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+ or DX5+TCR+ T Cells in Mice Conditioned with Fractionated Lymphoid Irradiation Protects Against Graft-Versus-Host Disease: "Natural Suppressor" Cells1
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Department of Medicine, Division of Immunology and Rheumatology, and
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
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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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An alternative approach to prevent GVHD is to use a nonmyeloablative host conditioning regimen that protects the host against donor T cell attack. This has been achieved by altering the APC of the host (12) or by using fractionated irradiation of the lymphoid tissues (total lymphoid irradiation, TLI) that induces regulatory "natural suppressor" T cells in the host (13, 14). However, it is not clear whether these host conditioning regimens can protect against severe GVHD induced by peripheral blood T cells, because in the case of human BM or mobilized hemopoietic progenitor transplantation there is a high level of contamination with these blood T cells (15, 16).
Our recent studies showed that allogeneic donor
NK1.1+ TCR
+ T cells
in the BM can protect mice against lethal GVHD induced by donor
NK1.1- TCR+ T cells
(17). Protection was dependent upon secretion of IL-4 by
NK1.1+ TCR + T cells
(17). The latter T cell subset differs from conventional T
cells in several ways, including the extremely high levels of secretion
of IL-4 and IFN-
, as well as the recognition of the CD1
Ag-presenting molecule, a class I MHC-like molecule that is
nonpolymorphic (18, 19). In addition, depletion of
peripheral NK1.1+
TCR+ T cells is followed by rapid
reconstitution within 48 h from rapidly dividing progenitors in
the BM, whereas depletion of conventional NK1.1-
TCR+ T cells involves a slower
reconstitution via the thymus (20).
In the current study we developed a nonmyeloablative conditioning
regimen that severely depletes peripheral T cells and thymocytes using
fractionated lymphoid irradiation with marrow shielding in combination
with anti-mouse thymocyte serum (ATS). After the regimen, the
splenic NK1.1+ TCR+ T
cells of C57BL/6 mice increased from 
2% of all
TCR+ T cells to >90% in association with
a marked shift in the cytokine secretion pattern favoring IL-4. A
similar observation was made in BALB/c mice using the DX5 marker
reported previously to be coexpressed on NK1.1+
TCR+ T cells, but not on conventional T
cells (21). The irradiated BALB/c host mice with the
predominant DX5+ TCR+
T cells were protected from acute lethal GVHD induced by the injection
of combined allogeneic BM and peripheral blood T cell transplants from
untreated C57BL/6 donors. Depletion of
DX5+TCR+ T cells occurred after treatment
of hosts with anti-asialo-GM1 Abs, a regimen previously shown to remove
the protection afforded by fractionated lymphoid irradiation
(22). Protection was also dependent on BALB/c host cell
secretion of IL-4. Adoptive transfer of splenic T cells with the
predominant NK1.1+
TCR+ T cell subset from C57BL/6 donor mice
conditioned with lymphoid irradiation also protected against
transplants from untreated donors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 CD45.2(H-2b) and BALB/c (H-2d) CD45.2 wild-type mice, 8–10 wk old, were purchased from the Department of Comparative Medicine, Stanford University (Stanford, CA). Male C57BL/6 IL-4-/- mice (C57BL/6J-IL4tm1Cgn) and male BALB/c IL-4-/- (BALB/cIL4tm2Nnt) were purchased from The Jackson Laboratory (Bar Harbor, ME). Male C57BL/6 CD45.1 mice were obtained from the colony of Dr. I. L. Weissman (Stanford University).
Irradiation
TLI was delivered to the abdomen, lymph nodes, thymus, and spleen with shielding of the skull, lungs, pelvis, and tail as described previously (13, 14). Irradiation was started on day -24 before transplantation, and 17 doses of 240 cGy each were administered. The last dose of TLI or a single dose of total body irradiation (TBI) was administered to BALB/c mice 24 h before cell infusions (13, 14). The irradiation was performed with a Philips x-ray unit (200 kV, 10 mA; Philips Electronic Instruments, Rahway, NJ) at a rate of 84 cGy/min with a 0.5-mm Cu filter.
Rabbit ATS and rabbit anti-asialo-GM1 antiserum
Rabbit ATS was purchased from Accurate Laboratories (New York, NY). Mice were injected i.p. with 0.05 ml ATS in 0.5 ml saline on days -12, -10, and -8 before BM transplantation, corresponding to days 12, 14, and 16 after starting TLI. Rabbit anti-asialo-GM1 antiserum was purchased from Wako Chemicals (Richmond, VA). In some experiments 20 µl antiserum was injected i.p. immediately after TLI and then again 24 h later.
