-15 respectively, did not experience a culture crisis period, and both lines
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TABLE 3.
Tf-1 Tf-1b2 TF-1b15
EC50 values (in pM) on Tf-1-derived cell lines in response to cytokines
Mean Number Mean Number Mean Number
GM-CSF
F42K
IL-2
IL-15
1.2 2 0.2 n=3 7.0 2 6.0 n=3 2.3 2 1.9 n=3
N/A N/A 96 2 11 n=4 138 2 13.5 n=3
N/A N/A 65.6 2 6.3 n=9 128 2 54 n=3
N/A N/A 184 2 38 n=7 336 2 28.6 n=3
Mean EC50 values in picomolar (pM) were measured from each of the assays and summarized with standard errors in this table, along with the number of curves used to generate the average of each cytokine required to reach half-maximal proliferation (EC50 ) value for each condition on each cell type.
maintained IL-2Rb expression without G418 selection (Fig. 4). This indicated that both IL-2 and IL-15 could sustain long-term culturing of Tf-1b and that these conditions were adequate to select and maintain expression of IL-2Rb. Tf-1, Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 cells were then assessed for distinctions in morphology, CD marker expression, and function. As evidenced here, infecting Tf-1 cells with a recombinant retrovirus does not grossly alter its original function as measured by proliferation induced by GM-CSF or other growth factors (Fig. 3), their morphology (Fig. 5), their ability to metabolize esterase, nor their surface expression of CD markers indicative of the parental line (Table 2). Conferring a new function upon Tf-1 cells, as evidenced in the Tf-1b cells, also does not grossly alter their original functions other than the IL-2Rb specific activities. Even more intriguing is the finding that if these cells must depend on the expression of the transfected IL-2Rb chain for their survival, they are also unaltered with regard to their original profile. This held true for the Tf-1b2 cells in all aspects studied here, and in most aspects studied here for the Tf-1b15 cells. The morphological staining and CD marker expression analysis did reveal that Tf-1b15 cells, which had been grown long-term in IL-15, do not maintain
the identical original profile of Tf-1 cells and may have undergone some subtle morphological and differentiation changes. Giemsa staining of Tf-1b15 demonstrated that the cells appeared slightly more differentiated than the other cell lines (Tf-1, Tf-1L, Tf-1b and Tf-1b2) as the nuclei were more condensed, the cytoplasm was enlarged, and the overall size was smaller. Since the more defined steps of differentiation are in part characterized by CD marker expression, samples from each cell type were screened for expression of various CD markers (summarized in Table 2). These data revealed that Tf-1b15 had downregulated a fraction of CD33, CD38 and HLA-DR expression in comparison to Tf-1, Tf-1L, Tf-1b and Tf-1b2. What possibilities may account for these subtle morphological and surface marker changes remains unclear, although the decreased viability of Tf-1b15 in comparison to Tf-1, Tf-1b, Tf-1b2 may have been an influence. Differentiation states may also be reflected in functional responses. In order to test this, Tf-1b15 cells were placed in a standard proliferative assay in parallel with Tf-1b2 and Tf-1. The results of these experiments demonstrate that Tf-1b2 and Tf-1b15 do not respond similarly to IL-15. Tf-1b15 cells require higher concentrations of IL-15 to reach the same level of proliferation as do Tf-1b2 cells in response to IL-15.
TABLE 4. Comparative statistical analysis of EC50 calculations from Tf-1-derived cell lines in response to cytokines P value GM-CSF Tf-1 vs Tf-1b2 Tf-1 vs Tf-1b15 Tf-1b2 vs Tf-1b15 F42K Tf-1b2 vs Tf-b15 IL-2 Tf-1b2 vs Tf-1b15 IL-15 Tf-1b2 vs Tf-1b15*
0.045 0.62 0.53 0.072 0.35
P value Tf-1b2 F42K vs IL-2 F42K vs IL-15 IL-2 vs IL-15* Tf-1b15 F42K vs IL-2 F42K vs IL-15* IL-2 vs IL-15*
0.051 0.064 0.019 0.88 0.025 0.042
0.015
P values were determined by t-test with unpooled variance using the Minitab software package. *Indicates P Q 0.05.
324 / Farner et al.
It should also be noted that Tf-1b15 cells also appear to require slightly elevated levels of IL-2 and F42K to achieve 50% maximal proliferation in comparison to the Tf-1b2 cells. However, this apparent increased requirement for IL-2 and F42K does not meet standard criteria for significance (P = 0.072 and 0.35). It is difficult to determine if such subtle differences in EC50 values in vitro are physiologically relevant. The alteration that occurred on Tf-1b15 cells that resulted in an increased EC50 value (decrease in responsiveness to IL-15) in response to IL-15 remains unclear. It is possible that expression levels of IL-15 receptors on Tf-1b15 influence its response to IL-15.20,21 Early characterizations of IL-15Ra indicate that this subunit confers high-affinity IL-15 binding to the IL-15 receptor complex. Although co-expression of IL-15Ra, IL-2Rb, gc results in high-affinity binding of IL-15, it is unclear if this necessarily results in proliferation to IL-15 that is also representative of high affinity. If it is, then this would indicate that Tf-1b15 cells may have decreased some portion of their IL-15Ra expression, as the EC50 value of IL-15 proliferation by Tf-1b15 cells is significantly higher than it is on Tf-1b2. Determining the relative level of IL-15Ra protein on these cell lines would clarify this issue. However, an antibody directed against IL-15Ra is not yet available. An analysis of the IL-15Ra mRNA levels would be possible, but it is unclear if an assessment of IL-15Ra mRNA would reliably translate to functional IL-15Ra protein expression on the cell surface. In conclusion, the ability of the Tf-1b15 cells to survive over an extended period of time with the support of IL-15 alone proves that IL-15 can sustain long-term cell growth and select for expression of transfected IL-2Rb chains. Furthermore, the subtle distinctions between Tf-1b2 and Tf-1b15 suggest that subtle distinctions between IL-2 and IL-15 functions may be found in long-term studies.
MATERIALS AND METHODS Cytokines Human recombinant IL-2 was provided by HoffmanLaRoche, Inc. (Nutley, NJ) and has a specific activity of 15 × 106 U/mg protein so that 1 U/mL = 4.4 pM. GM-CSF was provided by Immunex Corporation (Seattle, WA). F42K was kindly provided by Dr G. Ju (Hoffmann-LaRoche, Nutley, NJ), which is an IL-2 variant that does not interact with the IL-2Ra chain due to a point mutation that substitutes the lysine residue at the 42nd amino acid residue for phenylalanine.42–44 Recombinant human IL-15 was kindly provided by Dr M. Widmer (Immunex Research and Development Corp., Seattle, WA).
