|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-05-1513.
TRANSPLANTATION
From the Department of Internal Medicine, University of
Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City; and
the Department of Veterans Affairs Medical Center, Iowa City, IA.
Preconditioning with the nonmyeloablative regimen total lymphoid
irradiation (TLI) before hematopoietic cell transplantation facilitates
the establishment of mixed chimerism and protects against
graft-versus-host disease. We reported that the development of mixed
chimerism requires interleukin (IL)-4 and is associated with increased
host anti-donor TH2 cells, but the effect of TLI on the
differentiation of immunocompetent donor cells has not been
investigated. To examine the extent to which TLI preconditioning influences donor T cells, we measured responses of transgenic CD4+ cells specific for ovalbumin peptide (OVA-Tg)
following in vivo and in vitro antigen stimulation in a
TLI-preconditioned environment. OVA-Tg cells that were adoptively
transferred into TLI-preconditioned mice that express cross-reactive
antigens produced more IL-4 and less interferon- Total lymphoid irradiation (TLI)-preconditioned
mice accept major histocompatibility complex (MHC)-disparate
hematopoietic cell or bone marrow grafts if cells are given shortly
after the completion of TLI treatment,1-3 but not at all
if cell transfer is delayed beyond 7 days. When undergoing
transplantation within this window, mice develop a state of
bidirectional tolerance, which permits the engraftment of donor cells
leading to stable mixed chimerism and inhibits the development of
graft-versus-host disease (GVHD).4,5 This bidirectional
tolerance can be achieved and maintained in the long term without the
use of immunosuppressive therapy. Because TLI preconditioning combined
with hematopoietic cell transplantation offers the potential for
long-lived donor-specific tolerance and protection from GVHD without
global immunosuppression, much interest has centered on understanding
the mechanisms by which TLI induces acquired tolerance.
The development of mixed chimerism following hematopoietic cell
transfer into TLI-preconditioned mice requires interleukin (IL)-4;
treatment with anti-IL-4 at the time of hematopoietic cell transfer
significantly reduces the incidence of mixed chimerism in
TLI-preconditioned mice.6 IL-4 facilitates donor cell
engraftment and the development of mixed chimerism by promoting the
differentiation of host anti-donor TH2 CD4+
cells and preventing the differentiation of host anti-donor TC1 CD8
cells.6 The protection from GVHD conferred by TLI
preconditioning also requires IL-4.7 Whereas no
TLI-preconditioned mice die of GVHD following infusion of allogeneic
donor hematopoietic cells, all of the TLI-preconditioned mice die of
GVHD when IL-4 is completely eliminated through the use of IL-4
knockout host and donor mice.7
The development of GVHD is related to the presence of immunocompetent
donor CD4+ T cells in the inoculum,4,8 which
presumably give rise to cells reactive against host antigens. However,
the extent to which TLI preconditioning affects the activation and
differentiation of immunocompetent donor T cells following encounter
with their cognate antigen has not been extensively studied. We
reasoned that TLI preconditioning protects against the development of
GVHD in part by blocking the responses of "donor"
CD4+ cells that are activated within the TLI
environment. Indeed, we noted that normal CD4+ T cells that
were stimulated with anti-CD3 in the presence of spleen cells from
TLI-preconditioned mice proliferated less and produced less IL-2
compared with control stimulated cells.9,10
To begin characterizing how TLI preconditioning affects the
antigen-specific immune responses of immunocompetent CD4+ T
cells, we examined CD4+ cells from the DO11.l0 transgenic
mouse following antigen stimulation in vivo and in vitro within a
TLI-preconditioned environment. DO11.10 transgenic CD4+
cells (OVA-Tg) express a clonotypic T-cell receptor (TCR) reactive against OVA peptide11 and can be distinguished from
nontransgenic CD4+ cells using the monoclonal antibody
(mAb) KJ1-26.12 OVA-Tg have been used to examine
CD4+ T-cell differentiation.13,14 The present
study shows that when OVA-Tg are primed either in vivo in a
TLI-preconditioned mouse or in vitro with cells from a
TLI-preconditioned mouse, they differentiate preferentially into
IL-4-secreting OVA-Tg CD4+ cells. Host CD4+
cells from TLI-preconditioned mice that constitutively secrete IL-4
promote the development of these OVA-Tg TH2 cells by
providing endogenous IL-4 at the time of primary activation. The
results of this study suggest that upon encountering antigen within
TLI-treated mice, the activation and differentiation of immunocompetent
CD4+ T cells are redirected away from the TH1
pathway and toward the TH2 pathway.
Mice and TLI treatment
Groups of BALB/c and CB6F1 mice were given TLI treatment as
described previously.16 TLI treatment was started when
mice reached 12 to 15 weeks of age and weighed approximately 25 to 30 g. Radiation treatment was given in daily 275-rad fractions, 5 days per week, for a total of 17 doses. For each radiation treatment, the mice were anesthetized and placed individually into a jig that
allowed exposure of the peripheral lymphoid tissue while shielding
vital organs. The accumulated radiation dose was 4675 rad.
In vivo priming of OVA-Tg cells using adoptive transfer
Cell preparation, antigen stimulation, and immunologic assays Spleens were harvested from the following mice: adoptive transfer animals described above; TLI-preconditioned BALB/c wild-type (WT), BALB/c IL-4 /, and
CB6F1 mice; age-matched nonirradiated BALB/c WT and
CB6F1 mice; and DO11.10 mice. Single-cell suspensions were
prepared, and cells were resuspended at the appropriate concentration
in complete medium (RPMI supplemented with 10% fetal calf serum, HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid],
2-mercaptoethanol, L-glutamine, penicillin/streptomycin),
using trypan blue dye to exclude dead cells.
