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*
College of Physicians and Surgeons of Columbia University, Department of Pathology, New York, NY 10032; Departments of
Surgery and
Experimental Medicine and Pathology, Università di Roma "La Sapienza," Rome, Italy;
§
Department of Pathology, University of Oklahoma, Health Sciences Center, Oklahoma City, OK; and
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Campus Biomedico, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although immunologic tolerance to allogeneic and xenogeneic tissues has been induced in a variety of experimental models (2, 6), attempts to ablate specifically the immune response to HLA-incompatible transplants in human patients have failed thus far. However, two recent reports have described strategies for in vitro education of regulatory T cells that suppress in a specific manner the direct recognition by CD4+ T cells of MHC class II Ags expressed on allogeneic APCs (7, 8). In one of these studies, regulatory T cells with suppressor activity were generated by stimulation of CD4+ T cells with allogeneic monocytes in the presence of IL-10. These regulatory T cells inhibited specifically the reactivity of CD4+ Th cells through the secretion of IL-10 and TGF-ß (7).
In the other study, suppressor T cells were generated by multiple stimulations of human PBL with allogeneic APCs and shown to display the CD8+CD28- phenotype (8). These CD8+CD28- T cells recognized specifically HLA class I Ags expressed by the stimulatory APCs and suppressed the proliferative response of alloreactive CD4+ T cells against APCs used for priming. The suppressive effect was not mediated by lymphokines but instead required cell-to-cell interaction between CD4+ Th cells, CD8+CD28- T suppressor (Ts)3 cells, and allogeneic APCs expressing Ags against which the T cells were primed. In this system, Ts cells appeared to act by inhibiting costimulatory signals delivered by the allogeneic APCs, such as those provided by CD80/CD86 molecules (8).
This report demonstrates that xenospecific suppressor T cells can be also generated by multiple in vitro stimulations of human T cells with pig PBMCs. The CD8+CD28- population from these T cell lines (TCL) recognizes specifically xenogeneic MHC class I Ags and suppresses the proliferative response of Th cells to MHC class II Ags expressed by the xenogeneic APCs. Xenospecific Ts cells interfere with the expression of CD154, the CD40 ligand, on xenoreactive Th cells, further supporting the concept that the suppressor effect results from inhibition of costimulatory interactions between Th cells and APCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood was obtained from outbred pigs and from Yucatan miniature swine (Sinclair Research Center, Columbia, MO). MHC haplotypes were defined by RFLP using swine histocompatibility leukocyte Ag (SLA) class I- and class II-specific probes (9, 10, 11). For experiments aimed at the identification of MHC Ags recognized by xenospecific Ts cells, blood was obtained from three SLA homozygous lines named W, Z, and Q. Line Q is homozygous for a crossover haplotype that carries the SLA class I genes of strain W and the SLA class II genes of Z (9, 10, 11).
Human specimens
Blood was obtained from healthy blood donors typed for HLA class I and class II Ags by conventional serology and by genomic typing of in vitro amplified DNA with sequence-specific oligonucleotide probes.
Generation of xenoreactive and alloreactive T cell lines
Human and pig PBMCs were separated from buffy coats by Ficoll-Hypaque centrifugation. Responding human PBMCs (1 x 106/ml) were stimulated in 24-well plates with irradiated (1600 r) pig or human PBMCs (1 x 106/ml). Cells were cocultured for 7 days in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM, glutamine and 50 µg/ml gentamicin) (Gibco, Baltimore, MD). Responding cells were restimulated at 7-day intervals in medium containing 10 U/ml rIL-2 (Boehringer Mannheim, Indianapolis, IN).
Cell separation
NK cells were depleted from the alloreactive or xenoreactive TCLs before testing using goat anti-mouse magnetic beads (Dynal, Lake Success, NY) coupled with mAb anti-CD16 and CD56 (Becton Dickinson, San Jose, CA). Suspensions used in blastogenesis assays contained <2% CD16/CD56-positive cells, as indicated by flow cytometry. CD4+ and CD8+ T cells were separated from alloreactive and xenoreactive TCL by negative selection using Dynal CD4 and CD8 magnetic beads. T cell suspensions used as responders in blastogenesis assays were >98% positive for the CD4 and CD45RO markers. CD8+CD28- T cell suspensions were prepared by depletion of CD28+ T cells from purified CD8+ T cell suspensions. For this procedure, goat anti-mouse Dynal beads were coupled with mAb anti-CD28 (Becton Dickinson, San Jose, CA), according to the manufacturer’s instructions. The CD28-coupled beads were washed and incubated at 4 x 107 beads/ml with 1 x 107 CD8+ T cells for 20 min at 4°C, with gentle end-over-end mixing. Rosetted CD8+CD28+ T cells were detached from the beads by overnight incubation at 37°C and used in cell-mediated lysis experiments. Nonrosetted cells were collected, washed three times, and resuspended at 2.5 x 105 cells/ml in complete RPMI 1640 culture medium. The purity of the suspension was monitored by cytofluorographic analysis. The suspension was rerosetted with CD28 beads when necessary, to obtain a population contaminated by <2% CD28+ bright cells.
