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IL2-INDUCTION OF PROLIFERATION IN T CELLS INTERLEUKIN 2 INDUCTION OF PROLIFERATION IN
RESTING T LYMPHOCYTES REQUIRES CONTACT WITH MONOCYTES SUSANA PESOA1, ANDREA MARTIN2, ANA LIA MARIANI3, CARLOS VULLO1, HORACIO SERRA4 Laboratorio de Inmunogenética y Diagnóstico Molecular; 2 Departamento de Bioquímica Clínica, 4 Centro de Química Aplicada, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba; 3 Facultad de Bioquímica, Universidad Nacional de Misiones, Posadas
Resumen La proliferación de linfocitos T en reposo inducida por interleuquina 2 requiere del contacto con monocitos. Los linfocitos T (LiT) humanos en estado de reposo expresan un número significativo de receptores para IL-2 (IL-2R) con afinidad intermedia pero prácticamente no se detectan con baja y alta afinidad. IL-2 solamente induce proliferación de LiT en reposo en presencia de una pequeña proporción de monocitos autólogos. La proliferación requiere del contacto entre estos dos tipos celulares, ya que es inhibida por la presencia de una membrana semi-permeable o por anticuerpos anti-CD11b. Los monocitos no pueden ser reemplazados ni por linfocitos B autólogos ni sustituidos por el agregado de IL-1 recombinante o proveniente de extractos de monocitos activados. La falta de requerimiento antigénico en este proceso fue demostrada utilizando clones de LiT. La proliferación fue inhibida con Acm contra IL-2Ra. Citocinas como IL-4 o IL-6, solas o en presencia de monocitos, fueron incapaces de inducir la proliferación de LiT en reposo. La combinación de IL-2 y monocitos produjo la proliferación de todas las subpoblaciones de LiT (CD4+, CD8+, CD45RA+ y CD45RO+) al igual que clones específicos de antígeno. Esto también indujo la producción de ARNm y expresión de iL-2Ra, la aparición de IL-2R de alta afinidad y la proliferación de un gran número de LiT. Un mecanismo de activación similar ocurre en ratones, ya que la proliferación de células T murinas en reposo es dependiente de la presencia de IL-2 y contacto con monocitos. Esto sugiere que el mecanismo de activación de LiT se encuentra altamente conservado.
Abstract Resting
human T cells are known to express
significant numbers of intermediate
but none or barely detectable low and high affinity IL-2 receptors
(IL-2R). IL-2 alone failed to induce proliferation in these cells.
However, in presence of small proportion of autologous monocytes, as
low as 22 pM, IL-2 induced high levels of proliferation in resting T
cells. Introduction of a semi permeable membrane between the two
cell types or addition of an anti-CD11b mAb inhibited such induction
of proliferation by IL-2. Neither recombinant IL-1 nor
IL-1-containing cell-free extracts from activated monocytes
substituted for intact monocytes. Autologous B cells failed to
replace monocytes. Using antigen-specific cloned human T cells we
have shown a lack of requirement for antigen. The proliferation was
inhibited by anti-IL-2Ra mAb. IL-2 appears to be unique since neither IL-4 nor IL-6, alone or in
presence of monocytes, led to induction of proliferation in resting
T cells. Combination of IL-2 and monocytes induced proliferation in
all T cell subpopulations (CD4+,
CD8+,
CD45RA+
and CD45RO+)
and antigen-specific clones examined. It also induces mRNA and
surface expression of IL-2Ra, appearance of high affinity IL-2R and induction of proliferation in
large proportions of T cells. As in humans, the IL-2 induction of
proliferation in murine resting T cells required contact with
syngeneic monocytes, suggesting that such a mechanism of T cells
activation is highly conserved. Key words:
T lymphocytes, IL-2, IL-2 receptors, monocytes Antigen-specific
clonal expansion of T cells relies on successive and distinct
receptor-ligand interactions. Antigen-presenting cells (APC) provide
T cells not only with an antigen-specific stimulatory signal
(ligation of the TCR) and a series of co-stimulatory signals
(ligation of CD28, LFA1 and many other surface molecules) but also
with polarizing signals (secretion of IL-12, IL-18, PGE2). Those
interactions serve to select the T cells capable of responding to
the antigen and trigger a series of biochemical changes in T cells
leading to induction of IL-2 receptors (IL-2R) and, in some cases,
of IL-2. Binding of IL-2 to its newly acquired receptors on T cells,
leads to blastogenesis and entry of T cells into S phase1. The
IL-2R were originally classified into three isoforms, the high-,
intermediate-, and low-affinity receptors2.