T-cell-depleted control antigen-presenting cells (APCs) were generated by passage through a CS depletion column (Miltenyi Biotec, Auburn, CA) using Thy1.2-conjugated microbeads (Miltenyi Biotec); the cells contained less than 5% T cells by FACS analysis. To isolate naive OVA-Tg CD4+ cells, we subjected splenocytes from DO11.10 mice to a 2-step magnetic-bead separation using the anti-FITC MultiSort Kit (Miltenyi Biotec) and anti-CD4-FITC (RM4-4; Pharmingen), followed by anti-CD62L microbeads (Miltenyi Biotec), according to the manufacturer's specifications. The preparations contained more than 95% CD4 cells, and more than 90% of the CD4+ cells were CD62L+ cells. CD4+ cells were removed from TLI-spleen using CD4+ depletion Miltenyi Biotec Microbeads and CS columns (Miltenyi Biotec), per the manufacturer's instructions. CD4-depleted TLI-spleen cells contained less than 1% CD4+ cells. Spleen cells from adoptive transfer mice (1 × 106 cells/well) were cultured in complete RPMI medium in triplicate in 96-well round-bottom plates for 48 hours with increasing concentrations of OVA peptide 323-339 (American Peptide Company, Sunnyvale, CA) ranging from 0 to 1000 nM in a final volume of 200 µL/well. For primary antigen stimulation, 2 × 105 purified naive
OVA-Tg cells were cultured in complete RPMI medium in triplicate in 96-well round-bottom plates for 48 hours with increasing concentrations of OVA peptide 323-339 (0-1000 nM) and T-cell-depleted spleen from
untreated BALB/c mice, unfractionated spleen from TLI-preconditioned mice, or a 1:1 mixture of both as a source of APCs in a final volume of
200 µL/well. TLI-preconditioned BALB/c WT or BALB/c IL-4 To measure proliferation, we added 3H-thymidine in the
final 6 hours of incubation, harvested the cells at 48 hours after
stimulation, and measured 3H-thymidine incorporation in a
scintillation counter (Beckmann LS 5000 TD). For measurement of IL-2,
IL-4, and interferon (IFN)- In some experiments, cells were harvested at 24 hours after stimulation, and OVA-Tg cells were examined for the level of CD25 and CD69 surface expression using anti-CD25-RPE (BD Pharmingen), anti-CD69-FITC (BD Pharmingen), anti-CD4-Cy5 (in-house purification and conjugation to Cy5; Amersham, Piscataway, NJ), KJ1-26-biotin (in-house purification and conjugation to biotin; Sigma-Aldrich, St Louis, MO), and streptavidin-TR (Invitrogen Life Technologies, Carlsbad, CA), with 4-color FACS analysis (Coulter EPICS). For antigen restimulation studies, purified naive OVA-Tg
CD4+ cells (10 × 106 cells/well) were
cultured in 6-well plates with equal numbers of T-cell-depleted spleen
from untreated BALB/c mice, spleen from TLI-preconditioned mice, or a
1:1 mixture of both, plus 100 nM OVA peptide in 5 mL/well final volume
of complete medium for 5 days. Cells from the primary cultures were
centrifuged over a Lympholyte-M gradient (Cedar Lane Laboratories), and
OVA-Tg cells were further purified using anti-CD4-phycoerythrin and
KJ1-26-FITC and positive FACS sorting. Purified OVA-Tg
CD4+ cells (greater than 99%) were replated in 2-fold
dilutions (starting concentration 105/well) directly in
anti-IL-4 or anti-IFN- Cytokine ELISA and ELISPOT assays Cytokine-specific ELISAs were performed using the following cytokine capture and binding antibodies: IL-2, JES6-1A12 (American Type Culture Collection; permission from DNAX, Palo Alto, CA), and JES6-5H4-biotin (American Type Culture Collection; permission from DNAX); IFN- , R4-6A2 (American Type Culture Collection), and rabbit
anti-mouse IFN- (PeproTech, Rocky Hill, NJ); IL-4, BVD4-1D11
(American Type Culture Collection; permission from DNAX), and
BVD6-24G2-horseradish peroxidase (HRP; American Type Culture Collection, permission from DNAX); and IL-10, JES5-2A5 (American Type
Culture Collection, permission from DNAX), and SXC-1-biotin (American
Type Culture Collection, permission from DNAX). The IFN-
ELISA was developed using donkey anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA), and the IL-2 ELISA used goat anti-biotin-HRP (Sigma). Detection limits for all assays were as
follows: 10 to 28 pg/mL, IL-2; 5 to 38 pg/mL, IFN-; 1 to 4 pg/mL,
IL-4; and 35 to 50 pg/mL, IL-10. Transforming growth factor (TGF)-
was detected using the TGF- Emax Immunoassay system (Promega, Madison, WI) according to the manufacturer's specifications.
The cytokine ELISPOT assays were adapted from Taguchi et
al,17 as described.18 The IL-4 ELISPOT used
BVD4-1D11 for capture and BVD6-24G2-biotin and goat
anti-biotin-alkaline phosphatase (Sigma-Aldrich) for detection. The
IFN- Intracytoplasmic cytokine analysis Cells from primary cultures were centrifuged over Lympholyte-M. Recovered cells were cultured in 24-well plates at 2 × 106 cells/mL with 50 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the absence of APCs for 4 hours. Brefeldin A (10 µg/mL; Epicentre Technologies, Madison, WI) was added for the last 2 hours of culture. Cells were stained with anti-CD4+ and KJ1-26, then fixed and permeabilized using a commercial Fix & Perm kit (Caltag) according to the manufacturer's directions. Intracellular cytokine staining was performed with RPE-conjugated rat anti-mouse IFN- mAb (XMG-1.2;
BD Pharmingen), APC-conjugated rat anti-mouse IL-4 mAb (11B11; BD
Pharmingen), or conjugated rat immunoglobulin G1 (IgG1) isotype control
mAb. Analysis was performed with a Coulter EPICS FACS.
