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 Previous Article | Table of Contents | Next Article 
Blood, 15 February 2002, Vol. 99, No. 4, pp. 1449-1457
TRANSPLANTATION
Unique patterns of surface receptors, cytokine secretion, and
immune functions distinguish T cells in the bone marrow from those in
the periphery: impact on allogeneic bone marrow transplantation
Defu Zeng,
Petra Hoffmann,
Fengshuo Lan,
Philip Huie,
John Higgins, and
Samuel Strober
From the Department of Medicine, Division of Immunology
and Rheumatology, and the Department of Pathology, Stanford University
School of Medicine, CA.
 |
Abstract |
The "conventional" NK1.1 T cells from mouse blood
and marrow were compared with regard to surface receptors, cytokine
secretion, and function. Most blood NK1.1
CD4+ and CD8+ T cells expressed the naive
CD44int/loCD62LhiCD45RBhi
T-cell phenotype typical of those in the peripheral
lymphoid tissues. In contrast, most marrow NK1.1
CD4+ and CD8+ T cells expressed an
unusual CD44hiCD62LhiCD45RBhi
phenotype. The blood NK1.1 CD4+ T cells had a
naive T-helper cytokine profile and a potent capacity to induce lethal
graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host
bone marrow transplantation model. In contrast, the marrow
NK1.1 CD4+ T cells had a Th0 cytokine profile
and failed to induce lethal GVH disease, even at 20-fold higher numbers
than those from the blood. NK1.1 CD8+ T cells
from the blood but not the marrow induced lethal GVH disease.
Nevertheless, the marrow NK1.1 CD8+ T cells
induced potent antitumor activity that was augmented by marrow
NK1.1 CD4+ T cells and facilitated
hematopoietic progenitor engraftment. The inability of marrow
CD4+ and CD8+ T cells to induce GVH disease was
associated with their inability to expand in the blood and gut of
allogeneic recipients. Because neither the purified marrow
CD4+ or CD8+ T cells induced GVH disease, their
unique features are desirable for inclusion in allogeneic bone marrow
or hematopoietic progenitor transplants.
(Blood. 2002;99:1449-1457)
© 2002 by The American Society of Hematology.
| |
Introduction |
Mature T-cell receptor   +
(TCR+) T lymphocytes in the bone marrow (BM) are
located in an environment that is filled predominantly with
hematopoietic cells. This unique juxtaposition of immune T cells and
hematopoietic cells may require specialization of the marrow immune
cells as compared with those in the lymph nodes, spleen, and blood,
because vigorous immune responses including secretion of cytokines have
the potential to dysregulate hematopoiesis. T cells make up only 1% to
3% of nucleated cells in the marrow.1,2 The balance of
subsets among the marrow T cells is unusual, and NK1.1+
TCR+ T cells and
CD4CD8TCR+ T
cells are present at levels that are 10- to 30-fold higher than that in
the peripheral lymphoid tissues.2-5 The marrow
NK1.1+ TCR+ T cells appear to play a
regulatory role and can suppress immune responses such as the induction
of graft versus host (GVH) disease1 mediated by combined
marrow NK1.1 CD4+ and CD8+
TCR+ T cells.2-5 The
CD4CD8TCR+ marrow T cells
inhibit the mixed leukocyte reaction of peripheral T
cells.6
Although regulatory NK1.1+ T cells in the marrow can
suppress immune responses of NK1.1 T cells, it is unclear
whether the NK1.1 CD4+ and CD8+ T
cells in the marrow are similar or identical to those in the peripheral
lymphoid tissues. A recent study suggested that a high proportion of
marrow NK1.1 CD4+ T cells expressed the
activated/memory
(CD44hiCD62LloCD45RBlo) phenotype
as compared with CD4+ T cells in the
periphery.7 Our previous study indicated that marrow
NK1.1 CD4+ and CD8+ T cells
sorted in a single pool expressed a cytokine secretion pattern that
differed from that of peripheral T cells, and their capacity to induce
GVH disease was reduced as compared with T cells in the
blood.5 Furthermore, marrow NK1.1
TCR+ T cells may be derived from an extrathymic
T-cell developmental pathway in the BM itself.8,9 Several
laboratories have shown that marrow CD8+ T cells are able
to mediate vigorous graft versus tumor activity without GVH disease and
to facilitate engraftment of hematopoietic progenitor cells in
irradiated allogeneic hosts.10-12
In the current report, the surface phenotype, cytokine secretion
profile, and function of highly purified NK1.1
CD4+ or NK1.1 CD8+
TCR+ T cells in the BM were systematically compared
with the same T-cell subsets in the blood. The blood CD4+
and CD8+ T cells expressed the dominant naive T-cell
surface phenotype (CD44int/loCD62LhiCD45RBhi), and
the blood CD4+ T cells had the typical naive T-helper
cytokine secretion profile (high interleukin [IL]-2; low interferon
[IFN]- , IL-4, and IL-10) after activation in vitro. However, most
T cells in the marrow expressed an unusual
CD44hiCD62LhiCD45RBhi phenotype,
and the marrow CD4+ T cells had an unusual cytokine pattern
(high IFN-, IL-4, and IL-10) that was similar to the Th0 pattern.