Proliferation assays
Blastogenesis assays were performed on day 14 or 21, after two or three stimulations, respectively, of human T cells with allogeneic or xenogeneic PBMCs. TCLs were then tested for reactivity to stimulating APCs either as nonfractionated, NK-depleted suspensions (5 x 104cells/well) or as NK-depleted CD4+ T cell suspensions (2.5 x 104 cells/well). Responding cells were stimulated with irradiated allogeneic or xenogeneic PBMCs (5 x 104 cells/well). CD8+CD28- T cells tested for suppressor activity were added to the cultures (1.25 x 104 cells/well) at the initiation of the blastogenesis assay. To study the dose-dependent effect of CD8+CD28- T cells on Th cell proliferation, increasing concentrations of Ts cells were added to parallel cultures as indicated. Cultures were set up in 96-well trays in a total volume of 0.2 ml. In some experiments, murine mAbs to human IL-10 (at 1 µg/ml) or TGF-ß (at 5 µg/ml) from R&D Systems (Minneapolis, MN) were added to the cultures at the initiation of the assay. After 48 h of incubation, the cultures were pulsed with [3H]TdR and harvested 18 h later. [3H]TdR incorporation was determined by scintillation spectrometry in an LK Betaplate counter. Results were expressed as mean counts/min of triplicate reactions. Percent suppression was calculated as 1 - [(cpm in Th + Ts + APC cultures)/(cpm in Th + APC cultures)].
Diffusion chamber experiments
Xenoreactive CD4+ T cells (2.5 x 104 cells/well) and irradiated xenogeneic APCs (5 x 104 cells/well) were cocultured in the bottom compartment of a transwell system (Nalge Nunc International, Roskilde, Denmark). Xenospecific CD8+CD28- T cells (1.25 x 104 cells/well) were added either to the bottom compartment or cocultured with specific pig APCs in the top compartment of the transwell system. After 48 h, the semipermeable membranes were removed, and the proliferative response of Th cells was measured by [3H]TdR incorporation during the last 18 h of culture.
Flow cytometry
Human T cell subsets were defined using mAb CD4, CD8, CD28, CD45RO, and CD16/56 (Becton Dickinson). Cell suspensions were phenotyped before testing with a FACScan flow cytometer instrument (Becton Dickinson) equipped with a 15-mm argon laser. CaliBRITE flow cytometer beads and the FACSComp program (Becton Dickinson) were used for calibration of the cytometer.
To study the expression of CD154 on responding human Th cells, cells were incubated for 6 or 18 h in MLC and then stained with saturating amounts of mAbs CD3-Per CP (peridinin chlorophyll protein-conjugated anti-CD3 mAb), CD154-PE, and CD4-FITC or CD8-FITC (Becton Dickinson).
Cells were analyzed with CellQuest software on a 650 Apple Macintosh computer. Five parameter analysis (forward scatter, side scatter and three fluorescence channels) were used for list mode data analysis. The FL3 channel was used as fluorescence trigger, and FL1 and FL2 were used as analysis parameters.
The cytokine profile of xenoreactive Th and Ts cells was determined by
flow cytometry. CD4+ Th cells and
CD8+CD28- Ts cells were isolated from TCLs and
activated in 4-h cultures with 25 ng/ml PMA and 1 µg/ml ionomycin.
Brefeldin A (Sigma Chemical, St. Louis, MO) was added at 10 µg/ml for
the last 2 h of incubation to inhibit intracellular transport.
Cells were fixed and stained for detection of intracellular cytokines
using mAbs IL-2-FITC, IFN-
FITC, IL-4 PE (Becton Dickinson), and
IL-10 PE (R&D Systems).