The work done during the last fifteen years has allowed the
molecular characterization of the three distinct subunits (a, b
and g)
that constitute IL-2R complexes. Expression of IL-2Ra
alone or of both IL-2Ra and g
shows low affini-ties (kd~10-8M)
to IL-2 binding, the bg heterodimer complex exhibites intermediate affinities (kd~0.5-1.5x10-9M)
and the abg heterotrimer complex binds to IL-2 at still higher affinity (kd~10-11M)3. IL-2Rg is expressed on all human leukocytes, IL-2Ra and b
are differentially expressed on lymphocytes subpopulation, although
their expressions are enhanced by antigens and mitogens. CD8+ T and NK cells express IL-2Rb,
but little of IL-2Ra, while CD4+ T cells express faint amounts of IL-2Rb4. However,
IL-2 itself is shown to induce proliferation in a sizeable
proportion of resting human T cells, apparently bypassing the
requirement of antigen5-9.
This intriguing phenomenon was first reported several years ago but
its mechanism remained unknown. This is because the essential
requirements of such proliferation have not been clearly established
as several results of the studies of this process are controversial.
Some studies attributed such proliferation to a direct action of
IL-2 on resting T cells through IL-2Rb5,
7 whereas
others6,
8, 9
including our own10
demonstrated a requirement of monocytes. In some of these studies,
IL-1 was shown to replace monocytes in this process. This implied a
direct action of IL-1 on resting T cells. However, resting T cells,
unlike activated cells, express barely detectable IL-1 receptors11. The induction of proliferation is accompanied by the induction of IL-2Ra
and blocked by anti-IL-2Ra mAbs5, 8. However, the requirements for induction of IL-2Ra are unclear and the T cells preparations used in this study proliferated
in response to Con A and hence were not pure. In
order to understand its mechanism and possible physiological
significance, we decided to determine the key requirements of
IL2-induced proliferation in resting T cells. We demonstrate that
IL-2 in the presence of monocytes, but not alone, induces the
expression of both IL-2Ra
and high affinity IL-2 receptors in T cells and then causes their
proliferation. The induction of proliferation occurs irrespective of
lineage and specificity of the T cell. Materials
and Methods Interleukins:
Recombinants human IL-1 were purchased from Collaborative Research
Inc. Recombinant human IL-2 (1 unit/ml = 22 pM) was procured from
the Cetus Corporation. 125I-labelled
recombinant human IL-2 (38 µCi/µg) was bought from the New England
Nuclear. The recombinant human IL-4 and IL-6 were purchased from
Genzyme. Antibodies
and flow cytometric analysis: Purified rabbit anti-human-IL-2 antibody was purchased from Genzyme. An
anti-Tac mAb was a gift from Dr. Waldmann. The anti-DR mAb 7H.312,
anti-human B cell mAb 41H1613,
and mAb 49H.8 that binds to mouse NK cells14 were provided by Dr. Longenecker. The anti-CD45RO mAb UCHL115
and anti-CD45RA mAb 3AC516
were gifts
from Dr. Beverley and Dr. Ledbetter, respectively. The hybridoma
(M7/20) against mouse high affinity IL2 receptor17
was received from Dr. Strom. Hybridomas producing OKT3, OKT4, OKT8,
anti-OKM1, anti-MMA, anti-HLA-DR (L243), anti-IAd
(MK-D6), or anti-mouse monocytes (F4/80) mAb were obtained from
ATCC. Purified goat anti-mouse-Ig was purchased from Cappel
Laboratories. Anti-Tac, anti-OKM1, and 49H.8 mAbs were used after
purification on Protein A-Sepharose columns. For analysis on FACS,
cells were stained with appropriate mouse mAb and then with
FITC-conjugated goat anti-mouse antibody, fixed with 1%
paraformaldehyde and analyzed on a cytometer, as described18. PBMC
and T cell purification:
PBMC were isolated by Ficoll-Hypaque density gradient centrifugation
of freshly drawn heparizinzed blood or buffy coat. For purification
of T cells, the blood sample was first incubated (45 min, 37°C)
with carbonyl iron and then subjected to centrifugation on
Ficoll-Hypaque. The layer of mononuclear cells was recovered and
cells adherent to plastic or Sephadex G10 removed. The non-adherent
cells were incubated (45 min, 4°C) with a mixture of mAbs (anti-DR,
anti-OKM1, anti-Tac, and anti-B cell; unless stated otherwise)
washed and treated (40 min, 37°C) with Low-Tox-H rabbit C
(Cerdarlane). The recovered cells were 99.5% CD2+ and ~95% CD3+ and failed to proliferate in response to Con A (Sigma) or PHA (GIBCO)
unless supplemented with monocytes. These are referred to as
purified T cells. The T cells were treated with OKT8 or OKT4 plus C
to obtain CD4+
or CD8+
T cell subpopulations, respectively. Total or CD4+
T cells were treated with either mAb UCHL1 or mAb 3AC5 and then with
C to obtain CD45RA+
or CD45RO+
T cell subpopulation, respectively18.