Cell preparation
PBMC were isolated on density gradients (Lymphocyte-M; Cedarlane
Laboratories, Hornby, Ontario, Canada) and washed twice in ice-cold
RPMI 1640 (Life Technologies, Grand Island, NY). Femoral and tibial
bones taken from donor C57BL/6 mice were rinsed, and the residual
muscle on the bones was carefully removed. BM cells were prepared by
flushing the bones with RPMI 1640, and the cell suspension was filtered
through nylon mesh to remove aggregates and washed once before
transfusion. Mouse T cell-depleted BM cells and purified
CD4+ and
CD8+TCR+ T cells from
the spleen were sorted on a FACStar flow cytometer as described
previously (17). Sorted spleen T cells obtained after
TLI were first enriched on immunomagnetic bead columns
(Miltenyi Biotech, Friedrich, Germany) using biotinylated
anti-Thy1.2 mAb (5a-8; Caltag Laboratories, Burlingame, CA) and
streptavidin-conjugated microbeads. Enriched
Thy1.2+ cells were stained with
allophycocyanin-conjugated anti-TCR (H57-597; BD
PharMingen, San Diego, CA) before sorting on a Vantage flow
cytometer (BD Biosciences, Mountain View, CA) as described previously
(17).
Flow cytometry
Blood samples for chimerism analyses were hemolyzed with
ammonium chloride potassium carbonate to remove red cells. The white
cell pellets were washed twice with 0.05% sodium azide staining buffer
and incubated on ice for 15 min with saturating concentrations of mAb
mixtures as described previously (17). Biotinylated
anti-Gr-1 (RB6-8C5) and anti-Mac-1 (M1/70.15), and
allophycocyanin-conjugated anti-B220 (RA3-6B2; Caltag Laboratories)
as well as FITC-conjugated anti-H-2Kb (AF6-88.5),
FITC-conjugated anti-CD45.1 (A20), and PE-conjugated
anti-Thy1.2 (53-2.1) mAbs (BD PharMingen) were used for mouse
chimerism analyses. After incubation, cells were washed twice and
followed by streptavidin-Texas Red (Caltag) staining on ice. Background
staining for donor-type cells in normal BALB/c mice was 
0.5%. For
NK1.1+ or DX5+ T cell
staining from C57BL/6 and BALB/c mice, splenocytes were incubated with
a mAb mixture with PE-conjugated anti-NK1.1 (PK136), PE-conjugated
anti-DX5 (DX5), and allophycocyanin-conjugated
anti-TCR(H57-597) from BD PharMingen. All mouse cells were
incubated with CD16/32(2.4G2) (BD PharMingen) to block the FcRII/II
receptors, and propidium iodide was added to exclude dead cells.
Chimerism analysis used a lymphoid gate set by forward and orthogonal
light scatter (17).
Cytokine assays
Details of in vitro stimulation of sorted cell populations with
PMA and ionomycin, and analysis of IL-4 and IFN- in the 48-h
supernatants by ELISAs were described previously (17).
Histopathology of liver, skin, and intestines
Tissues were fixed in formalin and embedded in paraffin blocks, and sections were stained with hematoxylin and eosin.
Liver and gut lymphocyte preparations
After hosts were exsanguinated, livers were flushed by injection
of heparinized PBS in the right ventricle until the liver became pale.