Study of apoptosis
The ability of xenoreactive CD8+CD28- human Ts cells to induce apoptosis of pig PBMCs and of xenoreactive human CD4+ Th cells after 4 h of coincubation at 37°C was tested by flow cytometry with the use of annexin V as a marker for apoptotic cells. As positive controls, cells treated with camptothecin (Sigma) were used. The ratio of pig PBMC, human Th cells, and human Ts cells was 1:0.5:0.25, as also used in blastogenesis assays. After incubation, cells were stained with mAb anti-human CD3-PE or CD4-PE, washed, and subsequently stained with annexin V-FITC and propidium iodide (PI) (R&D Systems). To analyze the population of pig PBMCs, log FL2 (CD3-PE) vs side scatter parameters were used to gate out human CD3+ T cells. The percentage of apoptotic pig cells was determined from log FL1 (annexin-FITC) vs FL3 (PI) dot plots. To analyze the population of human CD4+ Th cells undergoing apoptosis, log FL2 (CD4-PE) vs side scatter parameters were used to gate on CD4-positive cells. Log FL1 (annexin V-FITC) vs FL3 (PI) dot plots of the gated population provided the percentage of apoptotic CD4+ Th cells.
Cytotoxicity assays
CD8+CD28- and CD8+CD28+ were isolated from activated CD8+ cells and tested for cytotoxicity in a 51Cr release assay. Target cells were pig PBMCs stimulated with PHA (2 µg/ml) 3 days before the cytotoxicity assay. The cytotoxicity assay was performed with different E:T ratios.
The percent cytotoxicity was calculated as % lysis = 100 x {[experimental release (cpm) - spontaneous release (cpm)]/[maximum release release (cpm) - spontaneous release (cpm)]}.
TCR spectratyping
Total RNA was extracted using Qiagen columns (Qiagen, Valencia, CA) from xenoreactive human CD8+CD28- Ts cells. RNA was reverse transcribed into cDNA in a reaction using Moloney murine leukemia virus reverse transcriptase primed with oligo(dT)18 (Clontech Laboratories, Palo Alto, CA), as recommended by the manufacturer.
Aliquots of the cDNA synthesis reaction were amplified in 50-µl reactions with each of the 24 Vß oligonucleotides (0.5 µM final concentration) and the Cß oligonucleotide (0.5 µM final concentration). Vß and Cß primers were previously described (12, 13). As an internal control for the amount of cDNA used per reaction, a tube containing sense and antisense primers for the first exon of Cß region was included. Two microliters of the Vß-Cß PCR products were subjected to elongation with a fluorophore-labeled Cß or Jß-specific primer (0.5 µM final concentration) (12). The size and fluorescence intensity of labeled runoff products were determined on a 377 DNA sequencer (Perkin-Elmer Applied Biosystem Division, Foster City, CA) and analyzed by ABI PRISM 377 GENESCAN Analysis Program (Perkin-Elmer Applied Biosystem Division) (13).
The relative intensity of each Vß family or Jß-Vß fragment was calculated as the peak area corresponding to each Vß family or Jß-Vß fragment divided by the sum of all area peaks (12).
Statistical analysis
Statistical analysis of the results was performed using BMDP statistical software. Analysis of variance to assess significance of group differences (ANOVA) followed by Tukey’s method for multiple comparison was applied. Correlation coefficients were obtained using Linear Regression Analysis. Student’s t test of significance was also used to access the differences between groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCLs were generated by priming T cells from a healthy volunteer
(SA) with PBMCs from an unrelated blood donor (BM) or with PBMCs from
an outbred pig (pig A). The allospecific TCL (SA-anti-BM) as well
as the xenospecific TCL (SA-anti-pig A) showed higher reactivity
against APCs from the original stimulator after removal of
CD8+CD28- Ts cells from the suspensions (Fig. 1
). Furthermore, when
CD8+CD28- T cells were added to the cultures
at the initiation of the blastogenesis assay, they inhibited
significantly (p < 0.05) the reactivity of
CD4+ Th cells against APCs used for priming. The
suppressive effect was species specific since
CD8+CD28- Ts cells primed to pig APCs did not
inhibit the response of CD4+ Th cells primed to human APCs.
Similarly, Ts cells primed to human APCs did not inhibit the response
of CD4 Th cells primed to pig APCs, indicating that Ts cells recognize
species-specific Ags (Fig. 1). Studies of an additional four
xenospecific and allospecific TCLs yielded similar results.
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, the strength of
the suppressive effect increased with the number of Ts cells,
indicating that suppression was dose dependent (r =
0.85, p < 0.008).
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Table II shows the results of independent
experiments in which TCL generated on three different occasions, by
priming PBMC from individual ES with APCs from strain Q, W, and Z, were
used.
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The reactivity of Th cells primed to APCs of strain Q to the specific
stimulator Q was inhibited efficiently by autologous Ts cells primed to
Q or to W (which shares MHC class I Ags with Q), but not by Ts cells
primed to Z (which is MHC class II identical, yet class I different
from the specific stimulator Q) (p < 0.05).