Cell lines Tf-1 cells were established from a patient with erythroleukaemia and requires GM-CSF or IL-3 for sustained
CYTOKINE, Vol. 9, No. 5 (May, 1997: 316–327)
proliferation and were kindly provided by T. Kitamura (University of Tokyo). This line has been classified as an immature precursor erythroid cell, although it expresses several myeloid markers and can be induced to differentiate into macrophages.23,24 Tf-1 cells are also brightly positive for CD33, CD34, and CD38, consistent with their designation as haematopoietic precursor cells. The THP-1 cell line has been characterized as a human monocyte line, and is known to be negative for gc mRNA expression.27 PHA blasts were generated by incubating Ficoll-hypaque separated normal donor peripheral blood lymphocytes in RPMI complete medium containing PHA at a concentration of 1 mg/ml.48
Antibodies The reagent to detect IL-2Rb expression by flow cytometry (Fig. 4) was the mAb 561 (IgG2a), a non-inhibitory antibody directed against the human IL-2Rb chain as described by Voss et al.49 The IgG2a isotype control antibody (UPC10) was purchased from Sigma Chemical Co. (St Louis, MO). The antibodies used for the CD marker expression were all obtained from Becton Dickinson.
Cloning strategy for IL-2Rb The cDNA for the IL-2Rb gene (kindly provided by Dr Rich Robb, Oncotherapeutics, NJ) was subcloned into the multiple cloning site of the retrovirus expression vector, pLXSN50 (kindly provided by Dr A. D. Miller, Fred Hutchinson Cancer Center, Seattle, WA) and designated pLXSN/IL-2Rb. In this system, gene expression is controlled by the 5' long terminal repeat of Moloney murine leukaemia virus (MMLV), while the eukaryotic dominant selection marker gene, neomycin phosphotransferase, is driven by the simian virus 40 (SV-40) early promoter.
Generation of recombinant retroviruses and gene transfer The amphotropic retrovirus packaging cell line, PA31751 was transfected by the calcium phosphate method50,52 with either the pLXSN/IL-2Rb construct or the pLXSN vector alone for 3 h at 37°C, then glycerol shocked for 1 min. Transfected cells were stably selected in the presence of G418 sulfate (Geneticin, GIBCO) at 1 mg/ml for 3–4 weeks. Viral supernatants from drug resistant PA317 cells were used to infect the Tf-1 cell line. Tf-1 cells infected with the pLXSN vector alone are designated Tf-1L, and the Tf-1 cells infected with the pLXSN/IL-2Rb vector are designated Tf-1b.
Culture conditions for Tf-1 , Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 Tf-1 cells were maintained in RPMI 1640 (supplmented with 1 mM HEPES, PenStrep/-glu, and 10% FCS) with 5 ng/ml GM-CSF for culture maintenance. Tf-1L and Tf-1b were also maintained in this same GM-CSF containing media, which also contained 0.5 mg/ml of G418. The Tf-1b2 cell line was generated from the Tf-1b cell line by culturing Tf-1b cells in 100 units/ml of IL-2 (without GM-CSF or G418) for at least 2 months, while the Tf-1b15 cell line was generated from the Tf-1b cells by culturing in 200 ng/ml of IL-15 for 2 months, also without GM-CSF or G418. Preliminary dose titration curves for IL-2 and IL-15 stimulation of Tf-1b had demonstrated that 100 units/ml
IL-2 and IL-15 Effects on the Tf-1b cell line / 325
IL-2 and 200 ng/ml IL-15 stimulate maximal proliferation by Tf-1b (J. de Jong, unpublished observation).
Isolation of RNA and Northern blotting RNA was extracted from each cell type as described by Chomczynski and Sacchi.58 Briefly, each cell pellet was resuspended in equal volumes of GITC solution (4 M guanidine thiocyanate, 25 mM NaCitrate, pH 7.0, 0.5% sarkosyl, 0.1% b-mercaptoethanol, and 0.2 M sodium acetate, pH 4.0) and buffer-saturated phenol. After vortexing, 1 2 volume of chloroform–isoamyl (24:1) was added and the suspension vortexed. Samples were centrifuged (12 000 × g) at 4°C for 30 min. The aqueous phase was transferred to a new tube, and 100% ethanol added to it. The suspension was mixed and placed on ice for 1 h at which time it was centrifuged at 4°C for 30 min at 12 000 × g. The ethanol was decanted and the RNA pellet resuspended in RNase-free water. The concentration of RNA was determined by spectrophotometry. mRNA (10 mg) was fractionated on a formaldehyde denaturing gel, were transferred to a nylon membrane, and probed with 32P-labelled probes. Probes were generated from cloned cDNA, purified through agarose gels and eluted using Geneclean glassmilk. The blots were exposed to film overnight at −80°C.
Immunofluorescent flow cytometry Immunofluorescent staining was performed as previously described.22 Antibodies from Becton Dickinson were used according to manufacturer’s instructions.
In vitro proliferative assay In vitro proliferation in response to various growth factors and cytokines was measured in 3-day in vitro [3 H]thymidine incorporation assays as previously described.22 In Figure 3, GM-CSF, IL-2 and IL-15 were used at a final concentration of 160 ng/ml. In Figure 6, GM-CSF, IL-2, F42K, and IL-15 were diluted from a starting concentration of 160 ng/ml.
Cytochemical staining The morphology of each cell line in comparison to the parental Tf-1 cell line was examined on cytocentrifuged slide preparations stained with Wright stain.54 Cytochemical assays were performed using a combined technique with a-naphthyl acetate as a substrate to detect ‘‘non-specific’’ (monocyte type) esterase and naphthol ASD choroacetate to detect ‘‘specific’’ esterase (granulocyte type) staining.55 Positive a-naphthyl acetate esterase staining is indicated by a red-brown colour, while positive naphthol ASD chloroacetate staining is indicated by a blue colour.
Acknowledgements Special thanks to John Hakimi and Bob Pilson (Hoffman-La Roche, Nutley, NJ) for the initial concept of growing Tf-1b on IL-2 long-term, and to Mike Widmer (Immunex Corp., Seattle, WA) for providing IL-15 as well as helpful discussions and insight. Thanks
to Dr S. Wu, Sally Drew, M. T., and Linda Sebree, H. T., (UW-Madison, Madison, WI) for technical assistance in generating the morphological data, and Ms Kristin Elmer (UW-Madison, Madison, WI) for flow cytometric analysis. Supported by American Cancer Society Grant No. IM-678 and National Institutes of Health Grants No. CA-32685, p30-CA14520-2, CM-87290, and RR03186. T. P. L. was a Fellow of the American Cancer Society.
