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,
Departments of
*
Pediatrics,
Medicine, and
Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-3 affected to a lesser degree and no effect
observed on IL-4 production. Thus, T cells in vivo can be distinguished
phenotypically by their susceptibility to anergic stimuli. Anergy so
induced affects selected T cell functions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subsequent studies analyzing the biochemical events in T cells exposed to anergic stimuli have suggested that these unresponsive cells maintain persistently high intracellular Ca2+ levels (8). Furthermore, these investigations have demonstrated that increasing the intracellular Ca2+ concentration with ionophores such as ionomycin is sufficient to establish the unresponsive state generated by other anergic stimuli. Other biochemical and molecular events that have been observed in these cells include altered tyrosine phosphorylation patterns of intracellular proteins (9, 10), defective activation of the Ras pathway (11, 12), and altered trans-activating activity of IL-2-specific proteins, such as activating protein-1 (13).
T cell clones have primarily been used in these cited studies and are thus cells that have been preactivated with Ag in vitro and maintained in culture. Little is known regarding the ability of generating this same anergic state in freshly isolated, naive T cells. Anergy induction has been shown to require an initial proliferative response that transforms a naive T cell to a preactivated in vitro memory cell (14, 15, 16). Previous studies have attempted to demonstrate that freshly isolated T cells can be rendered anergic. However, most studies have incubated these cells several days in culture, which may, in fact, change their phenotype (17, 18, 19). Indeed, such cells have been shown to possess a phenotype of an activated state, with low expression of CD62 (MEL-14) (20). Unfortunately, the analysis of Ag responses in naive, freshly isolated populations is limited by the low frequency of T cells specific for any one particular Ag. Ideally, such studies should be performed analyzing a cell population with one receptor specificity. This would allow for a specific Ag response to be evaluated after challenges with various stimuli.
We have proceeded with such studies in mice that possess a
transgene-derived Ag-negative TCR. This allows for the identification
and isolation of a specific T cell population in which the response to
a specific Ag can be monitored. The DO11.10, mouse whose transgenic
receptor is specific for a single polypeptide (323–339) contained
within hen egg OVA presented by I-Ad class II MHC
molecules, was used for these studies (21). To ensure that cells with
alternative TCRs would not affect these results, the transgene was
introduced into a mouse line deficient in the recombination-activating
gene-2 (RAG-2)3 gene (22),
preventing the formation of T cells with endogenous TCRs. Using these
mice, we have demonstrated that 1) naive T cells are resistant to
anergy induction; 2) the in vivo activated T cell is also susceptible
to anergy induction, indicating that this phenotypic change is not due
to the in vitro culture conditions; and 3) ionomycin-induced
unresponsiveness affects the production of IL-2, but other cytokines
expressed, such as Ag-induced IL-3 and IFN-, are affected to a
lesser degree, while IL-4 production remains unaffected.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DO11.10 TCR transgenic mice were described previously (21) and bred at the Washington University School of Medicine (St. Louis, MO) in a pathogen-free facility with monitored surveillance and serologic testing of sentinel mice every 3 mo. The RAG-2-/- mice were a gift from Dr. Y. Shinkai (22) and were used to establish DO11.10 TCR RAG-2-/-, H-2d mice. DO11.10 TCR RAG-2-/- mice were identified as those mice possessing T cells bearing the transgene-derived receptor using the Ab KJ1-26 and lacking cells expressing B cell surface markers as determined by FACS analysis of peripheral blood. Male and female mice used in the experiments discussed below were between 6 and 8 wk of age.
Flow cytometry
Cell surface immunofluorescence analysis of T cell populations was performed as described previously (10). In brief, 1 x 106 cells were incubated with the Ab at saturating concentrations at 4°C for 30 min, washed, and further incubated with FITC-, phycoerythrin-, or tricolor-conjugated appropriate secondary reagents for an additional 30 min. Control samples were prepared in the same manner, but without primary Ab or with an isotype-matched control. Samples were analyzed by a FACScan analyzer (Becton Dickinson, Mountain View, CA) using the CellQuest program. The mAbs used include KJ1-26, specific for an Id expressed on the transgene-derived TCR; DO11.10 (21); and MEL-14 (anti-CD62, PharMingen, San Diego, CA). Lymph node cells from OVA-primed mice were stained with KJ1-26 and biotinylated MEL-14, with a goat anti-mouse FITC secondary Ab (Caltag, San Francisco, CA) for KJ1-26, and with phycoerythrin-avidin secondary staining for MEL-14. CD62 low KL1-26-positive cells were separated by FACSVantge (Becton Dickinson). The resulting populations were >98% pure.