Livers were pressed through a nylon mesh to prepare single-cell
suspensions in 2 µM EDTA in PBS. The cell suspension was centrifuged
on Ficoll-Hypaque, and the interface layer was collected for lymphocyte
staining. Gut, including duodenum through rectum, was collected, cut
longitudinally, and rinsed thoroughly with cold RPMI 1640. The
rinsed gut was put into EDTA in PBS, minced into <5-mm segments, and
suspended by vortex 20–30 s, alternating with incubation at room
temperature for 2–4 min. After three repeats, cells in the
supernatant were filtered through a nylon mesh before separation on a
Ficoll-Hypaque gradient for collection of mononuclear cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ and
DX5+ TCR+ T cells after TLI
The spleen cells of C57BL/6 and BALB/c mice were examined for the
percentage of NK1.1+ or
DX5+ TCR+ T cells,
respectively, as well as the percentage of all
TCR+ T cells before and after fractionated
irradiation of the lymphoid tissues (TLI). Fig. 1
A shows flow cytometric
analyses of C57BL/6 spleen cells stained for the TCR marker vs
forward light scatter before and after 2, 8, or 17 irradiation
treatments of 240 cGy each, targeted to the thymus, spleen, and lymph
nodes while shielding the head, lungs, and hind limbs. In each case the
last irradiation dose was given 1 day before lymphoid tissues were
harvested. Whereas the TCR+ T cells were
33.2% of live nucleated cells (enclosed in box) before irradiation,
the percentage decreased to 25.6, 13.4, and 9.4% after 2, 8, and 17
treatments. Although the mean percentage of
TCR+ T cells in the spleen decreased
3-fold (30–9%) after 17 treatments, the mean absolute number
decreased 150-fold (32–0.2 x 106; Table I). Similar marked decreases in the
percentages and absolute numbers of TCR+ T
cells in the spleen of BALB/c mice after 2, 8, or 17 treatments of
irradiation were observed also (Fig. 1B and Table I).
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+ T cells in the spleen are rapidly
reconstituted from BM sources after peripheral depletion
(20), the percentage of NK1.1+
TCR+ T cells among gated
TCR+ T cells was determined before and
after lymphoid irradiation (with marrow shielding) of C57BL/6 mice
(Fig. 1
A). Whereas NK1.1+
TCR+ T cells accounted for only 1.3% of
all TCR+ T cells before irradiation, the
percentage increased dramatically after 2, 8, and 17 irradiation
treatments, such that 65.9% of T cells expressed the NK1.1 marker
after 17 treatments (Fig. 1A). As shown in Table I, the
increased mean percentage of NK1.1+ T cells among
all T cells was associated with a 4-fold decrease in the mean absolute
number of NK1.1+ T cells in the spleen. The mean
percentage of NK1.1+ T cells among all T cells
was calculated using the percentages shown in the boxes in the
right column of Fig. 1A for each
animal. However, there was a 30-fold greater reduction of
NK1.1- T cells compared with
NK1.1+ T cells that resulted in a marked change
in the balance of these T cell subsets in the spleen.
The lack of the NK1.1 marker in BALB/c mice (23) did not
permit a similar analysis of T cell subsets before and after
irradiation. However, we analyzed another marker, DX5, that has
previously been reported to be expressed on most
NK1.1+ T cells in the spleen (21).
Fig. 1A shows that a similar dramatic increase in the
percentage of DX5+
TCR+ T cells in the spleen of BALB/c mice
occurred after 17 irradiation treatments (1.2–64.4%). Table I shows
that the increased mean percentage of DX5+ T
cells was associated with a 4-fold decrease in the absolute number of
these cells in the spleen. A similar increase in the percentage of
NK1.1+ and DX5+ T cells
among all TCR+ T cells was observed in the
BM and peripheral blood (data not shown).
To determine whether further peripheral T cell depletion beyond that
achieved with irradiation would further increase the percentage of
NK1.1+ T cells in C57BL/6 mice, a group of mice
was given three doses of a T cell-depleting rabbit ATS on days 12, 14,
and 16, with the last of 17 doses of irradiation given on day 24 and
the first on day 0. Mice given both irradiation and ATS had 92.4%
NK1.1+ T cells among all T cells immediately
after the completion of the combined regimen (Fig. 1A).
Similarly, BALB/c mice given both irradiation and ATS had 91.8%
DX5+ T cells among all T cells (Fig. 1B). The mean increased percentages of
NK1.1+ and DX5+ T cells
among all T cells after irradiation and ATS was due to the more
complete depletion of NK1.1- and
DX5- T cells (350- to 1000-fold reduction)
compared with that of NK1.1+ and
DX5+ T cells (7- to 15-fold reduction; Table I).
In further phenotypic studies the expression of CD4 and CD8 markers on
NK1.1+ T cells in the C57BL/6 spleen before and
after irradiation was determined. Representative two-color flow
cytometric analyses of NK1.1 vs CD4 or CD8 markers on gated
NK1.1+ TCR+ T cells
before and after eight or 17 doses of irradiation are shown in Fig. 2A. The
CD4+NK1.1+ T cells remained
the major subset and increased from 62 to 76% of all
NK1.1+ T cells during the course of irradiation
(Fig. 2A). In contrast, the
CD8+NK1.1+ T cells
decreased from 14 to 4% during the same period (Fig. 2A).