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Interleukin 15 (IL-15) was initially isolated from the supernatant of the simian kidney epithelial cell line, CV-1/EBNA as it supported proliferation of the IL-2 dependent cell line, CTLL-2.1 It is expressed in virtually all tissues, including bone marrow-derived cells and placenta.1 Subsequent cloning documented that IL-15 did not have any appreciable homology to other proteins, but that it is structurally similar to IL-2.
From the 1Department of Human Oncology, 3Cellular and Molecular Biology, 4Pediatrics, 5Pathology and Lab Medicine, and 6 Genetics, University of Wisconsin and Wisconsin Comprehensive Cancer Center, Madison, WI 53792, USA; 2Experimental Biology Research, Virus Discovery Group, Abbott Laboratories, North Chicago, IL 60064, USA Correspondence to: Paul M. Sondel, K4/448 University of Wisconsin Comprehensive Cancer Center, 600 Highland Avenue, Madison, WI 53792 Received 14 May 1996; revised and accepted for publication on 16 November 1996 7 1997 Academic Press Limited 1043–4666/97/050316 + 12 $25.00/0/ck960171 KEY WORDS: CD34+/IL-15/Tf-1b cell line/retrovirus 316
Both IL-2 and IL-15 belong to the four alpha helix bundle cytokine family.1,2 IL-2 and IL-15 also share many functional similarities.1,3–8 Both induce proliferation of PHA-activated T cells from peripheral blood lymphocytes (PBL), including activation of both CD4+ and CD8+ subsets.1,3,4 Generation of cytolytic activity by PBL can be induced by IL-2 and IL-15 [the lymphokine activated killer (LAK) effect].1,3,5 Natural killer cells of both the CD56bright and CD56dim populations (expressing high-affinity abgc IL-2 receptors and intermediateaffinity bgc IL-2 receptors, respectively) proliferate in response to either IL-2 or IL-15.5 The CD56dim population can also be induced by both IL-2 and IL-15 to elicit a cytolytic response against NK-resistant target cells. B cells can undergo proliferation or differentiation in the presence of IL-2 or IL-15, depending on the culturing conditions.6 Other specific activities that IL-2 and IL-15 support have been documented, including their chemoattractant potential on lymphocytes,7 ability to expand and support the in vitro culture growth of tumour-derived activated cells (TDAC or CYTOKINE, Vol. 9, No. 5 (May), 1997: pp 316–327
IL-2 and IL-15 Effects on the Tf-1b cell line / 317
TIL),8 and induce anti-tumour activities in vivo in a tumour bearing mouse model system.9 The ability of IL-2 and IL-15 to elicit functional responses is mediated by the IL-2 and IL-15 receptor complexes. The IL-2 receptor is expressed in three forms, according to their affinity for IL-2.10–15 The high-affinity IL-2 receptor (IL-2R) is composed of the a, b, and gc subunits, and has a Kd of 2–50 pM. The intermediate-affinity IL-2R is composed of the b and gc subunits, and has a Kd of 0.5–2 nM. The a chain of the IL-2R binds IL-2 with low affinity (Kd = 2–20 nM), but is incapable of eliciting a signal of its own. Both the high- and intermediate-affinity IL-2Rs are functional as they contain the b and gc subunits.16–18 The b and gc subunits are important components of the IL-15R as well; signalling and subsequent functional responses to IL-15 cannot occur without these subunits in the IL-15R complex.1,19 The IL-15Ra chain can bind IL-15 with high-affinity alone, but cannot signal on its own.19–21 It is unclear at this time what purpose high-affinity binding of IL-15 by IL-15Ra plays in the immune system. Most of the functional characterizations of IL-15 to date have involved short-term treatment with IL-15. Responses of lymphoid cells transient IL-15 exposure (usually less than 7 days) are usually parallel to those of IL-2. Our hypothesis was that long-term growth stimulated by IL-2 vs IL-15 may reveal a differential effect on cell differentiation or function. Here we describe responses and characteristics of an immature
myeloid precursor (Tf-1b)22 to long-term IL-15 growth in comparison to long-term in vitro growth stimulated by IL-2. The parental line from which Tf-1b is derived (Tf-1), has been used extensively as a model for stem cells, expresses many haematopoietic markers, responds functionally to many haematopoietic growth factors, and can be induced to differentiate through the erythroid and myeloid lineages.23–25 Tf-1b does not express the IL-2Ra chain, but does express both IL-2Rb and gc chains, each involved in the response to IL-2 and IL-15. Thus, TF-1b is an appropriate model to compare the long-term effects of IL-2 and IL-15 exposure. These studies show that following 8 weeks of exposure to IL-15, Tf-1b cells are, in some aspects (expression of CD33, CD38, HLA-DR, morphology, and proliferation to IL-15), distinct from those cells exposed to IL-2 during the same period and indicate that although short-term effects of IL-2 and IL-15 are similar, some long-term effects of those cytokines are distinct.
RESULTS Tf-1 cells express IL-2Rb following retroviral infection The retroviral vector containing the gene for IL-2Rb (Fig. 1) was generated as described in Materials and Methods, and used to infect Tf-1 cells. Those Tf-1 cells infected with the vector containing the gene for IL-2Rb and expanded in the presence of G418 were
IL-2Rβ Chain (1.7 kb)
pA
ψ LXSN
LTR
SV
NEO
LTR
Multiple cloning site Figure 1.
Schematic of the IL-2Rb gene-containing retroviral vector.
The cDNA encoding IL-2Rb was subcloned into the multiple cloning site of pLXSN (see Materials and Methods for further details). LTR, retroviral long terminal repeat; C, packaging signal; SV, SV40 early promoter; NEO, neomycin phosphotransferase; pA, polyadenylation signal. Arrows indicate transcriptional start sites and direction of transcription.
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Tf-1β
THP-1
Tf-1L
120 000
Tf-1
PHA blasts
CYTOKINE, Vol. 9, No. 5 (May, 1997: 316–327)
100 000
Counts
80 000 1850 bp
β-actin
60 000 40 000 20 000 0
4500 bp
Tf-1
Tf-1β
Figure 3. Response of Tf-1 and Tf-1b to GM-CSF Q, IL-2 ; and IL-15 <.