In some experiments, the antibody bound cells were removed by
attachment to anti-mouse-IgG antibody-coated magnetic beads
(Dynabeads; Dynal, Great Neck, New York) rather than by lysis. Human
monocytes and B cells: PBMC were suspended (1x107/ml)
in RPMI 1640 supplemented with 10% autologous serum (heat
inactivated), and incubated (90 min, 37°C) in a plastic tissue
culture petri-dish. The dish was subjected to rotary motion and the
non-adherent cells removed by aspiration and, if desired, used for B
isolation. This step was repeated four times. The adherent monocytes
were detached by scraping with a soft plastic tip and consisted of
> 95% alpha-naphthyl acetate esterase positive cells. When
required, monocytes were activated by LPS. For cell-free extracts,
2x106
activated monocytes were lysed in 1 ml PBS by three cycles of quick
freeze and thaw. The lysate was centrifuged at 5.000xg
and the supernatant containing high levels of IL-1 activity19
used as monocyte extract. B cells were isolated from
monocyte-depleted E.– (SRBC
non-rosetting) PBMC by panning on anti-human-Ig antibody-coated
culture plates. Mouse
T cells and monocytes:
Lymph node cells from 6 to 10-week old Balb/c mice were suspended
(x106/ml)
in RPMI 1640 - 10% FCS) and incubated (90 min, 37°C) in a plastic
tissue culture dish. The non-adherent cells were recovered, 5x107
of them resuspended in 1 ml PBS, and passed at flow rate of
pre-swollen Sephadex G10 (2ml) in PBS. The unbound cells were
resuspended (1x107/ml) in 4°C BSS-BSA (1% BSA in basic salt solution) containing anti-IAd
(MK-D6), anti-IL2 receptor (M7/20), anti-monocytes (F4/80),
anti-mouse-Ig, and anti NK cell (49H.8) mAbs. Following incubation
(40 min, 4°C), cells were washed and treated (30 min, 37°C) with
C. The cells recovered were ~98% CD3+
and did not proliferate in response to Con A unless supplemetned
with monocytes. For isolation of monocytes, the spleen cells from
the same or syngeneic mice were suspended (1x107/ml)
in FCS-medium and incubated in a petri-dish, as for isolation of
human monocytes. The non-adherent cells were discarded and adherent
cells rcovered as described above. Cell
culture and DNA synthesis: Unless stated otherwise, cells were cultured in RPMI 1640 - 10%
autologous serum and incubated at 37°C with 7% CO2
and 90% humidity. Routinely, 0.8 to 1x105 T cells were cultured in 0.2 ml medium in flat bottom 96-well plates in
the presence of 5 to 10% autologous irradiated (3.000
rads) monocytes and IL-2. Monocytes isolated in the presence of
autologous serum were used to avoid the introduction of exogenous
antigens in the culture. Rate of DNA synthesis was measured on day 6
during a four-hour pulse with 3H-thymidine
(1µCi/well; 80Ci/mmole; NEN). Then values presented are average of
three or four replicates which, unless indicated otherwise, varied
within a span of 15% of the average. Antigen-specific
T cell clones:
Human monoclonal T cell lines, specific for either purified protein
derivative of tuberculin (PPD) or tetanus toxoid or keyhole limpet
haemocyanin, were derived from a donor who was immunized with first
two antigens. 1x106
PBMC were cultured in the presence of 50µg/ml antigen. On day 6,
80% of the medium was replaced with fresh medium containing the
antigen and 3x105
irradiated (3.000
rads) autologous PBMC. On day 9, IL-2 (final concentration 0.25 nM)
was added. On days 12 and 18, the medium, antigen and irradiated
PBMC were replenished, and on days 15 and 21 IL2 re-added, as
describe above. On day 22, the live cells from culture were
separated by centrifugation on Ficoll-Hypaque and cloned by limiting
dilution, as described20.
The cloned T cells were maintained in culture by weekly feeding. IL-2Ra and high affinity IL2 receptors:
The presence of IL-2 (free or cell-bound) interfered with binding of
both anti-Tac (IL-2Ra) mAb and 125I-IL2.
Therefore, prior to assay, the cells were washed and the
pre-existing IL-2/IL-2 receptor complexes minimized by incubation
(60 min-,
37°C; twice) and washings, as described8.
The IL-2Ra expression was monitored by flow cytometry after staining with anti-Tac
mAb. The high affinity IL2 bindings sites were assayed as described
previously10.