Multiparameter FACS analysis Splenocytes from TLI-treated and control mice were stained with anti-CD4-Cy5, anti-IL-2R-FITC (TM-B1; BD Pharmingen), and anti-CD44-biotin (IM7; BD Pharmingen), followed by streptavidin-TXR (Invitrogen Life Technologies). Rat IgG2b,  -FITC (BD Pharmingen) was
used as an isotype control for anti-IL-2R. In other experiments, splenocytes were stained with anti-CD4-RPE (H129.19; BD Pharmingen), anti-CD44-biotin (IM7; BD Pharmingen), and various antibodies against
mouse V TCRs-FITC followed by streptavidin TXR. The mouse antibodies
were against the following: V3 (KJ25); V5 (MR9.4); V6 (RR4-7);
V7 (TR310); V8.1, 8.2, 8.3 (F23.1); V8.1, 8.2 (KJ16.133.18);
V8.3 (IB3.3); V9 (MR10-2); and V11 (RR3-15). V antibodies
were from BD Pharmingen, except for V8.1, 8.2, and 8.3 (F23.1) and
V8.1 and 8.2 (KJ16.133.18), which were obtained from P. Marrack
(Howard Hughes Medical Institute, Denver, CO). All stained
cells were resuspended in RPMI complete medium with 5 µg/mL propidium
iodide to gate out dead cells. The stained cells were analyzed on a
Coulter EPICS FACS.
In vivo priming of OVA-Tg cells in TLI-preconditioned mice alters recall cytokine response We took advantage of the observation that OVA-Tg cells, which exhibit cross-reactivity to H-2b alloantigen,15 proliferate (not shown) and secrete cytokines (not shown) in a dose-dependent fashion following stimulation with CB6F1 cells. We therefore set up an adoptive transfer model in which we injected purified OVA-Tg T cells into either BALB/c or CB6F1 mice that had been preconditioned with TLI, and we measured the function of the cells one week later. OVA-Tg cells were detected in the spleens of TLI-preconditioned BALB/c and CB6F1 mice one week after adoptive transfer (Figure 1). There was no difference in the percentages of OVA-Tg cells in the spleens of TLI-preconditioned BALB/c versus CB6F1 mice (mean ± SE: 2.5% ± 0.2%, BALB/c, n = 3; 3.1% ± 0.3%, CB6F1, n = 7; P = .33). However, compared with spleens of the BALB/c mice, spleens from the CB6F1 mice contained a higher absolute number of OVA-Tg CD4+ cells (mean ± SE: 1.14 ± 0.26 × 106, BALB/c; 3.29 ± 0.33 × 106, CB6F1; P = .004).
When spleen cells were examined one week after adoptive transfer for
their ability to respond to OVA peptide (Figure
2), OVA-Tg cells from TLI-preconditioned
BALB/c mice secreted significantly more IL-2 and IFN-
In vitro priming of OVA-Tg with spleen from TLI-preconditioned mice promotes the maturation of OVA-Tg TH2 over TH1 cells To examine whether the TLI-preconditioned environment skews the development of CD4+ cells toward TH2, we stimulated purified naive CD4+ OVA-Tg in vitro with 100 nM OVA peptide and APCs from either untreated or TLI-preconditioned BALB/c mice for 5 days. Purified OVA-Tg cells were then restimulated with PMA and calcium ionophore. OVA-Tg cells that were primed in the presence of control APCs gave rise to more IFN--producing cells by
intracytoplasmic staining (Figure 3),
whereas OVA-Tg cells that were primed in the presence of TLI-spleen
gave rise to 5-fold more IL-4-producing cells and 75% less
IFN--producing cells. We saw few dual-producing IL-4 and IFN-
OVA-Tg cells, indicating that we were primarily detecting differentiation of either TH1 or TH2 cells.
Thus, the presence of TLI-spleen during primary antigen activation
promotes differentiation of TH2 OVA-Tg.
To confirm the differentiation of OVA peptide-reactive TH2
cells, we stimulated purified naive OVA-Tg in vitro as before with 100 nM OVA peptide plus control APCs, TLI-spleen, or a 1:1 combination in
primary cultures for 5 days. OVA-Tg cells were purified from the
primary cultures and restimulated with OVA peptide in secondary ELISPOT
cultures in the presence of control APCs. Figure
4 shows that most OVA-Tg cells from the
control primary cultures matured into IFN-
TLI-spleen cells alter the proliferative response and the pattern of cytokines produced by OVA-Tg CD4+ cells following primary antigen activation We then examined OVA-Tg responses in primary cultures. We first measured the ability of antigen-stimulated OVA-Tg to up-regulate surface CD69 and CD25, as an indirect indicator of TCR-ligand interaction. Purified naive OVA-Tg CD4+ cells were cultured for 24 hours with either no antigen or 100 nM OVA peptide and APCs from either untreated BALB/c mice or TLI-preconditioned BALB/c mice, and the levels of CD69 and CD25 expression were measured on OVA-Tg CD4+ cells by FACS. In 3 separate experiments, there were no differences in the percentages of OVA-Tg cells that coexpressed CD69 and CD25 (not shown), nor were there any differences in the levels of CD69 or CD25 expression (not shown) on OVA-Tg cells from primary cultures that contained TLI-spleen versus control APCs.We next examined OVA-Tg proliferative responses to OVA peptide in
primary cultures. OVA-Tg cells proliferated more when stimulated with
OVA peptide in the presence of control APCs as compared with TLI-spleen
(Figure 5A). The mean ± SE peak
proliferative response of antigen-activated OVA-Tg cells was
43 568 ± 5136 counts per minute (cpm) when control APCs were used
and 21 554 ± 3349 cpm when TLI-spleen cells were used in a total of
3 dose-response experiments. The addition of anti-CD28 did not enhance
the proliferation of OVA-Tg cells whether they were cultured with
control APCs or the TLI-spleen cells (Figure 5B). The addition of
exogenous IL-2 did not restore the proliferative response of OVA-Tg
cells from primary cultures with TLI-spleen cells to the control level
(not shown).