CD4+ T cell reactivity to strain Z was inhibited only by Ts
cells primed to Z, but not by Ts cells primed to strain Q or W which
are class I different from Z (p < 0.05) (Table II
). This indicates that CD8+CD28- Ts cells
are activated by SLA class I Ags on xenogeneic APCs and inhibit the
response of CD4+ Th cells against class II Ags expressed by
the same stimulating target cells. The MHC class II specificity of Th
cell reactivity was confirmed by the fact that human CD4+ T
cells primed to APCs from a strain Q swine reacted to APCs from strain
Z (class II identical with Q) but not from strain W (class II different
from Q).
To establish whether the suppressive activity of CD8+CD28- T cells requires the direct interaction of these cells with the APCs that trigger Th cell reactivity, cell-mixing experiments were performed. In these experiments, mixtures of APCs from strain Z and W were used to stimulate the reactivity of Th cells from TCL ES-anti-Q. The reactivity of Th cell anti-Q was tested in cultures with or without Ts cells primed to Q, W, or Z. In cultures without Ts cells, Th cells primed to Q proliferated vigorously, consistent with the specific recognition of MHC class II Ags shared by strains Q and Z. This response, however, was not inhibited by Ts cells primed to Q or W, indicating that Ts cells do not inhibit Th cell reactivity to SLA class II Ags unless the SLA class I Ags which they recognize are coexpressed by the same APCs. Indeed inhibition of the response to mixtures of APCs from W and Z was observed only in the presence of Ts cells primed to Z (p < 0.05), further demonstrating that the interaction of Ts cells and Th cells with the same APCs is required for suppression. This finding is consistent with the hypothesis that Ts cells interfere with the delivery of costimulatory signals by APCs to CD4+ Th cells (8).
It is possible, however, that in addition to interacting with APCs, Ts
cells and Th cells also "communicate" with each other, recognizing
TCR determinants or other structures in an MHC-restricted manner (14, 15). To explore this possibility, TCLs were generated by stimulating
PBMCs from two HLA-disparate individuals, AP (HLA-A30, B35,
DRß1*0701, 1301) and MN (HLA-A1, A32, B8, B44, DRß1*0101, 0301)
with APCs from the same outbred pig (pig B). The blastogenic response
of both TCLs (MN-anti-pig B and AP-anti-pig B) to pig APCs was
significantly stronger (p < 0.01) when
CD8+CD28- Ts cells were depleted from the cell
suspensions, indicating that CD4+ Th cell responses were
suppressed by autologous Ts cells (Fig. 2). The reactivity of CD4+ Th
cells from both lines to stimulating APCs was inhibited by
CD8+CD28- Ts cells from either of these lines
(p < 0.01). The difference between the
suppressor activity of Ts cells from MN-anti-pig B and
AP-anti-pig B was not statistically significant. These results were
confirmed in two additional experiments for which other TCL were used.
Hence, no MHC-restricted interaction between Th cells and Ts cells is
required for suppression to occur.
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A)
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B).
Cytofluorographic analysis of Ts cells from three different TCLs
(ES-anti-Q, ES-anti-W and ES-anti-Z) showed that they
produced high levels of IFN- and moderate amounts of IL-2, yet no
detectable levels of IL-4 and IL-10. Th cells from the same cultures
produced high levels of IL-2 and IFN-, moderate amounts of IL-4, and
no IL-10 (Fig. 4).
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We explored the possibility that the suppressive activity of
xenospecific Ts cells may be due to killing of pig APCs. Ts cells from
a human TCL (GC-anti-swine Z), which inhibited by 88% the response
of autologous Th cells to the specific stimulator, were tested for
their ability to induce apoptosis or lysis of pig APC. Flow cytometry
studies of apoptosis were performed by incubating Ts cells for 4 h
with pig APCs in the presence or absence of xenoreactive
CD4+ Th cells and then staining the cultures with annexin
V. The percentage of annexin V-positive APCs was not significantly
different in cultures with or without Ts cells, indicating that no
apoptosis of pig APCs was induced (Fig. 5A). Also, the percentage of
necrotic pig cells stained by PI was not significantly different
in cultures with or without human Ts cells. Furthermore, cell-mediated
lysis experiments in which PHA-activated pig lymphocytes were used as
targets showed lysis when CD8+CD28+ T cells
were used as effectors, but not when CD8+CD28-
T cells from the same line were tested. This demonstrates that Ts cells
do not kill xenogeneic APCs used for priming (Fig. 5B).