IL-2Rβ Tf-1 and Tf-1b were tested for their ability to proliferate in response to IL-2 and IL-15 in a standard 3-day proliferative assay as outlined in Materials and Methods. Total counts of incorporated [3H]thymidine are indicated. All cytokines were used in the assay at a concentration of 160 ng/ml. As demonstrated in this figure, Tf-1 cells respond by proliferation only to GM-CSF, while Tf-1b cells respond equally well to GM-CSF, IL-2 and IL-15. (q), control.
1400 bp
γc Figure 2.
Northern blot analyses of IL-2Rb and gc mRNA expression.
The blot was prepared as described in Materials and Methods. Was probed for b-actin, for IL-2Rb, and for gc . PHA blasts were used as a positive control for both IL-2Rb and gc expression, while THP-1 was used as a negative control for both IL-2Rb and gc expression. Molecular weight markers are indicated to the left of each panel.
designated Tf-1b, while those Tf-1 cells infected with the vector alone and expanded in the presence of G418 were designated Tf-1L. Expression of the gene at the mRNA level was determined by both RT-PCR22 and Northern blot (Fig. 2). As indicated in this figure, only the Tf-1b cells, and not the Tf-1L cells express the gene for IL-2Rb. PHA blasts are shown here as a positive control for IL-2Rb expression, while THP-1 cells, a monocytic cell line, are shown here as a negative control for IL-2Rb expression. The expression of gc was also evaluated here as IL-2 responsiveness cannot occur in the absence of this receptor component. As indicated in the bottom panel of Figure 2, Tf-1, Tf-1L, Tf-1b cells and PHA blasts express gc mRNA. THP-1 cells do not, which is consistent with previously published work.22,26,27
Tf-1b, but not Tf-1 or Tf-1L cells are responsive to IL-2 and IL-15 The functional expression of IL-2Rb in the Tf-1b cell line had been documented in an earlier study by determining that Tf-1b could respond by proliferation to IL-2.22 Here, we expand those studies to document IL-15 induced proliferation of Tf-1 cells. As indicated
in Figure 3, both Tf-1 and Tf-1b respond by proliferation to GM-CSF. In contrast, only Tf-1b cells respond by proliferation to IL-2 and IL-15. These results indicate that the transfection process resulted in expression of IL-2Rb which is reflected in the ability of the Tf-1b cells to proliferate to IL-2 and IL-15, and that the response of Tf-1 cells to IL-15 requires IL-2Rb expression as neither the Tf-1 parental line (nor the Tf-1L cell line, not shown) proliferate in response to IL-15.
IL-2 and IL-15 sustain long-term Tf-1b cultures and do not alter IL-2Rb expression One aspect of IL-15 characterizations that has not yet been studied is whether this new cytokine is capable of sustaining cell growth in long-term culture beyond the initial stimulus, and whether this ability parallels that of IL-2. In order to determine this, Tf-1b cells were grown in either IL-2 or IL-15 without GM-CSF since IL-2 or IL-15 alone should stimulate growth of these cells. Furthermore, growth stimulated by IL-2 or IL-15 requires expression of the IL-2Rb chain, therefore IL-2 and IL-15 should each function as ‘‘selecting’’ agents favouring growth of the IL-2Rb expressing transfectants. Thus, no G418 was added to these cultures either. After 8 weeks (20 passages) of culturing either in IL-2 or IL-15, the cells were still viable and replicating in vitro. No ‘‘crisis period’’ in cell growth was noted during this time. The resultant cells were renamed Tf-1b2, for those grown long term in IL-2, and Tf-1b15, for those grown long term in IL-15.
IL-2 and IL-15 Effects on the Tf-1b cell line / 319
Tf-1
Tf-1β
90
90 A
0 100
B
1
10
102
103
104
0 100
1
10
Tf-1β2
103
104
103
104
Tf-1β15
90
90 C
0 100
102
D
1
10
102
103
104
0 100
1
10
102
Figure 4. Flow cytometric analysis of IL-2Rb expression on Tf-1 (A), Tf-1b (B), Tf-1b2 (C), and Tf-1b15 (D) cells. Tf-1, Tf-1b, Tf-1b2 (grown in IL-2 alone) and Tf-1b15 (grown in IL-15 alone) were stained for expression of IL-2Rb using the anti-IL-2Rb antibody, 561, as outlined in Materials and Methods. The shaded areas represent staining with the isotype control for mAb 561, UPC10. The unshaded areas represent staining with mAb 561. As demonstrated by this figure, Tf-1 cells do not express IL-2Rb, as shown previously.10 Tf-1b, Tf-1b2, and Tf-1b15 all express IL-2Rb at roughly the same levels, indicating that selection of Tf-1b cells in IL-2 or IL-15 is sufficient to maintain IL-2Rb expression.
To confirm that culturing in IL-2 or IL-15 selected for IL-2Rb expression, Tf-1, Tf-1b, Tf-1b2, and Tf-1b15 cells were analysed by flow cytometry for their expression of IL-2Rb. As indicated in Figure 4, expression levels of IL-2Rb in Tf-1b, Tf-1b2, and Tf-1b15 are comparable, and the parental line Tf-1, does not express IL-2Rb. Hence, selection by IL-2 or IL-15 was adequate to maintain expression of IL-2Rb.
Long-term culture in IL-15 alters morphology, but not esterase activity Since IL-15 mRNA is expressed in virtually all human tissues (including those derived from bone marrow), it becomes of interest to determine if one of the functions of IL-15 is to support cellular differentiation which may in part be reflected in morphological changes. Samples from Tf-1, Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 were used to determine whether any changes in size, shape or morphological features could be observed, especially in the cells that had been grown in IL-2 or IL-15. This was also
an opportunity to determine if the retroviral infection itself had caused any alterations, and so Tf-1L was included in these studies. No gross alterations in morphology were detected in Tf-1L, Tf-1b, or Tf-1b2 in comparison to Tf-1, or to each other. This indicated that neither the retroviral infection process, nor growth in IL-2 rather than in GM-CSF, had altered the morphology of these cell lines. However, the Tf-1b15 cells were somewhat smaller in size, had enlarged cytoplasm, and smaller nuclei in comparison to Tf-1, Tf-1L, Tf-1b, and Tf-1b2. Figure 5 illustrates the morphological staining of Tf-1b and Tf-1b15 for comparison. These features are consistent with, but do not prove, some maturation or differentiation of the Tf-1b15 cells had occurred. Neither long-term selection in IL-2, nor the retroviral infection process itself, alters the morphology of these cells, although IL-15 seems to have a differentiating effect according to this morphological analysis. The parental line, Tf-1 was originally shown to be highly positive for esterase activity, a characteristic indicative of cells derived from the myeloid lineage.23
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TABLE 1.