The binding mixture (in 0.5 ml FCS-medium) contained ~4.5x106
cells, 2 mg/ml BSA and 100 pM 125I-IL2,
and was incubated at 37°C for 20 min. The radioactivity bound to
cells was determined in quadruplet after separation from free 125I-IL2 by centrifugation (2 min, 13,000xg)
through a silicon oil mixture. The non-specific 125I-IL2
binding was determined in parallel assays in the presence of a
100-fold excess of unlabelled IL2- and was subtracted to obtain
specific IL-2 binding. Results IL-2
induction of proliferation in resting human T cells requires
monocytes:
Addition of IL-2 to cultures of PBMC induces high levels of
proliferation in apparent absence of any nonself stimulus8, 10. The proliferation can be monitored by DNA synthesis which shows a peak
on day 6. Fig. 1A shows levels of IL-2 induced proliferation as a
function of monocytes depletion. Monocyte-depleted PBMC
(CI-treatment) exhibited lower proliferation in response to IL-2 or
PHA than total PBMC. Further purification of T cells by removal of
Sephadex-adherent cells and of DR+,
MMA+
and B cells eliminated any proliferation to PHA. These cells still
exhibited low but significant proliferation in response to IL-2.
Removal of OKM1+
cells in addition to the above cells completely abolished any
proliferation due to IL-2. Since OKM1 is expressed on both NK cells
and monocytes22,
23 and MMA
only on the latter24,
the proliferation in MMA-OKM1+
cells is likely due to NK cells. The purified T cells did not
proliferate despite the presence of 11 nM IL-2 which is far more
than sufficient for binding and signaling via IL-2Rbg.6, 7. Addition
of autologous monocytes (1-10%) restored the IL2-induction of
proliferation in purified resting T cells (Fig. 1B). Treatment on
monocyte preparations with anti-MMA plus C abolished their ability
to restore IL2-induction of proliferation in resting T cells.
Activated monocytes were only slightly more effective than untreated
monocytes. In contrast to monocytes, addition of similar
concentrations of autologous B cells failed to restore the induction
of proliferation in resting T cells by IL-2. Low
concentrations of IL-2 is sufficient for induction of proliferation
in T cells and its subpopulations: Resting T cells express 400 to 700 IL-2Rb/cell but
undetectable high affinity receptors, as indicated by measurement of
radioactivity and autoradiography (our unpublished results) after
binding of 125I-IL2.
Results in Fig. 2A show that, in presence of autologous monocytes,
as low as 1 unit/ml or 22 pM IL-2 was sufficient to induce
significant proliferation in resting T cells. Addition of anti-IL2
antibody at the beginning of cultures blocked the induction of
proliferation in an antibody-concentration dependent manner. From
these results we conclude that, in presence of monocytes, the
concentrations (such as 22 and 44 pM) of IL-2 partially saturating
for binding to high affinity receptors but not to IL-2Rb
are sufficient to induce proliferation in resting T cells. The
proliferative response rose linearly with increasing concentration
of IL-2, without noticeable change in the slope during transition
from 22 and 44 pM to 660 and 2.200
pM (saturating for binding to both high affinity receptors and IL-2Rb). In
presence of monocytes, low concentrations of IL-2 were able to
induce direct proliferation in CD4+, CD8+, CD45RA+, CD45R0+ T cells (not shown). Thus, induction of proliferation by IL-2 and
monocytes appears be a common property of most T cells and not that
of a unique subset (also see below results of T cell clones). Inhibition
by antibody blocking of CD11b or IL-2Ra:
OKM1
is an epitope on CD11b molecule which is expressed on monocytes but
not T cells22.
The IL-2Ra is barely
detectable on peripheral blood monocytes27
and resting T cell4
but induced in each upon activation. In an initial attempt to
determine the roles for these molecules, the effects of specific
mAbs that bind to them were examined. Fig. 2B shows that the
addition of anti-OKM1 or of anti-IL-2Ra mAb at the
beginning of the cultures inhibited the IL2-induction of
proliferation in resting T cells, in an antibody-concentration
dependent manner. This suggests that both CD11b and IL-2Ra
are parts of the mechanism of monocyte-dependent IL-2-induction of
proliferation in resting T cells. Neither
IL-4 nor IL-6 substitutes for IL-2: In addition to IL-2, IL-4 and IL-6 are T cell growth factors which also
induce proliferation and differentiation in B cells25.
As shown in Table 1, neither IL-4 (200 µ/ml) nor IL-6 (500 µ/ml)
alone induced any significant proliferation in resting T cells.
