0022-1767/87/1386-1813$02.00/0
THEJOURNAL
OF iMMUNOLCSY
Copyright 0 1987 by The AmericanAsmlatlon of lmmunologlsts
Vol. 138. 1813-1816. No. 6. March 15. 1987
Prlnted In U.S.A.
SPECIES-SPECIFICITYOFTCELLSTIMULATINGACTIVITIES
OFIL 2 AND
BSF-1 (IL 4): COMPARISONOF NOF2MAL AND RECOMBINANT,MOUSE AND
HUMAN IL 2 AND BSF-1 (IL 4)
TIM R. MOSMANN," TAKASHI YOKOTA,' ROBERT KASTELEIN,' SANDRA
NAOKO ARAI,' AND YUTAKATAKEBE'
M. ZURAWSKI,'
From the Departmentsof *Immunology and 'Molecular Biology, DNAX Research Instituteof Molecular and CellularBiology,
Palo Alto. CA94304
Mouse and human interleukin2 (IL 2) both cause interleukin 4 (IL 4) has been proposed (10, 11). For the
proliferation of T cells of the homologous species atpurposes of this paper we willrefer tomouse "BSF- 1"and
high efficiency. Human IL 2 also stimulates prolif- its human homologue as mouse and humanIL 4.
eration of mouse T cells atsimilar concentrations,
With the availability of better defined reagents,
we
whereas mouse IL 2 stimulates human T cells at a have reexamined the question of species cross-reactivity
lower (sixfold to 170-fold) efficiency.
In contrast, of mouse and human IL 2, and have also examined the
the T cell stimulating activities of mouse and human
species cross-reactivitiesof the T cell-stimulating activiB cell stimulatory factor 1 (interleukin 4: IL 4) ap- ties of mouse and human IL 4. We find that mouse and
pear tobe species specific over the range of concenhuman IL 2cross-react in both directions,although
IL mouse IL 2 is considerably less active onhuman thanon
trations tested:we detected no activity of mouse
4 on human T cells, or humanIL 4 on mouseT cells. mouse cells. In contrast, neitherof the two IL 4 proteins
causes detectable stimulation of T cells of the non-homologous species.
Mouse (1-3) and human (4,5) interleukin 2 (IL 2) both
stimulate the proliferation of homologous activated T
MATERIALS AND METHODS
lymphocytes. In addition to its activities on human lymCell lines. All cell lines, except where specified, were maintained
phocytes, human IL 2 is also known to stimulate prolif- in RPMI 1640 supplemented with 10%fetal bovine serum (GIBCO,
eration of mouse T lymphocytes (61,whereas the effect Grand Island. New York), 50 pM 2-mercaptoethanol, and homologous
IL 2 for all T cell lines. The JL-Epstein-Barr virus (EBV) human T
of mouse IL 2 on human T cells is not well known.
cell line and EBV-MANN B lymphoblastoid cell line were obtained
Mouse B cell stimulatory factor-1 (BSF-1)' has recently from M. Feldmann.The JL-EBV T cell line wasmaintainedin
been reported to stimulate proliferation or to maintaina medium containing 10%human AB serum (Gemini Biologicals, Tarstate of activation in T cells (7-9). This activity (T cell zana. CA),and wasperiodically stimulated with irradiated(4500 rad)
EBV-MANN cells. The HT2, LB2-1, and MB2-1 mouse T cell lines
growth factor 2; TCGF-2) causes increased survival and were maintained as described (7).