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C). Hence, Ts cell suppressive activity is not due to
killing of either Th cells or stimulatory APCs. Expression of CD40 ligand (CD40L) (CD154) on xenoreactive Th cells
We have explored the possibility that Ts cells interfere with the
costimulatory interaction between CD154 on Th cells (CD40L, T-BAM, p39,
or TRAP) and CD40 on xenogeneic APCs. For this, we studied the
expression of CD154 on xenoreactive CD4+ Th cells which
were stimulated with pig APCs in the presence or in the absence of Ts
cells. After 6 h of incubation, cells were stained with mAbs
anti-CD3, CD154, and either CD4 or CD8. Analysis of the results
obtained in independent experiments, using six different TCLs, showed
that the level of CD154 expression on CD4+ Th cells was
significantly higher (p < 0.01) in cultures
containing pig APCs than in cultures without stimulating cells (Fig. 6A and 6B).
However, expression of CD154 on CD4+ Th cells was
drastically reduced in the presence of Ts cells (Fig. 6C),
indicating that Ts cells prevent Ag-induced up-regulation of CD154 on
CD4+ Th cells. There was a statistically significant
difference between the level of CD154 expression on Th cell cultures
with and without Ts cells (p < 0.01) in all
six experiments. The up-regulation of CD154 was Ag specific, requiring
TCR activation, since it did not occur on CD4+ Th cells
challenged with APCs from an SLA class II-different pig (Fig. 6D). The expression of CD154 on xenoreactive
CD4+ Th cells was maximal after 6 h and decreased
significantly after 18 h of incubation with stimulating APCs (data
not shown). No expression of CD154 was observed on Ts cells at any time
point studied. Hence, Ts cell-induced events that result in Th cell
inhibition occur within the first 6 h of stimulation.
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The Vß gene usage of Ts cells from four human anti-pig TCLs
(MN-anti-pig B, ES-anti-Q, ES-anti-W, and ES-anti-Z)
was determined by spectratyping (Figs. 7
and 8). Ts cells from each of these
xenoreactive TCL showed a restricted TCR Vß gene usage. The
side-by-side comparison of the Vß repertoire expressed in
unstimulated and stimulated CD8+CD28- T cells
indicates that after two stimulations with xenogeneic APCs, there was
oligoclonal expansion of Ts cells, as illustrated in Figs. 7 and 8.
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and 8). The relative
intensities of Vß9 family in TCL MN-anti-pig B, ES-anti-Q,
ES-anti-W, ES-anti-Z were 0.22, 0.19, 0.05, and 0.07,
respectively, and the relative intensities of Vß23 were 0.48, 0.38,
0.30, and 0.23, respectively. Vß16 was highly represented in all TCL
generated from individual ES yet was absent in TCL obtained from
another human subject (MN). Vß5 was also expressed by Ts cells from
all TCL derived from responder ES, but with lower intensity than
Vß16. In two of the TCLs from individual ES (ES-anti-pig W and
ES-anti-pig Z), Vß14 was represented with high intensity (Fig. 8, B and C). Other Vß families such as Vß15 were
uniquely represented in one suppressor cell line (ES-anti-pig Z)
(Fig. 8C), while absent from the other lines (ES-anti-Q,
ES-anti-W, and MN-anti-pig B). Analysis of the CDR3 size
distribution revealed a unimodal or bimodal distribution for each Vß
family, except Vß23 that showed a multipeak gaussian-like
distribution (Fig. 9). The oligoclonality
of the Vß repertoire expressed by xenoreactive Ts cells was also
confirmed by analyzing the Jß-Vß fragments of Vß families (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4+ T cells producing TGF-ß, IL-4, and IL-10 were shown
to play an important role in protecting animals from experimental
autoimmune encephalomyelitis after oral feeding with Ag (21). IL-10 was
recently shown to induce in vitro differentiation of regulatory
CD4+ T cells with suppressor activity and inhibit
alloantigen-specific reactivity of CD8+ T cells (7, 23).
MHC class II-restricted CD8+ Ts cells that release IL-4 and
suppress Th1 cell proliferation were described in human leprosy (24, 25). In the mouse model, CD8+ Ts cells were also described,
yet these cells were restricted by nonclassical MHC class I Ags (Qa-1)
expressed by B cells and inhibited Th2 responses by production of IFN-
(18). In other studies, suppression was mediated by Qa-1-restricted
CD8+ T cells, which recognize TCR determinants on the
membrane of CD4+ Th cells (14, 15, 25). The mechanism of
antiidiotypic suppression involved Th cell lysis or induction of Th
cell apoptosis via ligation of Fas (15, 19).
An alternative mechanism of suppression seems to reside in inhibition of TCR-mediated cytotoxicity by CD8+CD28- and CD4+CD28- T cells, which express NK inhibitory receptors (26, 27, 28). The inhibitory effect of these killing-inhibitory receptors results from mobilization of protein tyrosine phosphatases on the cytoplasmic tail of killing-inhibitory receptor molecules (28).