To examine the cytokine response of OVA-Tg in primary cultures, we
stimulated OVA-Tg cells with increasing amounts of OVA peptide in the
presence of control APCs, TLI-spleen cells, or a 1:1 combination of
both, and determined cytokine production 48 hours after stimulation.
OVA-Tg cells secreted significantly more IL-4 and less IL-2 when
stimulated with OVA peptide in the presence of APCs from
TLI-preconditioned mice, and this cytokine pattern persisted even if
normal control APCs were added to the TLI-spleen cells (Figure
6). TLI-spleen cells alone did not
produce any IL-2, IFN-
Secretion of IL-4 by OVA-Tg cells from primary in vitro cultures depends on CD4+ TLI-spleen cells that constitutively secrete IL-4 TLI treatment results in a relative enrichment of CD4+ cells that produce IL-4.6 We examined whether the presence of the CD4+ cell within the TLI-spleen population had an effect on the responses of OVA-Tg cells in primary cultures. Highly purified naive OVA-Tg CD4+ cells were cultured with 100 nM OVA peptide and control APCs, unfractionated TLI-spleen cells, or TLI-spleen that had been depleted of CD4+ cells. The cultures were performed in either IL-4 (Figure 8, upper panel) or IL-2 (Figure 8, lower panel) ELISPOT wells. No IL-4-producing cells were detected in primary cultures in which OVA-Tg cells were stimulated in the presence of control APCs. In sharp contrast, IL-4-producing cells were detected in primary cultures when OVA-Tg cells were stimulated in the presence of unfractionated TLI-spleen cells. Deletion of CD4+ cells from the TLI-spleen cell population decreased the amount of IL-4-producing cells detected in primary cultures, but did not alter the number of IL-2-producing cells.
Surprisingly, we detected IL-4-producing spots even in primary
cultures that did not contain OVA peptide (Figure 8, upper panel; 0 nM
peptide). This finding was consistent in 3 separate experiments and
suggested that TLI-spleen contained cells that constitutively secreted
IL-4. To examine whether this constitutive production of IL-4 triggered
OVA-Tg cells to secrete IL-4 in the primary cultures, we cultured
highly purified naive CD4+ OVA-Tg cells directly in IL-4
ELISPOT wells containing 100 nM OVA peptide and APCs from control,
TLI-preconditioned WT mice, or TLI-preconditioned IL-4
Increased CD4+-natural killer T (TNK) cells in TLI-treated mice We previously reported that anti-CD3-stimulated spleen cells from 2-day post-TLI-treated mice secrete 20-fold greater amounts of IL-4 compared with IFN- and no measurable IL-10.6
We also reported that the CD4+ cells and not the
CD4CD8 cells were the source of the
IL-4.6 CD4+-TNK cells produce more IL-4 in
relation to other cytokines19 and are capable of secreting
high levels of IL-4 upon primary stimulation.20-22 To
determine whether the CD4+ spleen cells in TLI-treated mice
contained TNK cells, we measured CD44+ and IL-2R
expression in the CD4+ cells from the TLI-treated mice. We
used coexpression of CD44hi/IL-2R+ as a
marker for CD4+-TNK cells20,21,23,24 because
the BALB/c strain of mice does not express the standard NK marker,
NK1.1. We did not measure coexpression of CD25. TLI-treated mice
contained twice as many CD4+ spleen cells that coexpressed
CD44hi/IL-2R+ as did control unirradiated
mice (57.2% ± 3.4%, TLI; versus 28.8% ± 4.7%,
control; P = .0005). Figure
10A depicts a typical example of
CD4+/CD44hi/IL-2R+
cells from a TLI-treated BALB/c mouse and a BALB/c control mouse. To
further confirm that the
CD4+/CD44hi/IL-2R+
subset was TNK, we examined the V usage of
CD4+/CD44hi/IL-2R+ cells and
CD4+/CD44lo/IL-2R
cells. CD4+-TNK cells preferentially express V8
(especially 8.2), V7, or V2.21,25-28 We saw no
difference in the percentages of
CD4+/CD44hi/IL-2R+ or
CD4+/CD44lo/IL-2R populations
that expressed V3, 5, 6, 7, 9, or 11 (not shown). However, Figure
10B demonstrates that
CD4+/CD44hi/IL-2R+ cells from
TLI-treated mice expressed significantly more V8.1, 8.2, and 8.3 compared with the
CD4+/CD44lo/IL-2R cells
(P = .00007). In contrast, V8.3 expression was no
different between the groups (Figure 10B), indicating that the
CD4+/CD44hi/IL-2R+ cells from
TLI-treated mice are enriched for V8.1- and/or V8.2-expressing cells. Control mice did not show an increase in V8.1-, 8.2-, or
8.3-expressing cells in the
CD4+/CD44hi/IL-2R+ subset as
compared with the
CD4+/CD44lo/IL-2R
subset (P = .637, data not shown).