DNA synthesis of T cell lines, but the extentof proliferRecombinant lymphokines. The cDNA clone encoding mouse IL
2 (20) was modified and was expressed in E. coli (21). Purified
ation at saturating levels of BSF-1 does not reach the
recombinant mouse IL 2 was provided by Schering-Plough Corp.
levels attained afterIL 2 stimulation. RecombinantcDNA Recombinant
human IL 2 was expressedin E. coli by using a
clones coding for mouse BSF-1 have recently been iso- secretion vector (R. Kastelein. manuscript in preparation). Recomlated (10, 1 l), and the TCGF activity was confirmed by binant mouse (11) and human IL 4 (19) were expressed in COS
using recombinant BSF-1 (11).This lymphokine is now monkey cells from a n EBV-derived vector (Y. Takebe and N. Arai,
manuscript in preparation].
known to mediate effects on several cell types, namely,
Natural lymphokines. Mouse IL 2 was obtained from supernagrowth factor activity on B cells (12). T cells, and mast tants of Concanavalin A (Con A)-stimulated LB2-1 mouse T cells (7,
cells (7-9); induction of Ia antigens on B cells (13, 14); 20). and purified human IL 2 was obtained from the Biological
Response Modifiers Program, Frederick. MD. Mouse IL 4 was obactivation of resting B cells (15, 16); and enhancement tained from the supernatantsof Con A-stimulated MB2-1 T cells(7).
of IgG 1 (1 7)
and IgE (18)synthesis by lipopolysaccharide
Proliferation assays. Bioassays for IL 2 and TCGF-2 have been
described (7. 22). Mouse assays were carried out on the HT2 T cell
(LPS)-stimulated B lymphocytes.
and the JL-EBV T cell line or phytohemagglutinin (PHA)-stimA human cDNA clone homologous to mouse BSF-1 has line,
ulated human peripheral blood lymphocytes (PBL) were used for
now been isolated, and as with its mouse counterpart, human assays.In all cases, the
MTT colorimetric proliferation assay
the expressed human lymphokine has both B cell growth (22) was used. In this method, surviving and/or proliferating cells
factor (BCGF) and TCGF activities (19). The activity of are detected at the end of the assay by their ability to cleave the
tetrazolium salt MTT and produce a colored product. One unit of IL
both mouse and human "BSF-1" on T cells means that 2 or TCGF-2 activity is defined as the amount of factor that in 0.1
the name"BSF-1" is no longer appropriate, and the nameml stimulates a signal equal to 50% of the maximal signal in the
M T T assay by using 2000 HT2 cells/well for 24 hr for the mouse
assay, and 20,000 human T cells/well for 48 hr for the human
Received for publication July 9, 1986.
assay. Note that theIL 2 and IL 4 units cannotbe directly compared,
Accepted for publication November 20, 1986.
because the maximal signals and the shapesof the titration curves
The costs of publication of this article were defrayed in part by the differ.
payment of page charges. This article must therefore be hereby marked
aduertlsernent in accordance with 18 U.S.C. Section 1734 solely to indiRESULTS
cate this fact.
' Abbreviations used in this paper: BSF-1, B cell stimulatory factor-1:
IL 4. interleukin 4:
3-(4-5-dimethylthiazol-2-yl)-2.5-diphenyltetra- ZL 2 activity on mouse and human T cells. Several
zolium bromide: TCGF-2.T cell growthfactor 2.
mouse and humanIL 2 preparationswere tested for their
"IT.
1813
1814
SPECIES SPECIFICITY OF IL 2 A N D IL 4
effect on mouse and human T cells. Table I shows that particular, this rules out the possibility that the stimuhuman IL 2 was similarly effective in inducing prolifer- lation of the human T cells was indirect, due to producation of both T cell species, whereas mouse IL 2 was tion of human IL 2 by the target T cells in response to
much more effective on mouse T cells than human T some other componentof the samples.
cells. These results were obtained with several preparaMouse and human IL 4 activity on mouse and human
tions of both natural IL 2 and recombinant IL 2 synthe- T cells. Both mouse and human IL 4 stimulate T cells
sized in E. coli. Because the definitions of units are
from their respective species. This stimulation results in
arbitrary and differ in the type of target cell, number of increased survival and thymidine incorporation (9, 19).