In a previous study, we have shown that human CD8+CD28- Ts cells, which inhibit alloreactive CD4+ Th cells, recognize HLA class I Ags on the surface of allogeneic APCs used for priming (8). The suppression was mediated by down-regulation of CD80 and CD86 expression on the allogeneic APCs and, thus, by impairment of their ability to deliver the costimulatory signals required for the activation of CD4+ Th cells in response to HLA class II alloantigens.
The present study demonstrates for the first time that the xenospecific response of human CD4+ Th cells to pig MHC class II Ags can be also suppressed by CD8+CD28- T cells immunized in vitro against xenogeneic MHC class I Ags. The suppressive effect was not mediated by idiotypic interactions between xenoreactive Ts cells and Th cells, since Th cells primed to APCs from an individual pig were efficiently suppressed not only by autologous but also by allogeneic human Ts cells immunized against the same SLA class I Ags.
The possibility that suppression of CD4+ Th cells was
mediated by lymphokines secreted by CD8+CD28-
Ts cells is also unlikely, since the suppressive activity required the
interaction between Th cells and Ts cells with the same APCs. Thus, Th
cell inhibition occurred only when the immunizing SLA class I and class
II Ags were coexpressed on the membrane of stimulating APCs, but not
when these Ags were expressed by two distinct populations of APCs.
Furthermore, diffusion chamber experiments in which Ts and Th cells
were separated by semipermeable membranes showed that Th cell
reactivity to xenogeneic APCs was not inhibited, indicating that
suppression is not mediated by soluble factors. Cytofluorographic
analysis of CD8+CD28- Ts cells showed that
these cells produce IL-2 and IFN-, but not IL-4 and IL-10. Moreover,
experiments using mAbs against inhibitory cytokines, such as IL-10 and
TGF-ß, excluded their contribution to the suppressor effect. Hence,
neither the production nor the consumption of lymphokines by Ts cells
(22, 24, 29) can explain their inhibitory effect on Th cells in this
system.
In the allogeneic system, we demonstrated that Ts cells interfere with Th cell-induced up-regulation of B7 (CD80, CD86) expression on APC (8). The interaction between CD40 on APC and CD40L (CD154), a transiently expressed CD4+ T cell molecule, is essential for the induction of accessory molecules on APCs, in particular CD80, CD86, and 4–1BB ligand, and for the initiation of Ag-specific T cell reactivity (30, 31, 32, 33, 34, 35). However, blockade of either CD28/B7 or CD40L/CD40 pathways does not inhibit completely T cell-mediated alloimmune responses, indicating that, although interrelated, the CD28 and CD40L pathways serve as independent regulators of T cell responses (36).
We have explored the possibility that Ts cells interfere with the expression of CD40L (CD154) on activated CD4+ Th cells. Cytofluorographic analysis showed that up-regulation of CD154 expression on xenoreactive CD4+Th cells was induced by pig APCs, indicating that human CD154/pig CD40 interaction contributes to the strong proliferative response occurring on recognition by human TCRs of SLA class II Ags. Hence, in the human-pig system, xenoantigen-specific CD4+ Th cell responses involve not only the CD28/B7 and CD2/LFA1 costimulatory pathways, as previously described (37), but also the CD154/CD40 pathway. However, the expression of CD154 on xenoreactive CD4+ Th cells was significantly reduced in the presence of Ts cells. The molecular mechanism of CD154 down-regulation on xenoreactive CD4+ Th cells by Ts cells is currently under investigation. The possibility that Ts cells prevent up-regulation of CD40L on CD4+ T cells by killing the xenogeneic stimulating cells or by inducing Th cell apoptosis was ruled out since no evidence of Ts cell-induced cell death was found by either flow cytometry of 51Cr release studies. Proliferation of CD4+ T cells was not restored in the presence of cells expressing constitutively CD40L, suggesting that costimulation of xenogeneic APCs through the CD40-CD40L pathway is not sufficient to circumvent the suppressive effect of Ts cells (A. I. Colovai, manuscript in preparation). Down-modulation of CD154 by Ts cells may lead, however, to disengagement of Th cells from the targets, preventing full activation and proliferation of these cells.
T cell reactivity to allogeneic and xenogeneic MHC Ags bears resemblance to TCR activation by nominal Ags and pathogens, as it involves recognition of targets expressing novel MHC/peptide complexes. Since the generation of allo-or xenospecific Ts cells in vitro requires multiple rounds of stimulation, it is possible that the chronic exposure to Ag is also required in vivo for the induction of Ts cells. The oligoclonal expansion of a few TCR Vß families observed within the population of xenoreactive Ts cells is reminiscent of the skewed TCR repertoire displayed by T lymphocytes with HLA class I-specific NK-inhibitory receptors (27), a phenomenon suggested to result from chronic antigenic stimulation.