We reported that TLI preconditioning alters the phenotype of host T cells, eliminating CD8+ cells and sparing a subset of radioresistant CD4+ T cells.6 The remaining T cells secrete IL-4 upon in vitro activation. When host CD4+ cells are exposed to antigen during this early post-TLI phase, such as by adoptive transfer of semiallogeneic hematopoietic cells, they preferentially differentiate into anti-donor TH2 CD4+ cells.6 However, we had not investigated whether immunocompetent CD4+ cells behave similarly when exposed to antigen in a TLI-preconditioned environment. The purpose of this study was to examine the extent to which TLI
preconditioning affects the antigen-specific immune responses of
immunocompetent CD4+ cells. We used OVA-Tg CD4+
cells as a source of homogeneous immunocompetent "donor" T cells because they can be easily identified using anti-clonotypic mAbs. OVA-Tg cells cross-react to MHC alloantigens expressed on
CB6F1 cells, in addition to their nominal antigen, OVA
peptide. The property of cross-reactivity against CB6F1
allowed us to examine the differentiation of OVA-Tg following in vivo
priming by adoptive transfer into CB6F1 mice. The use of
adoptive transfer of the parent Seven days after adoptive transfer, the TLI-preconditioned CB6F1 mice contained higher numbers of OVA-Tg cells in their spleens than TLI-preconditioned BALB/c mice. The spleens from the TLI-preconditioned CB6F1 mice were larger than those of the TLI-preconditioned syngeneic controls (1.36 ± 0.11 × 108 versus 0.57 ± 0.13 × 108; P < .003), but similar in size to the unmanipulated control CB6F1 mice (P = .2). This indicates in vivo expansion of OVA-Tg cells in response to the cross-reactive MHC antigen as well as expansion of non-Tg cells. OVA-Tg cells from TLI-preconditioned CB6F1 mice differentiated primarily into TH2 cells (Figure 2). Fowler and Gress29 reported that donor TH2 cells do not cause acute GVHD and suppress GVHD following infusion of donor T cells without impairing engraftment. De Wit et al reported that although enhanced TH2-like cytokine responses have been associated with chronic GVHD,30 their presence is not sufficient for the development of chronic GVHD.31 We did not observe any features of acute or chronic GVHD, including splenomegaly, wasting, weight loss, hair loss, or death, in a subgroup of mice that were followed for up to 15 weeks (E.H.F., unpublished observations, August 2000). OVA-Tg cells that were primed with cognate antigen OVA in vitro in the presence of APCs from TLI-preconditioned mice also preferentially differentiated into TH2-like cells (Figures 3-4). We were best able to demonstrate immunoredirection toward IL-4-producing cells using the ELISPOT assay, which readily detected the higher frequency of antigen-reactive IL-4-producing OVA-Tg cells and was more sensitive than intracytoplasmic staining. The ELISPOT assay has been reported as more sensitive for detecting IL-4-producing cells.32 Besides their propensity to differentiate into TH2 cells, OVA-Tg cells that were primed in vitro in the presence of spleen cells from TLI-preconditioned mice exhibited other properties that distinguished them from OVA-Tg cells that were primed with control APCs. The former proliferated less, produced less IL-2, but produced more IL-4. On the other hand, OVA-Tg cells from both cultures expressed the same levels of surface CD25 and CD69. We previously reported that anti-CD3-activated CD4+ cells from TLI-preconditioned mice secreted more IL-4 and less IL-2 than cells from nonirradiated control mice.18 CD4+ cells from untreated mice that were stimulated with anti-CD3 in the presence of spleen cells from TLI-preconditioned mice also proliferated less and secreted less IL-2, but up-regulated CD69 and CD25 normally.10 The results herein extend these previous observations to immunocompetent CD4+ cells that are stimulated with cognate antigen. Thus, OVA-Tg CD4+ cells that encounter antigen in vitro in the presence of TLI-spleen cells exhibit an immunosuppressive phenotype characterized by decreased IL-2 production and proliferation, normal up-regulation of CD25/CD69 expression, and increased production of IL-4. The nature of the response of naive T cells to primary T-cell activation depends on several factors, including the strength of the TCR/Ag/MHC interaction (signal 1); the dose of peptide; the functional capacity of the APCs, which is largely dictated by costimulatory potential (signal 2); and the immediate cytokine environment. The up-regulation of both CD69 and CD25 depends on a strong TCR-ligand interaction33 and is relatively independent of costimulation.33-35 When the TCR-ligand interaction is weak, then up-regulation of CD69 and CD25 can occur in the presence of adequate costimulation.33 On the other hand, both IL-2 secretion and cell proliferation are relatively late events following cell activation and depend on B7 costimulation.33,36,37 Figures 5 through 7 show that naive OVA-Tg cells that are primed in the presence of spleen cells from TLI-preconditioned mice display a functional phenotype similar to that of naive CD4+ cells that are primed with altered peptide ligands. Naive T cells that are stimulated with altered peptide ligands proliferate less33,38,39 and produce less IL-2,33,40 but up-regulate CD25/CD69 normally if supplied with adequate costimulation.33 Furthermore, priming with altered peptide ligand induces naive CD4+ cells to produce IL-438,41 and promotes the differentiation of more TH2-like cells.38 For our in vitro studies, we used T-cell-depleted spleen as control APCs and unfractionated TLI-spleen for TLI APCs. In addition to the difference that the TLI-spleen contains 3% to 14% CD4+ cells, TLI-spleen also has a lower percentage of B cells and a higher