However, unlike IL-2, these lymphokines also failed to induce
proliferation in
presence of monocytes. The biological activities of IL-4 and IL-6
were confirmed in parallel assays by monitoring the induction of
proliferation and differentiation in B cells (not shown). The
results suggest that IL-2 may be unique in its ability to induce a
monocyte-dependent proliferation in resting T cells. IL-1
and monocyte extracts do not substitute for monocytes:
IL-1 is intricately implicated in T cell activation19, 26. Previous studies have shown that IL-1 or IL-1-like activity produced by
LPS-activated monocytes can replace monocytes in IL-2-induction of
proliferation in T cells. However, since resting T cells express
very low levels of IL-1 receptors, we re-examined the ability of
IL-1 to mediate this process. Results in Table 2 show that
recombinants human IL-1 failed to substitute for monocytes in
IL-2-induction of proliferation in resting T cell. The biological
activity of IL-1 preparations was confirmed in a parallel IL-1
bioassay (Table 2 lower section). Cell-free crude extracts from
LPS-activated monocytes also failed to replace monocytes. This
showed that IL-1, either alone or in the presence of some soluble
factors from activated monocytes, is insufficient to substitute for
intact monocytes in IL-2-induction of proliferation in resting T
cells. IL-2-induction
of proliferation occurs independently of antigen: IL-2
in combination of monocytes induces proliferation in resting T cells
in apparent absence of any nonself stimulus. For a conclusive
demonstration of a lack of requirement for the antigen, we examined
the response of several antigen-specific human T cell clones. The
cells used were specific for PPD or tetanus toxoid or keyhole limpet
haemocyanin. The T cells from each clone were passed through a
Sephadex G10 column to remove residual monocytes and then cultured
in the absence of feeder cells and antigen but in presence of IL-2.
After 7 days, IL-2Ra expression
on cells had declined considerably and they exhibited only low
levels of DNA synthesis in response to IL-2. At this stage, the T
cells were washed and tested for proliferation in response to IL-2
and monocytes. T cell from all the nine clones tested underwent
vigorous proliferation in the presence of IL-2 and monocytes. The
results of one of the PPD-specific clones depicted in Fig. 3 are
representative of others. The PPD-specific T cells failed to
proliferate in response to either antigen or monocytes alone but
showed little proliferation in response to IL-2. The latter was
apparently due to a few residual high affinity IL-2 receptors on T
cells. However, high levels of proliferation were induced by
incubation of T cells in presence of either antigen plus monocytes
or IL-2 plus monocytes, the latter combination causing much grater
proliferation that the former. These results demonstrate that
induction of proliferation by combination of IL-2 and monocytes is
an antigen-independent process and it occurs irrespective of the
clonal specificity of the T cells. Requirement
of contact between T cells and monocytes:
The inability of IL-1 and IL-1-containing monocyte extracts to
substitute for monocytes suggested the possibility for a role of
monocyte surface molecules in IL-2-induction of proliferation in
resting T cells. To test this idea, we introduced a 0.45 µ membrane
between T cells and monocytes. The membrane prevented any contact
between two types of cells but allowed flow of macromolecules
through its pores. As a control for the flow of the molecules across
the membrane, IL-2 was added on one side (top) of the membrane and
both T cells and monocytes on the other (bottom), and proliferation
of T cells monitored after the incubation. Results in Table 3 show
that combination of IL-2 and monocytes induced proliferation in T
cells regardless of whether IL-2 was added to the side of membrane
containing T cells and monocytes or across the membrane. However,
when T cells and monocytes were separated by the membrane, IL-2 was
unable to induce a similar proliferation. These results show a
requirement for contact between T cells and monocytes. Such contact
was necessary for IL-2-induction of proliferation in both CD4+
and CD8+
subpopulations of T cells. Similar results were obtained using
highly purified resting mouse T cells (Table 3). Induction
of IL-2Ra
and high affinity IL-2 receptors:
Previous studies have shown that IL-2 alone was sufficient to induce
IL-2Ra
and proliferation of resting T cells5. Our results so far do not agree with this claim since we have shown
that IL-2 alone is unable to induce IL-2Ra
mRNA in
resting T cells wich are rigorously depleted of monocytes10.
The surface expression of
IL-2Ra, determined
by flow cytometry after staining with an anti-IL-2Ra mAb, indicate that the combination of IL-2 and monocytes induce high
levels of IL-2Ra
in a large proportion (30 to 40%) of T cells two days prior to the
peak of DNA synthesis (not shown). The high affinity IL-2 receptor
was monitored by binding in the presence of 100 pM 125I-IL2.
Fig. 4 shows that a large number of high affinity IL-2 binding sites
were induced in T cells that were cultured in the presence of IL-2
plus monocytes but not in those cultured with either IL-2 or
monocytes. The appearance of high affinity receptors therefore
accompanied the expression of IL-2Ra. Discussion Resting
T cells express significant numbers of IL-2Rb
but barely detectable IL-2Ra5, 6.