In vitro cultures
In vitro cultured T cell lines were established by culturing 2 x 107 spleen cells from transgenic mice in DMEM with 5% FCS and OVA at a final concentration of 1 mg/ml for 10 days.
Induction of anergy with fixed APC
Preparation of fixed APC was performed under previously described conditions (1). Briefly, BALB/c splenocytes were irradiated at a dose of 2600 rad and were incubated for 1 h on ice in 0.44 ml of 0.9% NaCl containing 75 nM EDCI (Calbiochem, La Jolla). The cells were washed extensively in serum-free medium to stop the coupling reaction. Normal APC consisted of BALB/c splenocytes irradiated at a dose of 2600 rad. T cells (2 x 106 cells) were incubated with EDCI-treated or normal APC (10 x 106 cells) in the presence of 50 µM cOVA323–339 peptide in 24-well plates (Corning, Corning, NY) in a total volume of 2 ml of DMEM with 5% FCS for 16 to 20 h. Cells were harvested and washed with medium three times, and 2 x 104 recovered cells were stimulated with normal APC (5 x 105) alone or with APC plus OVA (1 mg/ml) or PMA (10 ng/ml; Sigma Chemical Co., St. Louis, MO) and ionomycin (1 µM; Calbiochem, La Jolla, CA) in DMEM with 5% FCS a final volume of 200 µl in 96-well round-bottom plates (Costar, Cambridge, MA). After 24 h of culture, supernatants were harvested, and the presence of cytokine was measured.
Induction of anergy with ionomycin
Cells (1 x 106) were incubated with ionomycin (Calbiochem) at a final concentration of 1 µM for 20 h in DMEM with 5% FCS in a volume of 2 ml. Recovered cells were stimulated in the same manner as fixed APC-stimulated T cells for cytokine production.
Proliferation and lymphokine assays
IL-2-dependent T cell proliferation was measured by cultured T
cells (2 x 104) in a final volume of 200 µl of
DMEM with 5% FCS containing IL-2 at a final concentration of 10 U/ml
in 96-well flat-bottom plates (Costar). The cells were incubated at
37°C for 24 h and harvested following a 6-h incubation with
tritiated thymidine (New England Nuclear, Boston, MA) at 1 µCi/well.
IL-2 activity was assayed using the IL-2-dependent CTLL line (23). IL-4
was measured using the IL-4-dependent cell line, 6I4, developed in this
laboratory. IL-3 was assayed using the IL-3-dependent line FDC-P3 (24).
IFN- activity was determined using the inhibition assay with the
indicator line WEHI-279 (25). Units are defined as reciprocal dilution
of culture supernatants that gave 50% of the maximum activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment of T cell clones with stimuli such as anti-CD3 Abs
or Ag presented by chemically fixed APC results in the generation of an
anergic state. T cells in this state are not capable of producing IL-2
upon challenge with Ag and functional APC. The effects of such
treatments on freshly isolated naive T cells have not been clearly
demonstrated. To facilitate this analysis, we used T cells from mice
possessing a transgene-derived TCR (DO11.10) that is specific for
OVA323–339 peptide presented by I-Ad.
Most of this mouse line’s peripheral T cells possess the transgenic
TCR, providing a naive T cell population whose function can be
analyzed. Freshly isolated, naive T cells from DO11.10 transgenic mice
and cell lines generated from in vitro activated T cells from this same
mouse strain were incubated with fixed APC plus peptide for 16 to
20 h. The cells were washed and rested for 3 days. Surviving T
cells were counted and stimulated with Ag and APC. The supernatants
derived from these cultures were then assayed for IL-2 production.