Figure 5.
Morphological and cytochemical staining of the cells.
Tf-1, Tf-1L, Tf-1b, Tf-1b2, and Tf-1b15 were used to determine whether any morphological changes in size, shape or morphologic features could be noted, especially in the cells that had been grown in IL-2 or IL-15. Samples from each cell line were Giemsa stained on the same day as described in Materials and Methods. (A) depicts the Giemsa staining on Tf-1b, and (B) depicts the Giemsa staining on Tf-1b15. No gross alterations in morphology were detected in Tf-1L, Tf-1b, or Tf-1b2 in comparison to Tf-1, or to each other. However, the Tf-1b15 cells (lower panel) were somewhat smaller in size and had enlarged cytoplasm, and smaller nuclei in comparison to Tf-1, Tf-1L, Tf-1b, and Tf-1b2.
Samples from each of the cell lines were also tested to determine if they had maintained this myeloid characteristic. All of the cells, including those grown long term in IL-2 or IL-15, maintained their positivity for esterase staining, indicating that long term growth in IL-2 or IL-15, as well as retroviral infection, has not altered this particular characteristic (data not shown).
Long-term culture of Tf-1b cells in IL-15 alters CD marker expression of CD33 , CD38 and HLA-DR Defined steps of differentiation are also characterized by the expression of certain CD markers.28–30 Flow cytometry was used then to determine if long-term culture in IL-2 or IL-15 had caused alterations in CD marker expression on the cells in comparison to the parental line, Tf-1. Descriptions of the CD markers tested here are summarized in Table 1. Since Tf-1 cells have many of the same markers as haematopoietic
Antibodies used in CD marker expression analysis
mAb
Isotype
CD3 CD5 CD7 CD10 CD11b CD14 CD16 CD19 CD33 CD34 CD38 CD45 HLA-DR
IgG1 IgG2a IgG2a IgG2a IgG1 IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
Antigenic specificity T cells T cells/thymocytes T cells/thymocytes/NK cells CALLA/lymphoid progenitors/granulocytes Granulocytes/precursor monocytes/NK cells Monocytes/granulocytes/macrophages NK cells/FcgRIII Pan B cells Monocytes/myeloid precursors/granulocytes Hematopoietic precursors/endothelial cells Plasma cells/thymocytes/activated T cells Leukocyte common antigen (LCA) MHC Class II
stem cells, it was possible that some step towards differentiation may have occurred that could not be detected by the gross cytochemical studies, but that may be reflected in CD marker expression. Each of the cells was subjected to a series of flow cytometric stainings in parallel, and the results of several separate experiments summarized in Table 2. For each cell line and each marker, data are presented as a percentage of positive cells, MFI (mean fluorescence intensity), and the level of expression measured by the fold increase of the MFI of the test antibody (i.e. CD11b, CD33, etc) over the MFI of the isotype control antibody on the same cells. As indicated in this table, the expression of CD11b, CD33, CD34, CD38 and HLA-DR on Tf-1, Tf-1L, Tf-1b, and Tf-1b2 were quite similar. This indicated that the retroviral transfection, selection in G418 and GM-CSF or selection in IL-2 had little effect on the expression of these markers as compared to expression by the parental line, Tf-1. This further supports that retroviral infection, acquisition of new protein expression, and long-term growth in IL-2 does not appreciably alter the original characteristics of the cell, other than the documented functional expression of the IL-2Rb molecule. However, this was not the case with Tf-1b15 cells, which were grown long-term in IL-15. The majority of the CD marker expression profile on Tf-1b15 cells is similar to that on the other cell lines, with the exception of CD33, CD38, and HLA-DR expression which are all decreased on Tf-1b15 cells. CD33, a marker expressed commonly on myeloid precursors and monocytes, but not on mature myelocytes,31,32 is less than half as intense on Tf-1b15 cells as on the other cell lines (MFI = 52 in comparison to MFI = 114 on the parental Tf-1 cell line). CD38, a marker expressed most commonly on lymphoid and myeloid precursor cells and 80 percent of monocytes,33,34 is less than 1/7 as intense on Tf-1b15 cells than it is on the other cell lines (MFI = 13 in comparison to MFI = 96 on Tf-1L). The MHC Class
IL-2 and IL-15 Effects on the Tf-1b cell line / 321
TABLE 2.
CD11b CD33 CD34 CD38 HLA-DR
Flow cytometric analysis of Tf-1-derived cell lines
%Positive MFI Fold Increase % Positive MFI Fold Increase % Positive MFI Fold Increase % Positive MFI Fold Increase % Positive MFI Fold Increase
Tf-1
Tf-1L
Tf-1b
Tf-1b2
Tf-1b15
26 7.98 2.7 100 113.96 31 94 32.23 11 96 124.29 33.8 100 243.85 70
53 13.0 3.7 100 119.73 29.3 97 36.35 12.7 99 96.34 23.6 100 240.55 64.8
40 9.82 3.4 100 148.45 49.9 98 35.64 12.5 99 108.79 36.5 100 282.41 93.2
42 10.32 2.7 100 164.42 35 95 37.89 9.9 99 100.74 21.5 100 248.79 53.5
27 3.87 1 99 52.48 13.7 86 41.16 10.7 78 13.33 3.5 97 125.46 32.7
CD5, CD10, CD14 and CD19 all Q1% Positive. CD3, CD7, CD16 and CD45 all Q5% Positive. Tf-1, Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 cells were stained with antibodies specific for the CD marker listed and described in Table 1. The values listed are given as ‘‘% Positive’’, which is the percentage of live cells in the population that stained positive with each respective antibody, ‘‘MFI’’, which is the Mean Fluorescence Intensity values for each respective antibody, and ‘‘Fold Induction’’ which is the MFI of the antibody staining divided by the MFI of the isotype control staining for that particular antibody.
II molecule, HLA-DR, is also decreased on Tf-1b15 cells in comparison to the other cell lines (MFI = 125 in comparison to MFI = 243 on Tf-1). Although expression of these three markers is co-regulated to some extent, dim or no expression of these markers generally indicates an immature state of the cell.34–41 It is also possible that the general decrease in surface expression of the CD markers on Tf-1b15 is a result of a decline in the robustness of this cell line. Indeed, analysing the percentage of dead cells as determined by uptake of propidium iodide in the flow cytometry analysis reveals that the Tf-1b15 cells are slightly less viable than Tf-1, Tf-1b, or Tf-1b2 (85% viable in comparison to 93–94%). This decrease in viability of Tf-1b15 may also account for its altered morphology.