Functional assays showed that both the purified blood
NK1.1 CD4+ and the NK1.1
CD8+ T cells induced lethal GVH disease in the C57BL/6
donor to BALB/c host combination. The former cells were at least
20-fold more potent than the latter. Neither the purified marrow
NK1.1 CD4+ nor the NK1.1
CD8+ T cells induced lethal GVH disease even at high cell
numbers. Nevertheless, the marrow NK1.1 T cells mediated
vigorous graft versus tumor activity and facilitated engraftment of
hematopoietic progenitor cells.
| |
Materials and methods |
Mice and monitoring of GVH disease
C57BL/6 (H-2b) and BALB/c
(H-2d) wild-type mice (CD45.2) were obtained
from the breeding facility of the Department of Comparative Medicine,
Stanford University. The C57BL/6 Rag2/ mice and
congenic C57BL/6 (CD45.1) mice were obtained from Jackson Laboratory,
Bar Harbor, ME. Only male mice were used at 8 to 12 weeks of age. Care
of all experimental animals was in accordance with institutional
guidelines. In BM transplantation studies, host BALB/c mice were given
8 Gy total body irradiation1 from a 250 kV x-ray
source and injected with C57BL/6 donor cells via the tail vein within
24 hours. Survival and appearance of mice were monitored daily, and
body weight was measured weekly. Mean body weights of surviving mice in
each group were determined at 100 days. Chimerism of hosts before and
after 100 days was respectively measured by staining peripheral blood
(PB) mononuclear cells from Ficoll-Hypaque gradients or spleen cells
with fluorochrome-conjugated anti-H-2b monoclonal
antibodies (mAbs) (Pharmingen, San Diego, CA) and analysis by one-color
flow cytometry.
BCL1 tumor cell passage and injection
BCL1 is a B-cell leukemia/lymphoma derived from a
BALB/c mouse with an IgM surface immunoglobulin
phenotype.13 This cell line was maintained by serial
passage in BALB/c mice as described previously.13 After
appropriate dilution, 60 BCL1 cells were coinjected with BM
cells into lethally irradiated BALB/c recipients.
Monoclonal antibodies; immunofluorescent staining and flow
cytometric analysis
BM cells were obtained from the femur and tibia and stained with
mAbs as described previously.2,5 Stainings were performed in the presence of anti-CD16/32 (2.4G2, Pharmingen) at saturation to
block FcRII/III receptors, and propidium iodide (Sigma Chemicals, St
Louis, MO) was added to staining reagents to exclude dead cells. Three-color FACS analysis was performed using a modified dual laser
FACS Vantage (Becton Dickinson, Mountain View, CA), and data were
analyzed using FACS/Flow Jo software (Becton Dickinson).14 The following conjugated antibodies were used for staining: fluorescein isothiocyanate (FITC)-anti-CD8 (CT-CD8) and streptavidin-Texas Red
purchased from Caltag, South San Francisco, CA; and allophycocyanin- and phycoerythrin (PE)- and FITC-anti-TCR (H57-597),
PE-anti-NK1.1 (PK136), FITC-anti-CD4 (RM4-5), FITC-anti-CD44 (IM7),
unconjugated anti-CD16/CD32 (2.4G2), FITC-anti-CD45.2 (104), FITC-
and PE-anti-H-2Kb (AF6-88.5), PE-anti-B220 (RA3-6B2),
PE-anti-Gr-1 (RB6-8C5), PE-anti-MAC-1 (M1/70), FITC-anti-CD62L
(MEL-14), and FITC-anti-CD45RB (16A) purchased from Pharmingen.