cells per well, and time of assay, the two assays cannot butthe level of proliferation induced by saturating
be compared directly. Instead, we have defined the spe- amounts of IL 4 does not reach the levels induced by IL
cies activity ratio as:
2. Figure 1A shows that mouse T cells respond in this
way to normal or recombinant mouse IL 4, but do not
Units (mouse IL 2 assayed on mouse)
respond detectably to recombinant human IL 4. SimiX units (humanon human)
larly, the results in Figure 1B show that human T cells
Units (mouse on human)
responded strongly to human IL 2 and also to human IL
X units (human on mouse)
4 witha lower saturation level, but not at all tomouse IL
This equation appliesonly to results in which
the same 4. Similar results were obtained with either a human T
two assays. cell line JL-EBV (Fig.1)or PHA-stimulated PBL blasts (T.
two IL 2 preparations are assayed in the same
The speciesactivity ratio should notbe affected by vari- Mosmann, unpublished). TableI1 shows the quantitation
ations between assays done on different occasions,
or by of the sensitivity of the two assays with each species of
the absolute titer of either IL 2 preparation. This ratio IL 4. The humanIL 4 preparation wasover 690-fold more
should have a value of 1 if both human and mouse IL 2 active than mouse IL 4 on human T cells,and themouse
have equal activity on mouseand human assaycells. In IL 4 was over 480-fold more active onmouse T cells. The
fact, thespecies activity ratio varied from 6 to 114 (Table species activity ratio (defined above) for IL 4 was greater
I), indicating that one or both of the IL 2 proteins was
considerably more active on homologous assay cells. To
determine which lymphokine showed the species specificity, we also tested preparations of known concentrations of purified recombinant mouse IL 2 and normal
human IL 2 on both mouse and human T cells. Human
IL 2 had a specific activity of 1.4 X lo5U/pg on mouse T
cells, and 5.7 X lo4 U/pg on human T cells, whereas
mouse IL 2 had specific activitiesof 1.6 X lo5 and 3.7 X
1O2 U/pg, respectively. These results indicate
that mouse
IL 2 shows considerable species
specificity, and accounts
for most or all of the magnitude of the species activity
ratio (Table I).
Inhibition of mouse IL 2 activity on human T cells by
0
monoclonal anti-mouse IL 2 antibody. We used the S4B6
16
14
12
10
8
6
4
2
anti-mouse IL 2 antibody (7) to prove conclusively that
Log 2 Dilution
human T cells responded to mouse IL 2. S4B6 is a rat
anti-mouse IL 2 monoclonal antibody that blocks the
biological activity of normal and recombinant mouse IL
2, but not rat or human IL 2. By using both mouse and
human T cells, the stimulation caused by mouse IL 2
could be inhibited by the S4B6 antibody(resultsnot
shown), indicatingthat the human
T cell stimulation was
due only to the mouse IL 2 present in the preparation. In
0.1
TABLE I
Specles spectfictty of mouse and human IL 2
Unlts TCGF/ml
1L 2 Specles
~ssayspedes
Mouse
Mouse
Human
Human
Human
Mouse
8.240
12,671
65,303
415
615
2.057
1.984
1.984
1,664
9.876
62,345
6,228
10.932
158,000
1.042
147
239
103
368
0
O'
rhlL 4
Specles
Human
Activity
Rat'oo
0
Natural IL 2
1,673
1.673
612
16.7
17.4
11.7
5,557
3.751
5.572
20,646
485.566 248.383
7,766
1.135
57,000
139,000
6.4
114.5
13.3
15.5
176.1
RecombinantIL 2
a
I
t
Units (mouse IL 2 assayed on mouse) x units (human on human)
Units (mouse on human) x units (human on mouse)
16
14
12
10
8
6
4
2
Log 2 Dilution
Ftgure 1. Activity of mouse and human IL 4 on mouse T cells. Dilutions of natural and recombinant mouse IL 4, and recombinant human
IL 4 were tested on HT2 mouse T cells (Panel A ) and JL human T cells
(Panel B )by using the MTT assay. A titration of IL 2 is shown in each
case for comparison. Lymphokine preparations were:
(A) recombinant(E.
colt) mouse IL 2 (Panel A ) , or natural human IL 2 (purified)(Panel B);(0)
natural mouseIL 4, supernatant of Con A-induced MB2-1 mouse T cells;
(0)recombinant mouseIL 4 expressed in COS cells; and 0)
recombinant
human IL 4 expressed in COS cells.