Surprisingly, naive T cells treated with fixed APC were capable of
producing amounts of IL-2 comparable to those produced by untreated T
cells (Fig. 1
A). In
contrast, in vitro cultured cell lines became anergic with exposure to
Ag presented by fixed APC, with a failure to produce IL-2 upon
rechallenge with Ag and functional APC. This experiment was performed
three times, and in each instance, IL-2 production, in response to Ag
stimulation, was maintained in the naive T cell population, while IL-2
production was virtually undetectable in the in vitro activated cell
line after ionomycin treatment. This unresponsive state induced in the
T cell lines was not due to decreased cell viability, as they were
still able to proliferate when the treated cells were incubated with
exogenous IL-2 (Fig. 1B). Naive, untreated T cells
that did not express significant amounts of the IL-2R, as expected, did
not proliferate in response to IL-2 exposure. However, naive T cells
treated with Ag and fixed APC did respond to IL-2 exposure (Fig. 1B). This indicates that the Ag presented by the
fixed APC induced a change in the naive cells, making them responsive
to the cytokine. Thus, naive T cells appear to be fundamentally
different from T cell clones in their responses to Ag presented by
fixed APC and their susceptibility to become anergic by such means.
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Previous studies have demonstrated that anergy induction via
chemically fixed APCs involves a rise in intracellular
Ca2+ (8). Furthermore, this anergic state can be
reproduced by raising intracellular Ca2+ levels in
susceptible T cell clones with ionophores such as ionomycin. However,
used in the context of additional stimuli, Ca2+ influx can
result in distinctly different responses in the cell. This is
illustrated in Table I. In vitro cultured
cell lines derived from TCR transgenic mice were stimulated with PMA,
ionomycin, and the combination of PMA and ionomycin. Alone, PMA and
ionomycin were insufficient to elicit significant IL-2 production,
while together they elicited a vigorous response. However, ionomycin
and PMA had drastically different effects on the cell when the treated
cells were subsequently challenged with Ag and appropriate APC. The
ionomycin-treated T cell line failed to generate measurable IL-2 in
response to Ag stimulation. PMA, in contrast, had no significant effect
on the capacity of T cells to respond to the secondary stimulation.
Thus, although both PMA and ionomycin failed to elicit any IL-2
production from the T cells when used alone, ionomycin, but not PMA,
induced a unique change in the state of the cell to make it
unresponsive to antigenic stimulation. This effect, however, was
dependent upon the state of the cell, as naive freshly isolated T cells
maintained their ability to produce IL-2 with Ag stimulation even after
ionomycin treatment (Table I). Naive cells, however, still appeared to
be influenced by ionomycin, as the magnitude of the secondary response
was greater than that of the response of the untreated population. This
indicates that ionomycin induces a distinctly different state in the
naive cell from that in the in vitro cultured T cell. Ionomycin was
used in all subsequent experiments to further characterize the
difference between naive and activated T cells. This method is cell
density independent and thus excludes the possibility that the
resistance of the naive cells to the fixed APC treatments is due to
differences in engagement at the TCR level. The differences observed in
this experimental system are more likely due to inherent differences in
the T cell populations in their susceptibility to anergy induction.
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Most studies examining the effects of anergy-inducing stimuli have used T cell lines maintained in culture for at least several days. Since freshly isolated T cells are resistant to anergy induction, one explanation is that the maintenance of cells in culture renders them susceptible. Alternatively, if ionomycin sensitivity is a property acquired with T cell activation, primed T cells isolated directly from the immunized host should possess the same sensitivity to ionomycin as that observed in the T cell line. To address the susceptibility of in vivo primed T cells to anergy induction, it was necessary to acquire a means by which uniform cell populations could be isolated from the host in both primed and unprimed states. Ideally, this would entail the identification of a cell population with a single TCR and would exclude those cells that may possess alternative receptors. To accommodate these needs, DO11.10 mice were bred with mice who were homozygous for the disruption of the RAG-2 gene, (RAG-2-/-) (22). This prevents the rearrangement of endogenous receptor genes and assures that all the T cells in the periphery possess a single, uniform TCR. These mice were used in the subsequent analysis.