percentage of non-T/non-B cells.6 We did not compare expression of MHC class II. However, OVA-Tg cells that were primed with TLI-spleen showed normal up-regulation of CD25 and CD69 (not shown), indicating that TLI-spleen APCs were capable of delivering signal 1. In addition, although increasing the dose of antigen can reverse the hypoproliferative phenotype of cells stimulated with altered peptide ligand,38,39 increasing the antigen dose does not restore immune response to the levels detected in controls, nor does it prevent the production of IL-4 in the primary cultures (Figures 5 and 7). Therefore, it is unlikely that the results herein can be explained by an alteration in the strength of TCR/Ag/MHC signaling to the OVA-Tg cells. We considered that the decreased ability of the naive OVA-Tg cells to fully proliferate or secrete IL-2 may be secondary to inadequate costimulation. OVA-Tg cells proliferate less and produce lower amounts of IL-2 if antigen stimulation is carried out in the presence of APCs that lack B7 molecules.37 However, the addition of anti-CD28 did not augment proliferation or IL-2 production by OVA-Tg cells that were primed with TLI-spleen (Figure 5 and data not shown) and did not affect IL-4 secretion in the primary cultures (Figure 7). Indeed, the results in Figure 7 argue that the B7/CD28 costimulatory pathway is intact in the TLI-spleen cells because naive CD4+ cells required B7/CD28 costimulation to produce IL-4 in primary cultures.37,42 The cytokine milieu at the time of initial antigen encounter greatly influences CD4+ subset maturation.43 IL-4 is absolutely required for the maturation of TH2 CD4+ cells,44 and the addition of IL-4 at the time of initial antigen exposure can enhance the differentiation of naive CD4+ cells into TH2 memory cells.34 When added to primary cultures, exogenous IL-4 stimulates OVA-Tg cells to transcribe IL-4 and to up-regulate IL-4 receptor.45 Exogenous IL-4 can also enhance IL-4R signaling,34 rendering the cell more sensitive to IL-4. Thus, naive OVA-Tg CD4+ cells can secrete low levels of IL-4 in primary cultures under the proper conditions, including in response to exogenous IL-4.34,41,46,47 Figures 8 and 9 demonstrate that spleen cells from TLI-preconditioned
mice constitutively secreted IL-4; CD4+ cells within the
TLI-spleen were the source of IL-4. When IL-4-secreting CD4+ cells were present in the TLI-spleen, OVA-Tg cells
produced more IL-4 in the primary cultures in response to increasing
doses of OVA peptide (Figure 8). The removal of this source of IL-4, by substituting TLI-spleen cells from IL-4 The CD4+ cells in the TLI-treated BALB/c mice showed
typical features of CD4+-TNK cells, including lower density
of surface CD4 and CD319 (data not shown), coexpression of
CD44hi/IL-2R The results of the experiments herein provide a mechanism by which TLI
preconditioning promotes bidirectional tolerance. TLI preconditioning
enriches for a population of CD4+-TNK cells whose
constitutive production of IL-4 induces the differentiation of
TH2 cells. The preferential development of TH2
cells from both host and donor CD4+ cells may prove to be
one important aspect for the development of bidirectional tolerance in
TLI-preconditioned mice that have undergone hematopoietic cell
transplantation. Indeed, donor TH2 cells have been found to
suppress GVHD following infusion of donor T cells.29 Lan
et al7 reported similar findings, in that the
TNK+ cells from TLI-treated mice protected the mice against
the development of lethal GVHD and provided the source of IL-4.
Although the authors did not directly examine cytokine production by
CD4+ NK+ TCR
We thank the University of Iowa fluorescence-activated cell-sorting CORE facility and Justin Fishbaugh for assistance with flow cytometry, the Diabetes Endocrine Research Center CORE for reagents, the Radiation Biology Facility, and Jacqueline Capper for manuscript preparation.
Submitted May 23, 2002; accepted October 14, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-05-1513.
Supported by a Merit Review Grant from the Department of Veterans Affairs (E.H.F.), and by US Public Health Service Grants CA45541 (E.H.F.) and DK25295 (E.H.F.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Elizabeth H. Field, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 601 Highway 6, 6W33, Veterans Affairs Medical Center, Iowa City, IA 52246; e-mail: liz-field{at}icva.gov.
1.
Slavin S, Strober S, Fuks Z, Kaplan HS.
Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts.
J Exp Med.
1977;146:34-48
2.
Gottlieb M, Strober S, Kaplan HS.
Allogeneic marrow transplantation after total lymphoid irradiation (TLI): effect of dose/fraction, thymic irradiation, delayed marrow infusion, and presensitization.
J Immunol.
1979;123:379-383 3. Field EH, Rouse TM, Gao Q, Chang B. Association between enhanced Th2/Th1 cytokine profile and donor T-cell chimerism following total lymphoid irradiation. Hum Immunol. 1997;52:144-154[CrossRef][Medline] [Order article via Infotrieve]. 4. Palathumpat V, Dejbakhsh-Jones S, Strober S. The role of purified CD8+ T cells in graft-versus-leukemia activity and engraftment after allogeneic bone marrow transplantation. Transplantation. 1995;60:355-361[Medline] [Order article via Infotrieve]. 5. Salam A, Waer M. Graft-versus-host reactivity and graft-versus-leukemia effect in murine allogeneic bone marrow chimeras conditioned with total body irradiation or total lymphoid irradiation. Transplantation. 1996;61:826-830[CrossRef][Medline] [Order article via Infotrieve]. 6. Field EH, Rouse T. Requirements for developing mixed chimerism in nonmyeloablative total-lymphoid irradiated mice: role of IL-4 and immunoredirection. Transplantation. 2002;74:1021-1029[CrossRef][Medline] [Order article via Infotrieve].