Treatment of these cells with IL-2, even at the concentrations that
are partially saturating for binding to IL-2Rb, fails to induce either IL-2Ra10
or proliferation (Fig. 1). As much as 0.22 µM IL-2 was unable to
induce proliferation in these cells (unpublished results). However,
unlike resting T cells, the NK cells present in OKM1+MMA-DR-PBMC
proliferated in direct response to IL-2 (Fig. 1A). This was in
agreement with findings of Ben Aribia et al.6
who showed that, despite the similar numbers of IL-2Rb
in IL-2Ra- NK and resting T cells, IL-2 alone induces proliferation in former but
not in the latter cells. This difference may in part be due to the
internalization of IL-2Rb-bound
to IL-2 in NK cells but not in resting T cells7, 28. In
the presense of a small proportion (1 to 10%) of autologous
monocytes, IL-2 induces the expression of both IL-2Ra
mRNA10
and high affinity IL-2 receptors in resting T cells (Fig. 4).
Subsequent interactions between IL-2 and its high affinity
receptors, as expected28,
leads to induction of proliferation in T cells (Fig. 1B). The
induction is inhibited by anti-IL-2Ra mAbs (5, 8; and Fig. 2B). The overall process requires low concentrations of IL-2
that are partially saturating for binding to the high affinity
receptors but not to IL-2Rb
(Fig. 2A). Since both peripheral blood monocytes27 and resting T cell6
express barely detectable IL-2Ra (hence none or barely detectable high affinity IL-2 receptors), the
target cell for the initial action of IL-2 remains unknown. It is
possible that during IL-2-induction of IL-2Ra
in T cells, the IL-2 first acts on monocytes and induces IL-1 which
then mediates the induction of IL-2Ra in T cells26. A direct action of IL-2 on resting T cells, via a small number of
undetectable high affinity receptors, which fails to induce
proliferation but established communications with monocytes cannot
be ruled out. Neither
recombinant IL-1 nor IL-1-containing cell-free extracts from
activated monocytes replaced monocyte requirement during
IL-2-induction of proliferation in resting T cells (Table 2). This
was not surprising in view of the fact that resting T cells express
only very low levels of IL-1 receptors11.
The observation is, on the other hand, consistent with our finding
that the induction of proliferation required contact between T cells
and monocytes (Table 3). We are in the process of identification of
surface molecules that establish the cell contact and transduce
activation signal in T cells. Since the contact and the induction of
IL-2Ra
or proliferation (Fig. 3) occur independently of antigen, the
precise nature of the T cell contact with monocytes is expected to
be different from that during antigen recognition1.
There are at least two known surface interactions, other than with
CD3/TCR, that lead to a non-clonotypic activation of T cells. One
involves triggering through CD2 by its natural ligand LFA-329 and the other through CD2830.
Some of these molecules are also involved in cell adhesion and
therefore play a role during antigen-dependent T cell activation1. We examined the effect of antibody-blocking of one of the components of
leukocyte adhesion molecule, CD11b, which is expressed on monocytes
but not on T cell. Antibodies against CD11b are known to inhibit
several monocyte functions including adhesion and aggregation but do
not normally affect T cell proliferation31. However, we found that anti-OKM1 mAb that specially binds to CD11b
sharply inhibited IL-2-induction of proliferation in T cells (Fig.
2B). It is possible that CD11b is involved in the contact between
monocytes and resting T cells. The antibody-blocking of CD11b, in
such a case, will be equivalent to having a membrane between T cells
and monocytes, as in Table 3. Alternatively, the binding of
anti-OKM1 to CD11b may generate a signal which inhibits the release
of unidentified molecules from monocytes that mediate the activation
of T cells. An anti-DR framework mAb was also shown to block
IL-2-induction of proliferation in resting T cells32.
We reproduced this result but then found that this mAb is able to
inhibit proliferation directly in both CD4+
and CD8+
T cells (unpublished results). Since induction of proliferation in
both human and mouse T cells (Table 3) required contact with
monocytes, the molecules involved in making the contact may be
related if not conserved. Previous
studies have shown that IL-1 can partially substitute for monocytes8.
We believe this is due to a partial activation of T cell during
their purification or to presence of contaminating monocytes in T
cells preparations. We have observed that IL-1 or IL-1-containing
extracts from activated monocytes, which fail to substitute for
monocytes during IL-2-induction of proliferation in resting T cells,
can partially replace the monocyte requirement IL-2-induction of
proliferation in previously activated quiescent T cells (unpublished
results). This difference is likely due to the presence of residual
IL-1 receptors on some of previously activated T cells but not on
resting T cells. It is possible that, in presence of IL-2, the
contaminating monocytes in T cells may allow a partial induction of
IL-1 receptors on resting T cells. The addition of IL-1 under these
conditions will produce IL-1-dependent effects normally not seen in
monocyte-free resting T cells. The above results nevertheless
suggest a role for IL-1 in IL-2-induction of proliferation in
resting T cells. A plausible mechanism for IL-2-induction of
proliferation may, therefore, first involve induction of IL-1
receptors on resting T cells by combination of IL-2 and monocytes
and induction of IL-1 production by monocytes in response to IL-2.