DO 11.10 TCR RAG-2-/- transgenic mice were immunized in
the footpad with OVA (100 µg) in CFA. The T cells from the draining
lymph nodes were isolated 7 days after immunization. The in vivo primed
T cell population was analyzed by surface immunofluorescence and
compared with cells from nonimmunized mice. Lymph node cells from naive
mice were stained with an anti-Id Ab (KJ1-26) and counterstained
with an anti-CD62 Ab (MEL-14). As shown in Figure 2, the vast majority of TCR-positive
cells in the naive mouse express high levels of CD62 (L-selectin). Upon
priming with Ag, the number of cells in draining lymph node as well as
in spleen increased significantly (Table II), and the expansion of Ag-reactive T
cells accounted for this increase as the cells expressed the
transgene-derived receptor, as measured by the Ab KJ1-26 (Fig. 2). A
significant fraction of T cells from immunized mice were now low for
CD62 expression, consistent with the findings of other studies
characterizing the surface phenotype of activated T cells (26, 27).
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). These cells were still viable and
capable of IL-2 production upon stimulation with PMA and ionomycin.
Thus, the unresponsive state elicited by ionomycin was not due a toxic
effect on the cells. When the in vivo primed, low CD62 T cells were
exposed to ionomycin and tested for their Ag reactivity, their capacity
to produce IL-2 in response to Ag was greatly reduced compared with the
that of the same T cell population not exposed to ionomycin. This
experiment was performed three times, and in each case, ionomycin
treatments of the in vivo primed, low CD62 population reduced the
amount of IL-2 production in response to Ag stimulation by at least 1
order of magnitude. This effect was simply not due to the effects of
the Abs used to isolate the cells, since similar treatments of the
naive population failed to affect their resistance to ionomycin. Thus,
in vivo activated T cells are sensitive to ionomycin-induced anergy
induction in a fashion analogous to that of the in vitro activated T
cell line.
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Further analysis was performed to determine whether other cytokine
production pathways were affected by ionomycin in the same fashion as
IL-2. In vitro cultured T cells were incubated with ionomycin for
16 h and stimulated with Ag and APC. Culture supernatants were
tested for IL-2, IL-3, IL-4, and IFN-. Similar to the previous
assay, ionomycin-treated cells produced no IL-2 upon restimulation with
Ag and APC. However, IL-4 production remained comparable to that of
untreated T cells. In contrast, the amounts of IFN- and IL-3
produced by ionomycin-treated cells were significantly lower than those
produced by nontreated cells, but were still readily detectable (Table IV). This experiment was performed three
times. In each instance, IL-4 production after Ag stimulation did not
decrease in response to ionomycin treatment, while the effects of
ionomycin on IFN- and IL-3 expression failed to reduce the response
more than 3- to 10-fold, with detectable activity remaining. This is in
contrast to IL-2 production, for which ionomycin treatments resulted in
no measurable biologic activity in the in vitro cell line after Ag
stimulation. Thus, each of these cytokines must be regulated in a
different fashion than IL-2, and anergy induction seems to be specific
for IL-2 and IL-2-dependent proliferation of T cells in vitro.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data presented above indicate that naive cells can be converted into ionomycin-susceptible cells with conventional immunization. This was demonstrated in TCR transgenic mice on a RAG-2-/- background in which all the T cells expressed only one receptor. After priming, this uniform T cell population expanded in significant numbers in response to the Ag. A large fraction of cells expressed low levels of CD62 (L-selectin), but a significant number of T cells remained high in CD62 expression. Since the low CD62 population alone cannot account for the entire increase in the number of T cells, some T cells were stimulated by Ag and maintained high CD62 expression. We observed similar heterogeneity for IL-2R and CD44 (pgp-1) expression (data not shown). It is not clear at present whether this heterogeneity represents a distinct T cell activation state. However, it is likely that this heterogeneity represents different stages of the T cell as it undergoes Ag-driven proliferation. Alternatively, the increase in the total number of high CD62 cells could be due to the migration of T cells into the lymphoid tissues that have not yet encountered Ag and lowered their expression of CD62. Thus, to restrict our analysis to an Ag-primed population, we have used exclusively low CD62 T cells and have demonstrated that the conversion from a naive to a primed T cell renders the cell susceptible to ionomycin-induced anergy. The relationship among surface phenotypic changes, cell cycle regulation, and susceptibility to anergy induction in primed mice requires further investigation to help elucidate a biochemical basis for the anergy susceptibility.