Elevated concentrations of IL-15 are required to stimulate Tf-1b15 cells in comparison to Tf-1b2 Although it was unclear whether Tf-1b15 had truly undergone differentiation, it was clear that some of its morphological and molecular features were altered in comparison to the original Tf-1 cell line. Tf-1b15 cells show larger cytoplasm, smaller nuclei, smaller overall size, and decreased expression of CD33, CD38, and HLA-DR. Tf-1b2, however, do resemble the original cell line, Tf-1, with the exception that Tf-1b2 cells can proliferate in response to IL-2 (or IL-15), whereas Tf-1 cannot. In the Tf-1 cell line, treatment with DMSO results in partial inhibition of proliferation, although this does not cause differentiation to the next defined step of the process.23 It was possible then that long-term growth in
IL-15 may have altered the ability of Tf-1b15 cells to proliferate in response to cytokines, in comparison to the Tf-1b2 cell line. Tf-1, Tf-1b2 and Tf-1b15 cells were each incubated with a range of concentrations of GM-CSF, IL-2, F42K (an IL-2 variant that does not bind the IL-2Ra)42–44 and IL-15 concentrations in a standard proliferative assay. One representative graph of this data is shown in Figure 6. The concentration of each cytokine required to reach half-maximal proliferation (EC50 values) for these responses are shown in Table 3 and the statistical comparisons are shown in Table 4. Tf-1, Tf-1b2 and Tf-1b15 cells respond to GM-CSF in a dose–response manner, with EC50 values of 1.2, 7 and 2.3 pM, respectively (Table 3). Statistical analysis demonstrates that these EC50 values reflecting the response to GM-CSF by these cell lines are similar. Tf-1 cells (Fig. 6A) do not respond to F42K, IL-2, or IL-15 as they do not express the IL-2Rb subunit, consistent with previously published results.1,45 The remaining proliferation analysis is thus focused on Tf-1b2 and Tf-1b15 cells, which do respond by proliferation to F42K, IL-2, and IL-15. Tf-1b2 cells (Figure 6B) and Tf-1b15 cells (Figure 6C) have similar proliferative responses to IL-2 [EC50 = 66 pM and 128 pM (P = 0.35)] as well as to the IL-2 variant, F42K, which does not bind to the IL-2Ra chain [EC50 = 96 pM and 138 pM (P = 0.072)]. The response of any Tf-1b derivative to IL-2 or F42K is predicted to be similar since Tf-1b cells do not express the IL-2Ra chain.22 Hence, the EC50 values for IL-2 and F42K responses by Tf-1b2 are similar [EC50 = 138 pM and 128 pM (P = 0.88)]. Kit225, which is a human T cell line bearing high-affinity abgc IL-2 receptors, has
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an EC50 value for IL-2 of 2 pM and an EC50 value for F42K of 68 pM (data not shown) which is consistent with the importance of the IL-2Ra chain in the response to IL-2, but not to F42K. These analyses demonstrate that alterations of proliferation by Tf-1b2 and Tf-1b15 to GM-CSF, IL-2, and F42K have not occurred in the process of long-term culture in IL-2 or IL-15 for Tf-1b2 and Tf-1b15 cells. However, the responses of Tf-1b2 and Tf-1b15 to IL-15 are not similar [EC50 = 184 pM vs 336 pM (P = 0.015)]. In fact, the response of Tf-1b15 to IL-15 requires twice as much IL-15 to reach maximal proliferation as is required for Tf-1b2. This result suggests that some subtle alteration of the Tf-1b cell lines’ quantitative ability to proliferate in response to IL-15 has occurred during long-term growth in the presence of IL-15.
DISCUSSION
Figure 6.
Response of Tf-1, Tf-1b2 and Tf-1b15 to cytokines.
These cells were tested for their ability to proliferate in response to GM-CSF (R), IL-2 (Q), F42K (W) (the IL-2 variant that does not bind to IL-2Ra), and IL-15 (T) in a standard 3-day proliferative assay as outlined in Materials and Methods. Total counts of incorporated [3H]thymidine are indicated. As demonstrated here, Tf-1 (A) cells respond by proliferation only to GM-CSF, and not to IL-2, F42K, or IL-15. Tf-1b2 (B) and Tf-1b15 (C) proliferate in response to all four cytokines.
IL-15 is a recently discovered cytokine with many functions similar to that of IL-2.1,3–9 However, many of the comparisons between IL-2 and IL-15 to date have been evaluated in short-term assays. It was our intent to determine if long-term exposure to IL-15 would be similar to long-term exposure of IL-2. We chose to perform these studies on the myelomonocytic Tf-1 cell line which has been characterized extensively.22–24 It has been established that Tf-1 cells express many haematopoietic stem cell markers, respond to haematopoietic stem cell growth factors, and can differentiate through myeloid and erythroid lineages.22–24,26 Because of these characteristics, Tf-1 has been used as a model for stem cell studies, as well as a target for retroviral infection.47 Tf-1b, the cell derivative of Tf-1, was required for these experiments as Tf-1 does not respond to IL-2 or IL-15 in the absence of IL-2Rb expression (Fig. 3).22 Tf-1b cells were generated by infecting Tf-1 cells with a retroviral construct containing the gene for IL-2Rb (Fig. 1) which is successfully transcribed (Fig. 2). Our prior report documents that Tf-1b cells could proliferate in response to IL-2, as they contained both the genes for IL-2Rb and gc which together generate the intermediate-affinity bgc IL-2 receptor.22 However, it was unclear if this single manipulation of Tf-1 cells would also confer IL-15 responsiveness on Tf-1b cells. As indicated in Figure 3, Tf-1b cells can also proliferate in response to IL-15. Since Tf-1b cells could respond by proliferation to IL-2 or IL-15, we then showed that IL-2 and IL-15 could sustain long-term cell growth of Tf-1b cells. Tf-1b2 and Tf-1b15 cell lines were generated from long-term culture in IL-2 or IL-15 respectively, did not experience a culture crisis period, and both lines
IL-2 and IL-15 Effects on the Tf-1b cell line / 323
TABLE 3.