The percentage of
CD4-CD8-NK1.1+
T cells was determined by staining with anti-NK1.1 mAb vs combined
anti-CD4 and CD8 mAbs. These double-negative
NK1.1+ T cells decreased slightly from 24 to
20% after irradiation (Fig. 2A).
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+ T cells in
the spleen of BALB/c mice. Fig. 2B shows that after TLI the
percentage of TCR+ T cells was 3%
(enclosed in box of left upper panel), and that 38% of
the T cells were DX5+ (right upper
panel). After two injections of anti-asialo-GM1 Abs, the
TCR+ T cells in the spleen of TLI-treated
mice were 1% (left lower panel) and the
discrete population of DX5+ cells among the gated
TCR+ T cells was no longer observed
(right lower panel). The percentage of
DX5+ cells among TCR+
T cells was reduced 10-fold in the latter mice. Thus,
anti-asialo-GM1 Abs effectively depleted
DX5+TCR+ T cells
compared with
DX5-TCR+ T
cells. Changes in T cell secretion of cytokines after TLI
Previous studies showed that just after the completion of TLI,
spleen T cells stimulated in vitro with anti-CD3 mAb secreted
markedly increased levels of IL-4 and reduced levels of IFN-
compared with T cells from unirradiated mice (24). In
addition, alloreactive T cell clones obtained within 3 wk after TLI
have been reported to be shifted toward a Th2 cytokine pattern
(25). Because NK1.1+ T cells secrete
very high levels of IL-4 (18, 19), we isolated the
NK1.1- and NK1.1+ T cell
from the C57BL/6 spleen after 17 treatments of TLI and compared their
capacities to secrete IL-4 and IFN- after stimulation in vitro with
PMA and calcium ionophore. The yield of sorted cells immediately after
TLI was too low for analysis; therefore, mice were rested for 7 days
before harvesting and sorting the spleen cells. At that time point,
there is a marked recovery in the absolute number of
NK1.1- TCR+ T cells.
Fig. 3A shows the two-color
profiles of the NK1.1+ and
NK1.1- TCR+ T cells
at that time point, and boxes R1 and R2 enclose the two populations of
sorted cells, respectively. After a 48-h stimulation, the mean
concentration of IL-4 in the supernatants from the sorted
NK1.1+ TCR+ T cells
was 2800 pg/ml, whereas the mean concentration in the supernatants
from the
NK1.1-TCR+cells was
100 pg/ml (Fig. 3B). Although the secretion of IL-4 was
markedly increased in NK1.1+ compared with
NK1.1- T cells, the secretion of IFN- was
slightly decreased (mean, 1600–1250 pg/ml; Fig. 3B).
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+ T cells after irradiation, sorted
TCR+ T cells were obtained from the spleen
of C57BL/6 and BALB/c mice before and after eight doses of irradiation.
At the latter point, the mean percentage of
NK1.1+ T cells among all T cells was 27% (Table I). Yields of sorted T cells immediately after eight doses of
irradiation were sufficient for analysis without resting the mice. Fig. 3C shows that the concentration of IL-4 from the sorted T
cells from C57BL/6 mice before irradiation was 75 pg/ml, whereas
after irradiation, the level increased 10-fold to 700 pg/ml. The
concentration of IFN- using cells harvested before irradiation was
1200 pg/ml, and increased 2-fold to 2200 pg/ml after
irradiation (Fig. 3C).
A similar increase in the concentration of IL-4 was observed with
sorted TCR+ T cells from BALB/c mice before
and after irradiation (Fig. 3C). Whereas the mean level
before irradiation was 200 pg/ml, after irradiation the mean level
rose to 800 pg/ml. Although a rise of the IL-4 secretion was
observed using C57BL/6 and BALB/c mice, the secretion of IFN- was
considerably different between the strains. Before irradiation, the
mean level of IFN- in the BALB/c mice (550 pg/ml) was about
one-half that of the C57BL/6 mice. After irradiation the level in
BALB/c mice did not change appreciably and was 4-fold below that of
the irradiated C57BL/6 mice. This resulted in a reversal of the ratio
of concentrations of IL-4:IFN- after irradiation in the two strains;
in C57BL/6 the ratio was 1:4, whereas in BALB/c mice it was
1:0.75. Before irradiation of C57BL/6 and BALB/c mice the ratios
were 1:16 and 1:3, respectively.