Other investigators have noted previously that naive cells appeared to be uninfluenced by the effects of fixed APC treatments (28). These studies were unable to clearly demonstrate differences between primed and naive cells in IL-2 production following exposure to fixed APCs and concluded that the means by which anergy is induced may only affect certain T cell subtypes. Our assay systems, however, were able to detect clear differences between naive and primed T cells in their susceptibility to anergy induction. By using cell sorting for a low CD62 population to isolate in vivo primed T cells and by allowing the in vitro activated cell line to incubate for a sufficient amount of time in culture, we were able to analyze more uniform populations that were devoid of cells that may be at different stages of activation and thus may possess different susceptibilities to anergic stimuli. In addition, the use of ionomycin as the means of inducing anergy ensured that the T cells assayed were uniformly treated so that the response assessed was not dependent upon the degree of engagement of the cell’s receptor with a fixed APC, which may fail to induce anergy. Our data suggest that selecting for cells expressing low CD62 expression is a means of identifying a T cell population that is sensitive to the effects of anergic stimuli.
Ionomycin treatments have different effects on different cytokine
production pathways. IL-2 production is dramatically reduced with
subsequent Ag stimulation, while IL-4, in contrast, appears to be
unaffected. Ionomycin reduced the responses of IFN- and IL-3
production to Ag, but its effects on these two cytokines were
incomplete. The T cell lines used in these experiments were only
stimulated for 7 days and were thus heterogeneous. Cells within this
mixture predominantly consist of CD4 Th cells expressing Th1 (IFN-)
or Th2 (IL-4) cytokines at various stages of differentiation. Since
IL-2 can be generated by cells that produce either Th1 or Th2 (29)
cytokines, ionomycin must have influences on more than one population,
since no significant IL-2 activity can be measured following such
treatments.
One possibility accounting for the different effects of ionomycin on the expression of different cytokines is that ionomycin may render activated T cells unresponsive to Ag-induced protein kinase C (PKC) activation. Previous investigations have demonstrated the requirement for PKC activation for IL-2 expression, while other cytokines, such as IL-4, lack this dependence (30, 31, 32) This lack of kinase activation is not irreversible, since PMA and ionomycin can still induce IL-2 from anergized T cells. It is unclear whether a cytokine’s dependence on PKC activation directly determines its sensitivity to anergic stimuli. The specific biochemical events required for anergy induction remain to be defined and thus may involve additional pathways distinct from PKC activation and Ca2+ influx (33) Despite this possibility, our investigations have demonstrated that T cells can possess different susceptibilities to anergy induction, and the use of T cells that resist anergy induction would provide a useful tool for further investigation of the biochemical nature of this process.
Our findings presented in this report do not address the critical steps of tolerance induction in vivo or whether prior activation is required to induce tolerance in vivo. Furthermore, the physiologic role of anergy defined by a lack of IL-2-producing capacity in vivo is not clearly understood. The observations identify two distinct states for the T cell: the naive, anergy-resistant state and the primed, anergy-susceptible state. Further characterization of the two states and the molecular event that maintains each state will provide a better understanding of immune regulation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert J. Hayashi, Department of Pediatrics, Washington University School of Medicine, Box 8116, 660 South Euclid, St. Louis, MO 63110.
3 Abbreviations used in this paper: RAG-2, recombination-activating gene-2; PKC, protein kinase C.
Received for publication June 17, 1997. Accepted for publication September 12, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and lack of zap 70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
. J. Immunol. 139:767.[Abstract]
. J. Exp. Med. 174:547.
(PKC) and 14–3–3
in T cells: 14–3–3 overexpression results in inhibition of PKC translocation and function. Mol. Cell. Biol. 16:5782.[Abstract]
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