The former may require contact between T cells and monocytes.
Interactions between IL-1 and its receptor on T cells may lead to
induction of IL-2Ra, as previously shown in cloned T and NK cells lines26,
33. The
association of newly synthesized IL-2Ra
with IL-2Rb (either pre-existing or also newly synthesized) will result in
appearance of high affinity receptors, enabling the T cells to
internalize IL-228
and
proliferate in response to IL-2. The
combination of IL-2 and monocytes is directly able to induce
proliferation in all T cell subpopulations and clones examined (Fig.
2, 3, Table 3). The combination induced IL-2Ra
expression in at least 20% of T cells under conditions in which DNA
synthesis was pharmacologically inhibited (unpublished data). This
therefore represents a major endogenous mechanism of T cell
activation. As indicated by its requirement, it is distinct from
other well-known mechanisms29, 30. Such antigen-independent mechanism of proliferation may be involved in
the formation34 and maintenance35
of T cell repertoire in vivo. The mechanism is operative in both man
and mouse and therefore appears to be conserved. Acknowledgements:
We wish to thank all those listed in Materials and Methods, who
generously provided various antibodies and cells. The excellent
technical assistance of Ms. Nora Gómez and Mr. Pablo Romagnoli is
thankfully acknowledged References
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1999; 11: 173-81. Received: 9-VIII-1999 Accepted: 4-XI-1999 Postal
address: Dr. Horacio M.
Serra, CEQUIMAP, Facultad Ciencias Químicas, Universidad Nacional
de Córdoba, Pabellón Argentina - Ala 1 Ciudad Universitaria, 5000
Córdoba, Argentina Fax: (54-0351) 4334162
e-mail: hserra@bioclin.fcq.unc.edu.ar Fig.
1. IL2-induction of proliferation in resting T cells is
monocyte-dependent.
(A) PBMC were treated, as indicated, to obtain T cell
populations with varying proportions of monocytes and NK cells. 8x104
cells were
cultured in medium containing 10% autologous serum in the presence
of either 1% PHA or 11 nM IL2, and 3H-thymidine incorporation monitored on day 3 or 6, respecti-vely. The
incorporation in cultures without PHA and IL 2 was < 1.000
cpm. (B) 105
purified T cells were cultured with the indicated addition. The
concentrations were 1.1 nM IL2 and 104
autologous irradiated (3.000
rads) either monocytes (with or without activation with LPS) or B
cells per culture. 3H-thymidine
incorporation was monitored on day 6. The incorporations by either
monocytes or B cells, with or without IL2, were < 600 cpm. In
both (A) and (B), the results are typical of several experiments
performed. Fig.
2.– IL2-induction or proliferation in T cells. (A) 105
total T cells were cultured in presence of 104 autologous monocytes (irradiated) and the indicated concentrations of
IL2 (1unit/ml = 22pM). Cultures contained either 2 µl/ml or 4 µl/ml
of anti-IL2 antibody. 1 µl antibody neutralized 10 units of IL2
dissolved in 1 ml FCS-medium. (B) 105 purified T cells were cultured in the presence of 0.22 nM IL2, 104
autologous (irradiated) monocytes and the indicated concentrations
of a purified mAb. The 3H-thymidine
incorporation was monitored on day 6. Fig.
3.– IL2-induction of proliferation in T cells is
antigen-independent. Five days after feeding, live cells from a
culture of PPD-specific human T cell clone were recovered by
centrifugation on Ficoll-Hypaque. The cells were washed, passed
trough a Sephadex G10 column and the non-adhering cells cultured in
FCS-medium in the presence of 1.1 nM IL2. On day 7, cells had lost
over 90% of the responsiveness to IL2. At this stage, cells were
washed and replicates of 8x104
cell cultured in 0.2 ml FCS-medium in the presence of medium alone
(none) or 50 µg/ml PPD or 1.1 nM IL2 or 8x103
autologous (irradiated) monocytes or the indicated combination of
the two. 3H-thymidine
incorporation was measured on day 4. Fig.