Protection against GVHD after TLI
NK1.1+ TCR+ T
cells from the BM of C57BL/6 donor mice inhibit the ability of donor
NK1.1- TCR+ T cells
to induce severe GVHD in lethally irradiated BALB/c hosts
(17). Inhibition of GVHD is dependent on the secretion of
IL-4 by NK1.1+ TCR+ T
cells (17). We theorized that the predominance of
DX5+ TCR+ T cells and
the increased secretion of IL-4 by all TCR+
T cells in BALB/c mice conditioned with TLI would protect against GVHD
induced by C57BL/6 T cells after allogeneic BM transplantation.
Accordingly, BALB/c mice were conditioned with either single-dose
lethal (800 cGy) TBI or 17 doses of TLI and given a single i.v.
injection of C57BL/6 BM cells with or without C57BL/6 PBMC.
Fig. 4A shows that the BALB/c
mice given 800 cGy TBI and an i.v. injection of C57BL/6 BM cells
(3 x 106) all survived at least 100 days
without clinical signs of GVHD. As shown above, addition of 0.5 x
106 C57BL/6 PBMC to the marrow resulted in the
death of all hosts by day 48 (Fig. 4A). Three i.p.
injections of ATS on days -12, -10, and -8 failed to protect the
irradiated hosts, and all died by day 44 (Fig. 4A). Control
hosts given irradiation without cells all died by day 14 (Fig. 4A). BALB/c hosts given sublethal (450 cGy) instead of
lethal (800 cGy) TBI uniformly died after the infusion of the combined
C57BL/6 marrow and PBMC transplantation with or without the addition of
ATS (data not shown).
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B). However, the
addition of 0.5 x 106 C57BL/6 PBMC to the
marrow cells resulted in the death of 60% of hosts by 100 days (Fig. 4B). Nevertheless, survival of the latter hosts was
significantly improved (p < 0.001, by log-rank
test) compared with that of hosts given TBI and the same cell inoculum.
Injection of ATS during the second week of TLI (days -12, -10, and
-8) protected the hosts further such that the combined infusion of
marrow and PBMC failed to kill any hosts during a 120-day observation
period (Fig. 4C). Control hosts given the same preparatory
regimen without a cell infusion or with only a marrow cell infusion all
survived at least 120 days also (Fig. 4C).
CD1-/- BALB/c hosts given the same preparatory regimen
and C57BL/6 BM and PBMC had increased mortality, and 70% died by day
35 (data not shown).
Histopathological analyses of the tissues of six host wild-type mice
that received either the TBI or TLI and ATS preparatory regimen and a
combined infusion of BM cells and PBMC were performed when hosts
developed clinical GVHD or when protected hosts were sacrificed >100
days after the cell infusions. In all cases microscopic analysis of the
liver, small and large intestines, and skin was performed using
hematoxylin and eosin staining to look for evidence of GVHD. All hosts
given TBI developed typical microscopic changes in GVHD in the skin,
including epidermal hyperplasia, a dermal inflammatory infiltrate,
subepidermal blistering, and necrotic keratinocytes (Fig. 5A). In the large intestine
changes included crypt apoptosis and inflammation, and atrophy of
mucin-containing glandular cells (Fig. 5B). These changes
were not observed after 100 days in six of six hosts given the TLI and
ATS regimen as shown in representative tissue secretions from a host in
Fig. 5, E and F. The epidermis was one to two
cells thick (Fig. 5E), and plump mucin-containing cells
lined the intestinal crypts (Fig. 5F). The microscopic
appearance of the skin and intestines of the latter hosts was similar
to that of control hosts given the TLI and ATS regimen and injected
with BM cells without PBMC (Fig. 5, C and D).
Examination of tissue sections from the liver and small intestines
showed minimal abnormalities in hosts given either TBI or TLI and ATS
and an injection of BM cells and PBMC (data not shown).