MHC class I-restricted Ts cells may play a physiologic role in regulating the immune response of Th cells against self or nonself peptide/MHC class II complexes. The finding that there is cross-talk between the MHC class I and class II pathways of peptide processing supports the notion that the same APCs present both helper- and suppressor-inducing peptides (38). It is possible that recognition by Ts cells of MHC class I-bound peptides helps control local inflammation caused by Ag-specific Th cells. Identification of suppressor-inducing peptides may be useful for induction of unresponsiveness to auto-, allo-, or xenoantigens. Furthermore, understanding of the mechanism of Ts cell-mediated down-regulation of CD154 expression on activated Th cells may contribute to the development of new immunotherapeutic strategies.
This issue becomes particularly important in view of the recent finding that Th cells condition the APCs to directly stimulate T killer cells by CD154-CD40 signaling, rather than by delivering short range acting lymphokines (39, 40, 41, 42). The emerging picture from our studies is that Ts cells down-regulate the immune response by interfering with CD154-CD40 signaling, thus preventing the up-regulation of costimulatory (B7) molecules on APCs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nicole Suciu-Foca, Department of Pathology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, P&S 14-401, New York, NY 10032. E-mail address:
3 Abbreviations used in this paper: Ts cell, T suppressor cell; PI, propidium iodide; TCL, T cell lines; SLA, swine histocompatibility leukocyte antigen; CD40L, CD40 ligand.
Received for publication March 5, 1998. Accepted for publication July 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes in miniature swine. J. Immunol. 149:841.[Abstract]
ß T-cell repertoire: characteristics of a polyclonal and naive but completely formed repertoire. Blood 91:340.-irradiation. J. Immunol. 156:4476.[Abstract]
chain plays a critical role in antigen-specific suppressor cell function. Proc. Natl. Acad. Sci. USA 88:8700.This article has been cited by other articles:
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  J. Zheng, Y. Liu, G. Qin, P.-L. Chan, H. Mao, K.-T. Lam, D. B. Lewis, Y.-L. Lau, and W. Tu Efficient Induction and Expansion of Human Alloantigen-Specific CD8 Regulatory T Cells from Naive Precursors by CD40-Activated B Cells J. Immunol., September 15, 2009; 183(6): 3742 - 3750. [Abstract] [Full Text] [PDF]
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 R. H. Friedline, D. S. Brown, H. Nguyen, H. Kornfeld, J. Lee, Y. Zhang, M. Appleby, S. D. Der, J. Kang, and C. A. Chambers CD4+ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance J. Exp. Med., February 16, 2009; 206(2): 421 - 434. [Abstract] [Full Text] [PDF]
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 A Tulunay, S Yavuz, H Direskeneli, and E Eksioglu-Demiralp CD8+CD28-, suppressive T cells in systemic lupus erythematosus Lupus, July 1, 2008; 17(7): 630 - 637. [Abstract] [PDF]
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 S. A. Greenberg Proposed immunologic models of the inflammatory myopathies and potential therapeutic implications Neurology, November 20, 2007; 69(21): 2008 - 2019. [Abstract] [Full Text] [PDF]
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 H. Ben-David, A. Sharabi, M. Dayan, M. Sela, and E. Mozes The role of CD8+CD28 regulatory cells in suppressing myasthenia gravis-associated responses by a dual altered peptide ligand PNAS, October 30, 2007; 104(44): 17459 - 17464. [Abstract] [Full Text] [PDF]
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 D. Baeten, S. Louis, C. Braud, C. Braudeau, C. Ballet, F. Moizant, A. Pallier, M. Giral, S. Brouard, and J.-P. Soulillou Phenotypically and Functionally Distinct CD8+ Lymphocyte Populations in Long-Term Drug-Free Tolerance and Chronic Rejection in Human Kidney Graft Recipients J. Am. Soc. Nephrol., January 1, 2006; 17(1): 294 - 304. [Abstract] [Full Text] [PDF]
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 K. Lietz, R. John, E. Burke, M. Schuster, T. B. Rogers, N. Suciu-Foca, D. Mancini, and S. Itescu Immunoglobulin M-to-Immunoglobulin G Anti-Human Leukocyte Antigen Class II Antibody Switching in Cardiac Transplant Recipients Is Associated With an Increased Risk of Cellular Rejection and Coronary Artery Disease Circulation, October 18, 2005; 112(16): 2468 - 2476. [Abstract] [Full Text] [PDF]