4.– Induction of high affinity IL2 receptor. Purified T
cells were cultured in the presence of medium or 1.1 nM IL2 or 10%
autologous monocytes or 1.1 nM IL2 plus 10% autologous monocytes. On
day 6, the high affinity IL2 binding sites were estimated in the
presence of 100 pM 125I-IL2,
as described in Materials and Methods. The results shown are
specific IL2 per 106
cells. TABLE
1.– IL4 OR IL6 fails to substitute for IL2
3H-Thymydine
incorporation (CPM)
Addition
T
CD4+
CD8+
CD45RA+
CD45RO+ Monocytes 470
870
548
428 305 IL2
1,073
3,135
2,034
2,160
1,890 Monocytes
+ IL 2
29,132
49,761
33,112
35,894
27,430 IL4
625
679 478
754 456 Monocytes
+ IL 4
976 632
594
389
403 IL6
295
ND
ND
ND ND Monocytes
+ IL 6
347 ND
ND
ND
ND 8
x 104
total cell or their subpopulation (< 98% cells expressing
subpopulation specific marker) were cultured with or without 104 autologous (irradiated) monocytes. Where indicated, the cultures
contained 1.1 nM IL2 or 200 µ/ml IL4 or 500 µM/ml IL6. The 3H-Thymidine
incorporation was measured on day 6. The incorporation by monocytes
without any lymphokine was < 360 cpm. TABLE
2.– IL1 or monocyte extract fails to replace monocytes
3H-Thimidine
incorporation (CPM) Addition
to T cells Experiment
1
Experiment 2 None
317
257 IL1
(alpha-beta) ND
304 IL2
1,438
1,131 Monocytes
795
504 IL2+Monocytes
57,549
31,422 IL2+IL1
(alpha)
2,684
ND IL2+IL1
(beta) 3,021
ND IL2+IL1
(alpha+beta) 3,521
2,143 IL2+Monocyte
extract 3,478
ND Addition
to indicator T cells
IL2 production (units/ml)
None
0.51 ± 0.07
PHA 0.99 ± 0.10
IL1 (alpha+beta) 0.33 ±
0.12 PHA+IL1 (alpha) 14.75 ± 1.18 PHA+IL1 (beta) 12.96 ± 0.80 PHA+IL1 (alpya-beta) 12.99 ± 2.01 Upper section: replicates
of 105 T cells were cultured with indicated additions. Their concentrations
were 20 µ/ml recombinant IL1 alpha or beta, 20 µ/ml each
recombinant IL1 alpha plus beta, 1.1 nM IL2, 104
autologous (irradiated) monocytes per culture, and cell-free extract
from 2x104
LPS-activated monocytes per culture. The preparation of the extract
is described in Materials and Methods. Lower section: The
IL1 biossay was based upon the ability to complement pHA in inducing
IL2 production from a mouse splenic lymphoma T cell line LBRM-33-1A525. The
IL1 concentration tested was 20 µ/ml. IL2 production was monitored
by CTLL proliferation assay. One unit IL2 led to incorporation of
2x105
cpm of 3H-Thymidine
in 104 CTLL cells
in 16 hours. TABLE
3.– A requirement for T cell contact with monocytes
3H-Thymidine
incorporation
Top Bottom
(CPM ± S.D) –
T cells + IL2
1,437
±
291 –
T cells + monocytes
1,101
±
295 –
T cells + monocytes + IL2
38,371
±
5,262 IL2
T cells + monocytes
29,003
±
3,013 IL2
+ monocytes
T cells
2,132
±
612 IL2
+ T cells
monocytes
3,028
±
851 –
CD4+T
cells + IL2
2,209
±
691 –
CD4+
T cells + IL2
56,363
±
4,422 IL2
CD4+
T cells + monocytes
53,168
±
8,720 IL2
+ monocytes
CD4+
T cells
2,922
±
1,092 –
CD8+
T cells + IL2
2,915
±
943 –
CD8+
T cells + monocytes + IL2
33,956
±
3,509 IL2
CD8+
T cells + monocytes
27,015
±
1,178 IL2
+ monocytes
CD8+
T cells
1,593
±
181 –
mouse T cells
313
±
87 –
mouse T cells + IL2
2,675
±
277 –
mouse T cells + monocytes + IL2
23,100
±
1,814 IL2
mouse T cells + monocytes
17,864
±
2,270 IL2
+ monocytes
mouse T cells
1,758
±
131 IL2
+ mouse T cells
monocytes
1,856
±
156 Cells
were cultured in 24-well plates. Each well was partitioned into top
and bottom compartments by introduction for a 0.45 µ membrane at
its bottom (manufactured by Costar). 10.6
total or (CD4+
or CD8.+) subpopulation of T cells were cultured in 2 ml medium in the presence
or absence of 1.2-1.6 x 10.5 autologous (irradiated) monocytes and/or 1.1 nM IL2, as indicated. On
day 5.5, cells from the two compartments were mixed together and
four replicates of 105 µl culture pulsed for six hours with 1 µCi
of 3H-Thymidine
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