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To determine the extent of chimerism in long term surviving BALB/c
hosts, PBMC were harvested at 100 days after the allogeneic cell
infusion and stained for a donor-type surface marker using
anti-H-2Kb mAb vs T cell (Thy-1.2), B cell
(B220), and granulocyte (Gr-1) and macrophage (Mac-1) markers. Fig. 6A shows the two-color flow
cytometric analysis of PBMC from a host given TLI, ATS, and an infusion
of only C57BL/6 BM cells. Mixed chimerism was observed for T cells, B
cells, monocytes, and granulocytes, and donor cells from each lineage
(right upper quadrants) accounted for 5.1, 36.5, and 8.6%
of the PBMC, respectively. A similar pattern of mixed chimerism was
observed in BALB/c hosts given TLI, ATS, and a combined injection of
C57BL/6 marrow cells and PBMC (Fig. 6B). Hosts conditioned
with the myeloblative TBI regimen and given an injection of only
C57BL/6 marrow cells were complete chimeras for all lineages tested
(Fig. 6C). Table II summarizes
the mean percentages of donor-type cells within the PBMC harvested from
the different groups of surviving hosts at 100 days. All hosts given
TLI or TLI and ATS developed mixed chimerism after the injection of
allogeneic BM cells with or without donor PBMC (Table II). In contrast,
all hosts given TBI and a marrow cell infusion were complete
chimeras.
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To determine the effect of the host conditioning regimens on the
early expansion of donor T cells in the lymphoid tissues and on the
early invasion of donor T cells into the nonlymphoid tissues, BALB/c
hosts given either TLI and ATS or TBI and ATS, and an infusion of
C57BL/6 combined BM (3 x 106) and spleen
(6 x 106) cells were euthanized 12 days
after the cell injection. Spleen cells were used as a source of
peripheral T cells instead of PBMC to increase the number of peripheral
T cells infused to 2 x 106/host. A
variety of tissues were harvested from the hosts, and mononuclear cells
were enriched from each tissue as described in Materials and
Methods. Staining of donor T cells derived from the injected BM or
spleen was distinguished using congenic C57BL/6 donor mice expressing
either the CD45.1 marker (spleen cell donors) or the CD45.2 marker (BM
donors). BALB/c hosts expressed the CD45.2 marker. Fig. 7 shows the two-color flow cytometric
analysis of the harvested mononuclear cells stained for TCR vs
the CD45.1 marker. Almost all donor cells in hosts conditioned with TLI
or TBI and ATS were CD45.1 TCR+ T cells
(enclosed in boxes). Because both BM donors and hosts were CD45.2, the
latter T cells were derived from the injected donor spleen cells in all
host tissues tested, including peripheral blood, spleen, liver, gut,
and BM. In all tissues the percentages of donor T cells among the
mononuclear cells was markedly increased in hosts given TBI compared
with those given TLI (Fig. 7). This was particularly striking in the
blood, liver, and gut, where the differences were 15-, 14-, and
40-fold, respectively.
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shows the mean percentages and
absolute numbers of donor T cells derived from the injected C57BL/6
spleen cells in the mononuclear cells harvested from the different
tissues on day 12. The differences in the absolute number of cells in
the hosts conditioned with TLI or TBI reflect the differences in donor
T cell expansion and invasion into the host tissues. The largest
differences in absolute numbers were observed in the blood
(300-fold), gut (200-fold), and liver (1500-fold; Table III).
Thus, the ability of the TLI and ATS regimen to protect against GVHD
compared with TBI is reflected in the markedly reduced early expansion
of donor T cells in these hosts, especially in the nonlymphoid tissues,
despite the development of stable mixed chimerism. It is of interest
that the differences in donor T cell expansion in spleen and BM in TBI
and TLI conditioned hosts (3-fold) were considerably less than those
observed in the nonlymphoid tissues (200- to 1500-fold).
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To determine whether resistance of TLI-conditioned BALB/c hosts to
GVHD is dependent on host secretion of IL-4, experiments were performed
in which wild-type and IL-4-/- BALB/c hosts
were given TLI and BM cells from C57BL/6
IL-4-/- donors. These donors were used to
eliminate the contribution of donor IL-4 in vivo, because donor marrow
NK1.1+ T cells inhibit GVHD via IL-4 secretion
(17). Whereas 90% of the treated wild-type hosts given
IL-4-/- marrow cells survived 120 days,
IL-4-/- hosts died more rapidly
(p < 0.007, by log-rank test), and only 33%
survived during this time interval (Fig. 8B). In additional experiments
wild-type and IL-4-/- BALB/c hosts were given
TLI and marrow cells and PBMC from C57BL/6
IL-4-/- donors. Again, the survival of
IL-4-/- hosts was significantly reduced
(p < 0.03) compared with that of wild-type
hosts (Fig. 8B).