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G. Vlad, R. Cortesini, and N. Suciu-Foca License to Heal: Bidirectional Interaction of Antigen-Specific Regulatory T Cells and Tolerogenic APC J. Immunol., May 15, 2005; 174(10): 5907 - 5914. [Abstract] [Full Text] [PDF]
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 J. S. Manavalan, S. Kim-Schulze, L. Scotto, A. J. Naiyer, G. Vlad, P. C. Colombo, C. Marboe, D. Mancini, R. Cortesini, and N. Suciu-Foca Alloantigen specific CD8+CD28- FOXP3+ T suppressor cells induce ILT3+ ILT4+ tolerogenic endothelial cells, inhibiting alloreactivity Int. Immunol., August 1, 2004; 16(8): 1055 - 1068. [Abstract] [Full Text] [PDF]
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 S. Mocellin, E. Wang, M. Panelli, P. Pilati, and F. M. Marincola DNA Array-Based Gene Profiling in Tumor Immunology Clin. Cancer Res., July 15, 2004; 10(14): 4597 - 4606. [Abstract] [Full Text] [PDF]
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A. Ribas, J. A. Wargo, B. Comin-Anduix, S. Sanetti, L. Y. Schumacher, C. McLean, V. B. Dissette, J. A. Glaspy, W. H. McBride, L. H. Butterfield, et al. Enhanced Tumor Responses to Dendritic Cells in the Absence of CD8-Positive Cells J. Immunol., April 15, 2004; 172(8): 4762 - 4769. [Abstract] [Full Text] [PDF]
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 A. Ribas, L. H. Butterfield, J. A. Glaspy, and J. S. Economou Current Developments in Cancer Vaccines and Cellular Immunotherapy J. Clin. Oncol., June 15, 2003; 21(12): 2415 - 2432. [Abstract] [Full Text] [PDF]
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 K. Sato, N. Yamashita, M. Baba, and T. Matsuyama Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells Blood, May 1, 2003; 101(9): 3581 - 3589. [Abstract] [Full Text] [PDF]
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Y. N. Naumov, E. N. Naumova, K. T. Hogan, L. K. Selin, and J. Gorski A Fractal Clonotype Distribution in the CD8+ Memory T Cell Repertoire Could Optimize Potential for Immune Responses J. Immunol., April 15, 2003; 170(8): 3994 - 4001. [Abstract] [Full Text] [PDF]
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W. Chen, M. S. Ford, K. J. Young, M. I. Cybulsky, and L. Zhang Role of Double-Negative Regulatory T Cells in Long-Term Cardiac Xenograft Survival J. Immunol., February 15, 2003; 170(4): 1846 - 1853. [Abstract] [Full Text] [PDF]
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T. J. Dengler, D. R. Johnson, and J. S. Pober Human Vascular Endothelial Cells Stimulate a Lower Frequency of Alloreactive CD8+ Pre-CTL and Induce Less Clonal Expansion than Matching B Lymphoblastoid Cells: Development of a Novel Limiting Dilution Analysis Method Based on CFSE Labeling of Lymphocytes J. Immunol., March 15, 2001; 166(6): 3846 - 3854. [Abstract] [Full Text] [PDF]
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E. J. Wagar, M. A. Cromwell, L. D. Shultz, B. A. Woda, J. L. Sullivan, R. M. Hesselton, and D. L. Greiner Regulation of Human Cell Engraftment and Development of EBV-Related Lymphoproliferative Disorders in Hu-PBL-scid Mice J. Immunol., July 1, 2000; 165(1): 518 - 527. [Abstract] [Full Text] [PDF]
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M. Vukmanovic-Stejic, B. Vyas, P. Gorak-Stolinska, A. Noble, and D. M. Kemeny Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes Blood, January 1, 2000; 95(1): 231 - 240. [Abstract] [Full Text] [PDF]
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J. Li, Z. Liu, S. Jiang, R. Cortesini, S. Lederman, and N. Suciu-Foca T Suppressor Lymphocytes Inhibit NF-{kappa}B-Mediated Transcription of CD86 Gene in APC J. Immunol., December 15, 1999; 163(12): 6386 - 6392. [Abstract] [Full Text] [PDF]
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H. Suzuki, Y. W. Zhou, M. Kato, T. W. Mak, and I. Nakashima Normal Regulatory {alpha}/{beta} T Cells Effectively Eliminate Abnormally Activated T Cells Lacking the Interleukin 2 Receptor {beta} in Vivo J. Exp. Med., December 6, 1999; 190(11): 1561 - 1572. [Abstract] [Full Text] [PDF]
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