7.
Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S.
Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells.
J Immunol.
2001;167:2087-2096 8. Palathumpat V, Dejbakhsh-Jones S, Holm B, Strober S. Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease. J Immunol. 1992;149:808-817[Abstract]. 9. Field EH, Becker GC. Blocking of mixed lymphocyte reaction by spleen cells from total lymphoid-irradiated mice involves interruption of the IL-2 pathway. J Immunol. 1992;148:354-359[Abstract]. 10. Field EH, Ye ZX, Rouse TM. Mechanisms of tolerance induction: splenocytes from total lymphoid irradiated mice inhibit the IL-2 pathway in TCR-activated CD4 cells. J Immunol. 1996;157:3796-3803[Abstract].
11.
Murphy KM, Heimberger AB, Loh DY.
Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.
Science.
1990;250:1720-1723
12.
Haskins K, Kubo R, White J, Pigeon M, Kappler J, Marrack P.
The major histocompatibility complex-restricted antigen receptor on T cells, I: isolation with a monoclonal antibody.
J Exp Med.
1983;157:1149-1169
13.
Hosken NA, Shibuya K, Heath AW, Murphy KM, O'Garra A.
The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model.
J Exp Med.
1995;182:1579-1584
14.
Hsieh CS, Heimberger AB, Gold JS, O'Garra A, Murphy KM.
Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic system.
Proc Natl Acad Sci U S A.
1992;89:6065-6069
15.
Garside P, Ingulli E, Merica RR, Johnson JG, Noelle RJ, Jenkins MK.
Visualization of specific B and T lymphocyte interactions in the lymph node.
Science.
1998;281:96-99 16. Field EH, Becker GC. The immunosuppressive mechanism of total lymphoid irradiation. I. The effect on IL-2 production and IL-2 receptor expression. Transplantation. 1989;48:499-505[Medline] [Order article via Infotrieve]. 17. Taguchi T, McGhee JR, Coffman RL, et al. Detection of individual mouse splenic T cells producing IFN-gamma and IL-5 using the enzyme-linked immunospot (ELISPOT) assay. J Immunol Methods. 1990;128:65-73[CrossRef][Medline] [Order article via Infotrieve]. 18. Field EH, Rouse TM. Alloantigen priming after total lymphoid irradiation alters alloimmune cytokine responses. Transplantation. 1995;60:695-702[Medline] [Order article via Infotrieve].
19.
Arase H, Arase N, Nakagawa K, Good RA, Onoe K.
NK1.1+ CD4+ CD8
20.
Yoshimoto T, Paul WE.
CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3.
J Exp Med.
1994;179:1285-1295
21.
Emoto M, Emoto Y, Kaufmann SH.
IL-4 producing CD4+ TCR alpha beta int liver lymphocytes: influence of thymus, beta 2-microglobulin and NK1.1 expression.
Int Immunol.
1995;7:1729-1739
22.
Chen H, Paul WE.
Cultured NK1.1+ CD4+ T cells produce large amounts of IL-4 and IFN- gamma upon activation by anti-CD3 or CD1.
J Immunol.
1997;159:2240-2249 23. Bendelac A, Schwartz RH. CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation. Nature. 1991;353:68-71[CrossRef][Medline] [Order article via Infotrieve].
24.
MacDonald HR.
NK1.1+ T cell receptor-alpha/beta+ cells: new clues to their origin, specificity, and function.
J Exp Med.
1995;182:633-638
25.
Arase H, Arase N, Ogasawara K, Good RA, Onoe K.
An NK1.1+ CD4+8
26.
Lantz O, Bendelac A.
An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4
27.
Bendelac A, Killeen N, Littman DR, Schwartz RH.
A subset of CD4+ thymocytes selected by MHC class I molecules.
Science.
1994;263:1774-1778
28.
Ohteki T, MacDonald HR.
Major histocompatibility complex class I related molecules control the development of CD4+8 29. Fowler DH, Gress RE. Th2 and Tc2 cells in the regulation of GVHD, GVL, and graft rejection: considerations for the allogeneic transplantation therapy of leukemia and lymphoma. Leuk Lymphoma. 2000;38:221-234[Medline] [Order article via Infotrieve]. 30. De Wit D, Van Mechelen M, Zanin C, et al. Preferential activation of Th2 cells in chronic graft-versus-host reaction. J Immunol. 1993;150:361-366[Abstract]. 31. Williamson E, Garside P, Bradley JA, More IA, Mowat AM. Neutralizing IL-12 during induction of murine acute graft-versus-host disease polarizes the cytokine profile toward a Th2-type alloimmune response and confers long term protection from disease. J Immunol. 1997;159:1208-1215[Abstract]. 32. Ekerfelt C, Ernerudh J, Jenmalm MC. Detection of spontaneous and antigen-induced human interleukin-4 responses in vitro: comparison of ELISPOT, a novel ELISA and real-time RT-PCR. J Immunol Methods. 2002;260:55-67[CrossRef][Medline] [Order article via Infotrieve].
33.
Rogers PR, Grey HM, Croft M.