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+ T cells from TLI-treated wild-type
donor C57BL/6 spleen cells to inhibit GVHD induced by sorted
TCR+ T cells from untreated wild-type
C57BL/6 spleen cells in BALB/c hosts conditioned with TBI. Fig. 8A shows that control BALB/c hosts given TBI and 1.5 x
106 T cell-depleted marrow cells from untreated
C57BL/6 donors all survived at least 100 days. T cell-depleted marrow
cells were used to eliminate any regulatory effects of donor marrow T
cells on GVHD in these hosts. The addition of 0.1 x
106 sorted spleen T cells from untreated
wild-type donors to the marrow cells resulted in the death of 90% of
hosts by 90 days. In contrast, the addition of 0.1 x
106 sorted spleen T cells from TLI-treated
wild-type donors failed to induce GVHD, and all hosts survived 100 days
(Fig. 8A). When BALB/c hosts were given T cell-depleted
C57BL/6 marrow cells and a mixture of sorted spleen T cells from
untreated and treated wild-type donors, the rapidity of death was
significantly reduced compared with that in the group given only marrow
and sorted spleen T cells from untreated donors
(p < 0.5), as judged by the log-rank test. In
contrast, addition of spleen T cells from treated
IL-4-/- instead of treated wild-type donors
resulted in more rapid death of the hosts compared with the group given
marrow and untreated spleen T cells (Fig. 8A). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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after in vitro
activation with anti-CD3 mAb compared with normal spleen cells
(24). Thus, a change in the composition of T cells and
their cytokine profile after TLI was theorized to contribute to
protection against GVHD (24). Protection was also reported
to be dependent upon the presence of asialo-GM1+
cells, because depletion of the latter cells by injection of
anti-asialo-GM1 Abs into hosts given TLI markedly increased their
sensitivity to lethal GVHD after subsequent allogeneic BM
transplantation (22). Taken together these studies
suggested that T cells expressing asialo-GM1, a marker shared with NK
cells, may play an important role in ameliorating GVHD.
In the current study the role of NK1.1+
TCR+ T cells was investigated, because this
subset shares markers with NK cells, secretes high levels of IL-4, and
has recently been shown to protect against GVHD induced by
NK1.1- TCR+ T cells
(17). Immunofluorescent staining and multicolor analysis
of the spleen cells in C57BL/6 and BALB/c mice showed a progressive
increase in the percentage of NK1.1+
TCR+ T cells and
DX5+TCR+ T cells,
respectively, as the number of treatments of fractionated lymphoid
irradiation increased. After 17 treatments, the latter cells rose from
1% in unirradiated controls to 60–70% among the residual
TCR+ T cells. Addition of ATS to the
irradiation regimen resulted in a further increase to >90% of all
TCR+ T cells. Among the
NK1.1+ T cells, the predominant subset was
CD4+, and few CD8+ cells
were observed.
The marked change in the T cell subset composition occurred without an
expansion in the absolute number of NK1.1+ or
DX5+ T cells and was instead due to a more
profound depletion of NK1.1- and
DX5- T cells. This remarkable alteration in
subsets is probably explained by the ability of the BM that is shielded
during TLI to restore NK1.1+ T cells by virtue of
a rapid expansion of progenitor cells in response to peripheral
depletion (20). In contrast, replenishment of
NK1.1- or DX5- T cells is
likely to be delayed from progenitors in the thymus that are damaged
during thymic irradiation (28). Injection of
anti-asialo-GM1 Abs into BALB/c host given TLI rapidly depleted the
DX5+ TCR+ T cells and
indicated that these cells are asialo-GM1+. The
latter cells are likely to be the asialo-GM1+
cells reported previously to protect against GVHD after TLI
(22).
As expected, the sorted NK1.1+
TCR+ T cells in the spleen after TLI
secreted considerably higher levels of IL-4 after in vitro activation
compared with the sorted NK1.1-
TCR+ T cells. The increased proportion of
NK1.1+ and DX5+ T cells
among all T cells after TLI resulted in an altered cytokine secretion
pattern by all sorted TCR+ T cells from
C57BL/6 and BALB/c mice, such that the levels of IL-4 secretion and the
ratio of IL-4 to IFN- secretion were markedly increased. This is
likely to explain the high levels of IL-4 secreted in response to
anti-CD3 mAb activation of whole spleen cells after TLI as well as
the polarization toward Th2 alloreactive T cell clones reported
previously (24, 25).