SPECIES SPECIFICITY O F IL 2 AND IL 4
TABLE I1
Species specificitu of mouse and human IL 4
1815
cause changes in the species activity ratio (21). Regardless of the precise quantitation, the results presented
Units TCGF/mi
here clearly indicate that both normal and recombinant
Species Acmouse IL 2 do stimulate human T cells, andmouse IL 2
1 ~ 4 s p e ~ i e Mouse
~Mouse
Human
Human
tivlty Ratio"
~
~
~ Mouse
n
~ Human
Human
Mouse
s
~
~
i
~
~
is less activethan human IL 2 on human cells.
>1.5x lo5
Mouse and humanIL 4 mediate very similar effectson
e10
e10
4.166
3.495
>3.1 X lo5
e10
(10
6.290
4,887
the
T cells of their respective species. In both cases, IL 4
e10
>1.7X lo5
e10
7,696
2.235
resultsinstimulation
of T cells, measuredeither by
a See Corresponding footnote
in Table 1.
thymidine incorporation or the MTT colorimetric assay.
The stimulation due to
saturating amounts of IL 4 is
than 100,000 in contrast to IL 2, which gave values
characteristically lower than that due to IL 2, and the
ranging from 6 to 176 (Table I).
slope of the dose-response curve for IL 4 is significantly
Lack of inhibition of IL 4 activity by heterologous IL
more shallow. In addition, both human and mouse IL 4
4. Because mouse and human IL 4 had no measurable
have BCGF activity. In spite of these similar activities in
effect on T cells of the heterologous species, we tested
the species of origin, we have not detected any crossdilutions of heterologous IL 4 for the ability to inhibit the
reaction of mouse or human IL 4 on the heterologous
stimulation of T cells with minimal amounts of homolospecies. In this regard, it is interesting that thetwo IL 4
gous IL 4. No inhibition was seen in either
direction
proteins share reasonable homologyover most of the
(results not shown).In addition, another of the activities
molecule, but thereis a region of approximately 40 amino
of mouse IL 4 is a synergy with IL 3 to induce rapid
acids in the middle of the sequence that has very little
growth of certain mast cell lines (7-9). Human IL 4 also
homology between mouse and humanIL 4 (19).The lack
had no detectableeffect in thismouse assay.
of activity and cross-blocking ability of mouse and human 1L 4 on the heterologous species can be most easily
DISCUSSION
explained by the assumption that neither IL 4 molecule
The ability of human IL 2 to cross-react on mouse T binds to the heterologous receptor with sufficient affincells and effectively stimulate proliferation has been ity.
The resultsdescribed here on thespecies specificity of
known for some time (6,).Early reports suggested that
mouse IL 2 did not cross-react on human
T cells,and this mouse and human IL 2 and IL 4 should be useful in
information has been widely accepted. The results pre- several experimental systems in vitro in which it is deIn
sented here clearly indicate that mouse IL 2 does stimu- sirable to use lymphokines across species barriers.
late humanT cells, although larger amountsof mouse IL addition, the information onthe cross-reactions between
2 are required. Thecurrent availability of purified natural species will be very useful in future studies on the conand recombinant lymphokines has greatly assisted the tributions of various regions of the molecules to the bioanalysis of this effect. Because the mouse and humanIL logical activity.
2 proteins share considerable homology, the cross-reacAcknowledgments. We thank Gerard Zurawski for
tion in both directions is perhaps not surprising, especially because it is already known that one of the major stimulating discussions, Marc Feldmann for providing
sequencedifferences between the two molecules, the the JL-EBV T cell clone, Frank Lee for assistance with
poly-glutamine stretch in mouse IL 2, can be removed transfections, and Holly Cherwinski for valuable technical assistance.
without losing activity (21).
Although both normal and recombinant mouse IL 2
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