Modulation of naive CD4 T cell activation with altered peptide ligands: the nature of the peptide and presentation in the context of costimulation are critical for a sustained response.
J Immunol.
1998;160:3698-3704
34.
Kubo M, Yamashita M, Abe R, et al.
CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.
J Immunol.
1999;163:2432-2442
35.
Jaiswal AI, Dubey C, Swain SL, Croft M.
Regulation of CD40 ligand expression on naive CD4 T cells: a role for TCR but not co-stimulatory signals.
Int Immunol.
1996;8:275-285 36. McKnight AJ, Perez VL, Shea CM, Gray GS, Abbas AK. Costimulator dependence of lymphokine secretion by naive and activated CD4+ T lymphocytes from TCR transgenic mice. J Immunol. 1994;152:5220-5225[Abstract].
37.
Schweitzer AN, Sharpe AH.
Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production.
J Immunol.
1998;161:2762-2771 38. Tao X, Grant C, Constant S, Bottomly K. Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR binding. J Immunol. 1997;158:4237-4244[Abstract]. 39. Sloan-Lancaster J, Steinberg TH, Allen PM. Selective loss of the calcium ion signaling pathway in T cells maturing toward a T helper 2 phenotype. J Immunol. 1997;159:1160-1168[Abstract]. 40. Sloan-Lancaster J, Allen PM. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu Rev Immunol. 1996;14:1-27[CrossRef][Medline] [Order article via Infotrieve]. 41. Tao X, Constant S, Jorritsma P, Bottomly K. Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell differentiation. J Immunol. 1997;159:5956-5963[Abstract]. 42. Rulifson IC, Sperling AI, Fields PE, Fitch FW, Bluestone JA. CD28 costimulation promotes the production of Th2 cytokines. J Immunol. 1997;158:658-665[Abstract]. 43. O'Garra A. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity. 1998;8:275-283[CrossRef][Medline] [Order article via Infotrieve]. 44. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol. 1994;12:635-673[CrossRef][Medline] [Order article via Infotrieve]. 45. Nakamura T, Kamogawa Y, Bottomly K, Flavell RA. Polarization of IL-4- and IFN-gamma-producing CD4+ T cells following activation of naive CD4+ T cells. J Immunol. 1997;158:1085-1094[Abstract].
46.
Liu L, Rich BE, Inobe J, Chen W, Weiner HL.
Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4.
Int Immunol.
1998;10:1017-1026
47.
Oosterwegel MA, Mandelbrot DA, Boyd SD, et al.
The role of CTLA-4 in regulating Th2 differentiation.
J Immunol.
1999;163:2634-2639 48. Schmitt E, Hoehn P, Huels C, et al. T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-gamma and is inhibited by transforming growth factor-beta. Eur J Immunol. 1994;24:793-798[Medline] [Order article via Infotrieve].
49.
Gray JD, Hirokawa M, Ohtsuka K, Horwitz DA.
Generation of an inhibitory circuit involving CD8+ T cells, IL-2, and NK cell-derived TGF-beta: contrasting effects of anti-CD2 and anti-CD3.
J Immunol.
1998;160:2248-2254 50. Field EH, Matesic D, Rigby S, Fehr T, Rouse T, Gao Q. CD4+CD25+ regulatory cells in acquired MHC tolerance. Immunol Rev. 2001;182:99-112[CrossRef][Medline] [Order article via Infotrieve].
51.
Taylor PA, Lees CJ, Blazar BR.
The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality.
Blood.
2002;99:3493-3499
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  A. B. Pillai, T. I. George, S. Dutt, and S. Strober Host natural killer T cells induce an interleukin-4-dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells that protects against graft-versus-host disease Blood, April 30, 2009; 113(18): 4458 - 4467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kashiwada, G. Cattoretti, L. McKeag, T. Rouse, B. M. Showalter, U. Al-Alem, M. Niki, P. P. Pandolfi, E. H. Field, and P. B. Rothman Downstream of Tyrosine Kinases-1 and Src Homology 2-Containing Inositol 5'-Phosphatase Are Required for Regulation of CD4+CD25+ T Cell Development J. Immunol., April 1, 2006; 176(7): 3958 - 3965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Hashimoto, S. Asakura, S. Miyake, T. Yamamura, L. Van Kaer, C. Liu, M. Tanimoto, and T. Teshima Stimulation of Host NKT Cells by Synthetic Glycolipid Regulates Acute Graft-versus-Host Disease by Inducing Th2 Polarization of Donor T Cells J. Immunol., January 1, 2005; 174(1): 551 - 556. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
20°C for future cytokine-specific enzyme-linked immunosorbent
assays (ELISAs).
) or TLI-preconditioned BALB/c mice (TLI, 
).
After 48 hours, proliferation was measured by 3H-thymidine
incorporation. (B) OVA-Tg cells were cultured as in (A) with OVA
peptide and APCs from either untreated (BALB/c, solid symbols) or
TLI-preconditioned (TLI, open symbols) BALB/c mouse spleen without
(circles) or with (squares) anti-CD28. Proliferation was measured by
3H-thymidine incorporation. Symbols represent the mean ± SD of triplicate wells for one representative experiment from 3 separate experiments. APCs alone did not proliferate in response to
increasing OVA peptide (not shown).
), whole spleen
from TLI-preconditioned BALB/c mice (
) in IL-4 (upper) and
IL-2 (lower) ELISPOT plates. Cytokine-producing cells were detected as
described in "Materials and methods." The data represent a single
experiment. The experiment depicted in the upper panel was repeated
twice with similar results.
F1 combination
allowed us to avoid the use of adjuvants for the in vivo priming.