Although BALB/c hosts conditioned with either lethal (800 cGy) or
sublethal (450 cGy) TBI with or without addition of ATS succumbed to
acute GVHD after an injection of combined C57BL/6 BM and PBMC, hosts
conditioned with TLI or TLI and ATS showed marked resistance to GVHD.
Even in the case of sublethally (450 cGy) irradiated hosts, infusion of
allogeneic peripheral blood cells resulted in the death of all
recipients by 14 days (29). Histopathologic analysis of
the liver, gut, skin, and pancreas of the hosts given TLI and ATS
showed no microscopic evidence of GVHD at 100 days after
transplantation. Nevertheless, these hosts were all mixed chimeras,
with donor-type cells found in all lineages tested, including T cells,
B cells, granulocytes, and macrophages. Hosts conditioned with TBI that
subsequently developed severe GVHD after combined marrow and spleen
cell transplantation showed a marked early expansion of donor-type T
cells derived from the injected spleen cells in lymphoid and
nonlymphoid tissues. The most dramatic expansion was observed in the
gut and liver, with 35–70% of mononuclear cells extracted from these
tissues on day 12 being made up of donor spleen-derived
TCR+ T cells. In contrast, minimal early
expansion of donor T cells was observed in the same tissues in hosts
conditioned with TLI and ATS, and only 2% of mononuclear cells from
the gut and liver were donor T cells. It is likely that the inability
of the donor T cells to invade and expand especially in the host
nonlymphoid tissues is responsible for protection after the TLI and ATS
regimen.
Because the increase in the percentage of
DX5+asialo-GM1+TCR+
T cells in BALB/c hosts given TLI was associated with increased IL-4
secretion and protection against GVHD, we compared the severity of GVHD
in wild-type and IL-4-/- hosts conditioned with
TLI. These hosts received BM cells with or without blood mononuclear
cells from IL-4-/- C57BL/6 donors to eliminate
donor contribution of IL-4. The survival of the wild-type hosts was
significantly improved compared with that of the IL-4-/-
or CD1-/- hosts, indicating that host CD1-reactive cells
that express/secrete IL-4 contribute to protection against GVHD.
In additional experiments, sorted splenic T cells from C57BL/6
wild-type donors conditioned with TLI inhibited the ability of
untreated wild-type donor splenic T cells to induce GVHD in wild-type
BALB/c hosts conditioned with TBI. The inhibitory activity of the T
cells from TLI-treated donors was expected based on our previous
studies of natural suppressor T cells found after TLI
(27).
The current study shows that this inhibitory activity is probably
mediated by the secretion of IL-4 by the adoptively transferred T
cells, because sorted T cells from IL-4-/-
C57BL/6 mice given TLI failed to inhibit GVHD and worsened the survival
of BALB/c hosts. The cellular mechanisms by which IL-4 protects against
GVHD are not clear. One possibility is that IL-4 regulates the
interaction between donor T cells and host APCs such that donor T cell
activation is impaired or shifted to a Th2 pattern with reduced GVHD
potency (30, 31). In vitro PMA and ionomycin activation of
sorted chimeric donor and host cells obtained from TLI-treated hosts
100 days after BM transplantation showed secretion of a Th2 pattern of
cytokines (high IL-4 and low IFN-; data not shown). It is possible
that secretion of IFN- by host T cells also plays a role in
protection. However, we did not address this issue in the current
study.
In conclusion, the T cells with the predominant NK1.1+ and DX5+ asialo-GM1+ subsets found after fractionated lymphoid irradiation can transfer protection to hosts against GVHD induced by donor alloreactive T cells. Cloned NK1.1+ T cell lines have been reported to protect against GVHD (32). In addition, protection is lost after depletion of asialo-GM1+ or IL-4-secreting cells. The experimental findings indicate that the residual host NK1.1+ and DX5+asialo-GM1+ T cells prevent the rapid expansion and invasion of donor T cells in the host nonlymphoid target tissues of GVHD and affect the patterns of host and donor T cell cytokine secretion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Samuel Strober, Division of Immunology and Rheumatology, Department of Medicine, CCSR Building, Room 2215-C, Stanford Medical Center, MC5166, 300 Pasteur Drive, Stanford, CA 94305-5166. E-mail address: sstrober{at}stanford.edu
3 Abbreviations used in this paper: BM, bone marrow; GVHD, graft-vs-host disease; ATS, anti-mouse thymocyte serum; TLI, total lymphoid irradiation; TBI, total body irradiation.
Received for publication December 20, 2000. Accepted for publication June 14, 2001.
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