Tf-1 Tf-1b2 TF-1b15
EC50 values (in pM) on Tf-1-derived cell lines in response to cytokines
Mean Number Mean Number Mean Number
GM-CSF
F42K
IL-2
IL-15
1.2 2 0.2 n=3 7.0 2 6.0 n=3 2.3 2 1.9 n=3
N/A N/A 96 2 11 n=4 138 2 13.5 n=3
N/A N/A 65.6 2 6.3 n=9 128 2 54 n=3
N/A N/A 184 2 38 n=7 336 2 28.6 n=3
Mean EC50 values in picomolar (pM) were measured from each of the assays and summarized with standard errors in this table, along with the number of curves used to generate the average of each cytokine required to reach half-maximal proliferation (EC50 ) value for each condition on each cell type.
maintained IL-2Rb expression without G418 selection (Fig. 4). This indicated that both IL-2 and IL-15 could sustain long-term culturing of Tf-1b and that these conditions were adequate to select and maintain expression of IL-2Rb. Tf-1, Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 cells were then assessed for distinctions in morphology, CD marker expression, and function. As evidenced here, infecting Tf-1 cells with a recombinant retrovirus does not grossly alter its original function as measured by proliferation induced by GM-CSF or other growth factors (Fig. 3), their morphology (Fig. 5), their ability to metabolize esterase, nor their surface expression of CD markers indicative of the parental line (Table 2). Conferring a new function upon Tf-1 cells, as evidenced in the Tf-1b cells, also does not grossly alter their original functions other than the IL-2Rb specific activities. Even more intriguing is the finding that if these cells must depend on the expression of the transfected IL-2Rb chain for their survival, they are also unaltered with regard to their original profile. This held true for the Tf-1b2 cells in all aspects studied here, and in most aspects studied here for the Tf-1b15 cells. The morphological staining and CD marker expression analysis did reveal that Tf-1b15 cells, which had been grown long-term in IL-15, do not maintain
the identical original profile of Tf-1 cells and may have undergone some subtle morphological and differentiation changes. Giemsa staining of Tf-1b15 demonstrated that the cells appeared slightly more differentiated than the other cell lines (Tf-1, Tf-1L, Tf-1b and Tf-1b2) as the nuclei were more condensed, the cytoplasm was enlarged, and the overall size was smaller. Since the more defined steps of differentiation are in part characterized by CD marker expression, samples from each cell type were screened for expression of various CD markers (summarized in Table 2). These data revealed that Tf-1b15 had downregulated a fraction of CD33, CD38 and HLA-DR expression in comparison to Tf-1, Tf-1L, Tf-1b and Tf-1b2. What possibilities may account for these subtle morphological and surface marker changes remains unclear, although the decreased viability of Tf-1b15 in comparison to Tf-1, Tf-1b, Tf-1b2 may have been an influence. Differentiation states may also be reflected in functional responses. In order to test this, Tf-1b15 cells were placed in a standard proliferative assay in parallel with Tf-1b2 and Tf-1. The results of these experiments demonstrate that Tf-1b2 and Tf-1b15 do not respond similarly to IL-15. Tf-1b15 cells require higher concentrations of IL-15 to reach the same level of proliferation as do Tf-1b2 cells in response to IL-15.
TABLE 4. Comparative statistical analysis of EC50 calculations from Tf-1-derived cell lines in response to cytokines P value GM-CSF Tf-1 vs Tf-1b2 Tf-1 vs Tf-1b15 Tf-1b2 vs Tf-1b15 F42K Tf-1b2 vs Tf-b15 IL-2 Tf-1b2 vs Tf-1b15 IL-15 Tf-1b2 vs Tf-1b15*
0.045 0.62 0.53 0.072 0.35
P value Tf-1b2 F42K vs IL-2 F42K vs IL-15 IL-2 vs IL-15* Tf-1b15 F42K vs IL-2 F42K vs IL-15* IL-2 vs IL-15*
0.051 0.064 0.019 0.88 0.025 0.042
0.015
P values were determined by t-test with unpooled variance using the Minitab software package. *Indicates P Q 0.05.
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It should also be noted that Tf-1b15 cells also appear to require slightly elevated levels of IL-2 and F42K to achieve 50% maximal proliferation in comparison to the Tf-1b2 cells. However, this apparent increased requirement for IL-2 and F42K does not meet standard criteria for significance (P = 0.072 and 0.35). It is difficult to determine if such subtle differences in EC50 values in vitro are physiologically relevant. The alteration that occurred on Tf-1b15 cells that resulted in an increased EC50 value (decrease in responsiveness to IL-15) in response to IL-15 remains unclear. It is possible that expression levels of IL-15 receptors on Tf-1b15 influence its response to IL-15.20,21 Early characterizations of IL-15Ra indicate that this subunit confers high-affinity IL-15 binding to the IL-15 receptor complex. Although co-expression of IL-15Ra, IL-2Rb, gc results in high-affinity binding of IL-15, it is unclear if this necessarily results in proliferation to IL-15 that is also representative of high affinity. If it is, then this would indicate that Tf-1b15 cells may have decreased some portion of their IL-15Ra expression, as the EC50 value of IL-15 proliferation by Tf-1b15 cells is significantly higher than it is on Tf-1b2. Determining the relative level of IL-15Ra protein on these cell lines would clarify this issue. However, an antibody directed against IL-15Ra is not yet available. An analysis of the IL-15Ra mRNA levels would be possible, but it is unclear if an assessment of IL-15Ra mRNA would reliably translate to functional IL-15Ra protein expression on the cell surface. In conclusion, the ability of the Tf-1b15 cells to survive over an extended period of time with the support of IL-15 alone proves that IL-15 can sustain long-term cell growth and select for expression of transfected IL-2Rb chains. Furthermore, the subtle distinctions between Tf-1b2 and Tf-1b15 suggest that subtle distinctions between IL-2 and IL-15 functions may be found in long-term studies.
MATERIALS AND METHODS Cytokines Human recombinant IL-2 was provided by HoffmanLaRoche, Inc. (Nutley, NJ) and has a specific activity of 15 × 106 U/mg protein so that 1 U/mL = 4.4 pM. GM-CSF was provided by Immunex Corporation (Seattle, WA). F42K was kindly provided by Dr G. Ju (Hoffmann-LaRoche, Nutley, NJ), which is an IL-2 variant that does not interact with the IL-2Ra chain due to a point mutation that substitutes the lysine residue at the 42nd amino acid residue for phenylalanine.42–44 Recombinant human IL-15 was kindly provided by Dr M. Widmer (Immunex Research and Development Corp., Seattle, WA).
Cell lines Tf-1 cells were established from a patient with erythroleukaemia and requires GM-CSF or IL-3 for sustained
CYTOKINE, Vol. 9, No. 5 (May, 1997: 316–327)
proliferation and were kindly provided by T. Kitamura (University of Tokyo). This line has been classified as an immature precursor erythroid cell, although it expresses several myeloid markers and can be induced to differentiate into macrophages.23,24 Tf-1 cells are also brightly positive for CD33, CD34, and CD38, consistent with their designation as haematopoietic precursor cells. The THP-1 cell line has been characterized as a human monocyte line, and is known to be negative for gc mRNA expression.27 PHA blasts were generated by incubating Ficoll-hypaque separated normal donor peripheral blood lymphocytes in RPMI complete medium containing PHA at a concentration of 1 mg/ml.48
Antibodies The reagent to detect IL-2Rb expression by flow cytometry (Fig. 4) was the mAb 561 (IgG2a), a non-inhibitory antibody directed against the human IL-2Rb chain as described by Voss et al.49 The IgG2a isotype control antibody (UPC10) was purchased from Sigma Chemical Co. (St Louis, MO). The antibodies used for the CD marker expression were all obtained from Becton Dickinson.
Cloning strategy for IL-2Rb The cDNA for the IL-2Rb gene (kindly provided by Dr Rich Robb, Oncotherapeutics, NJ) was subcloned into the multiple cloning site of the retrovirus expression vector, pLXSN50 (kindly provided by Dr A. D. Miller, Fred Hutchinson Cancer Center, Seattle, WA) and designated pLXSN/IL-2Rb. In this system, gene expression is controlled by the 5' long terminal repeat of Moloney murine leukaemia virus (MMLV), while the eukaryotic dominant selection marker gene, neomycin phosphotransferase, is driven by the simian virus 40 (SV-40) early promoter.
Generation of recombinant retroviruses and gene transfer The amphotropic retrovirus packaging cell line, PA31751 was transfected by the calcium phosphate method50,52 with either the pLXSN/IL-2Rb construct or the pLXSN vector alone for 3 h at 37°C, then glycerol shocked for 1 min. Transfected cells were stably selected in the presence of G418 sulfate (Geneticin, GIBCO) at 1 mg/ml for 3–4 weeks. Viral supernatants from drug resistant PA317 cells were used to infect the Tf-1 cell line. Tf-1 cells infected with the pLXSN vector alone are designated Tf-1L, and the Tf-1 cells infected with the pLXSN/IL-2Rb vector are designated Tf-1b.
Culture conditions for Tf-1 , Tf-1L, Tf-1b, Tf-1b2 and Tf-1b15 Tf-1 cells were maintained in RPMI 1640 (supplmented with 1 mM HEPES, PenStrep/-glu, and 10% FCS) with 5 ng/ml GM-CSF for culture maintenance. Tf-1L and Tf-1b were also maintained in this same GM-CSF containing media, which also contained 0.5 mg/ml of G418. The Tf-1b2 cell line was generated from the Tf-1b cell line by culturing Tf-1b cells in 100 units/ml of IL-2 (without GM-CSF or G418) for at least 2 months, while the Tf-1b15 cell line was generated from the Tf-1b cells by culturing in 200 ng/ml of IL-15 for 2 months, also without GM-CSF or G418. Preliminary dose titration curves for IL-2 and IL-15 stimulation of Tf-1b had demonstrated that 100 units/ml
IL-2 and IL-15 Effects on the Tf-1b cell line / 325
IL-2 and 200 ng/ml IL-15 stimulate maximal proliferation by Tf-1b (J. de Jong, unpublished observation).
Isolation of RNA and Northern blotting RNA was extracted from each cell type as described by Chomczynski and Sacchi.58 Briefly, each cell pellet was resuspended in equal volumes of GITC solution (4 M guanidine thiocyanate, 25 mM NaCitrate, pH 7.0, 0.5% sarkosyl, 0.1% b-mercaptoethanol, and 0.2 M sodium acetate, pH 4.0) and buffer-saturated phenol. After vortexing, 1 2 volume of chloroform–isoamyl (24:1) was added and the suspension vortexed. Samples were centrifuged (12 000 × g) at 4°C for 30 min. The aqueous phase was transferred to a new tube, and 100% ethanol added to it. The suspension was mixed and placed on ice for 1 h at which time it was centrifuged at 4°C for 30 min at 12 000 × g. The ethanol was decanted and the RNA pellet resuspended in RNase-free water. The concentration of RNA was determined by spectrophotometry. mRNA (10 mg) was fractionated on a formaldehyde denaturing gel, were transferred to a nylon membrane, and probed with 32P-labelled probes. Probes were generated from cloned cDNA, purified through agarose gels and eluted using Geneclean glassmilk. The blots were exposed to film overnight at −80°C.
Immunofluorescent flow cytometry Immunofluorescent staining was performed as previously described.22 Antibodies from Becton Dickinson were used according to manufacturer’s instructions.
In vitro proliferative assay In vitro proliferation in response to various growth factors and cytokines was measured in 3-day in vitro [3 H]thymidine incorporation assays as previously described.22 In Figure 3, GM-CSF, IL-2 and IL-15 were used at a final concentration of 160 ng/ml. In Figure 6, GM-CSF, IL-2, F42K, and IL-15 were diluted from a starting concentration of 160 ng/ml.
Cytochemical staining The morphology of each cell line in comparison to the parental Tf-1 cell line was examined on cytocentrifuged slide preparations stained with Wright stain.54 Cytochemical assays were performed using a combined technique with a-naphthyl acetate as a substrate to detect ‘‘non-specific’’ (monocyte type) esterase and naphthol ASD choroacetate to detect ‘‘specific’’ esterase (granulocyte type) staining.55 Positive a-naphthyl acetate esterase staining is indicated by a red-brown colour, while positive naphthol ASD chloroacetate staining is indicated by a blue colour.
Acknowledgements Special thanks to John Hakimi and Bob Pilson (Hoffman-La Roche, Nutley, NJ) for the initial concept of growing Tf-1b on IL-2 long-term, and to Mike Widmer (Immunex Corp., Seattle, WA) for providing IL-15 as well as helpful discussions and insight. Thanks
to Dr S. Wu, Sally Drew, M. T., and Linda Sebree, H. T., (UW-Madison, Madison, WI) for technical assistance in generating the morphological data, and Ms Kristin Elmer (UW-Madison, Madison, WI) for flow cytometric analysis. Supported by American Cancer Society Grant No. IM-678 and National Institutes of Health Grants No. CA-32685, p30-CA14520-2, CM-87290, and RR03186. T. P. L. was a Fellow of the American Cancer Society.
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