APC-anti-CD4 and APC-anti-CD8a were gifts from Dr Irving Weissman's
laboratory at Stanford. FITC-anti-BCL1-Id (6A5.1) mAb was
obtained from hybridoma supernatants and conjugated as described
previously.12
Sorting of blood and BM T cells
The CD4+, CD8+, and
CD4+/CD8+TCR+
(NK1.1+ or NK1.1) T cells from the blood or
marrow were obtained by flow cytometry using FACStar or FACS Vantage
(Becton Dickinson) after enrichment of blood and marrow T cells on
immunomagnetic bead columns (Miltenyi, Auburn, CA) as described
previously.2,5 The stringently T-cell-depleted (TCD)
marrow cells (CD4CD8/)
were also obtained by flow cytometry as described
previously.5
Liver and gut lymphocyte preparation
After hosts were exsanguinated, livers were flushed with
heparinized phosphate-buffered saline in the right ventricle until the
liver became pale. Livers were pressed through a nylon mesh to prepare
single-cell suspensions in 2 µM ethylenediaminetetraacetic acid in
phosphate-buffered saline. 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 ethylenediaminetetraacetic acid in phosphate-buffered
saline, minced into smaller than 5 mm segments, and suspended by vortex
20 to 30 seconds. After 3 repeats, cells in the supernatant were
filtered through a nylon mesh before separation on a Ficoll-Hypaque
gradient for collection of mononuclear cells.
In vitro secretion and measurement of cytokines
Sorted T cells (1 × 105) from the blood or BM
were stimulated in vitro with 20 ng/mL phorbol myristate acetate (Sigma
Chemicals) and 1µM ionomycin (Calbiochem, San Diego, CA) in 10%
fetal bovine serum and RPMI complete medium in 96-well round bottom
plates and harvested at the peak time point (48 hours) as described
previously.2,5,15 Supernatants were assayed for the
secretion of IL-2, IFN-, IL-4, and IL-10 using commercial
enzyme-linked immunosorbent assay kits (Biosource, Camarillo,
CA).2,5,15
Histopathology of skin and large intestine
Histopathologic specimens from the skin and large intestines of
hosts were obtained at 40 to 60 days or 100 days after transplantation and fixed in formalin before embedding in paraffin blocks. Tissue sections were stained with hematoxylin and eosin and examined at 400× magnification.
Statistical analysis
Differences in survival of groups of hosts given BM transplants
were analyzed using the log-rank test. Differences in donor-type T-cell
recovery in tissues of hosts were analyzed using the 2-tail Student t test.
| |
Results |
Different patterns of surface receptor expression on
CD4+ and CD8+ T cells from blood
and BM
Our previous studies showed that NK1.1+ T cells make
up about 30% to 50% of TCR+ T cells in the marrow
but only about 1% in the blood.2,5 NK1.1+ T
cells have been previously reported to be
CD44hiCD45RBhi.4 In the current
study, we studied the expression of CD44 on the NK1.1+ and
NK1.1 CD4+ and CD8+ T cells from
blood and marrow by multicolor flow cytometry after staining with mAbs.
As shown in Figure 1A, 26.4% of blood
mononuclear cells were CD4+TCR+ T cells.
The mean (± SE) percentage in 4 mice was 23% ± 2%. After gating
on the CD4+TCR+ cells, only 0.8% of the
cells were found to be NK1.1+, and 99.2% were
NK1.1 (Figure 1B; mean 0.7% ± 0.1% and
99.3% ± 0.1%, respectively). TCR+CD4+ cells were gated into
NK1.1+ and NK1.1 cells for single-color
analysis of CD44. Blood NK1.1 CD4+ cells were
found to be mostly CD44int/lo (Figure 1C, open profile,
left panel). CD4+TCR+ cells accounted for
0.4% of cells in the marrow (Figure 1A), and 25.4% of them were
NK1.1+, and 74.6% were NK1.1 (Figure 1B;
mean 27% ± 1% and 73% ± 2%, respectively). In contrast to
blood NK1.1 CD4+ cells, most of the marrow
NK1.1 CD4+ cells were CD44hi
(Figure 1C, shaded profile, left panel). About 16.3% of PB mononuclear cells were CD8+TCR+ cells (Figure 1A;
mean 13% ± 2%). Only 0.9% of the latter cells were
NK1.1+ (mean 1.0% ± 0.2%), and there were insufficient
PB NK1.1+ CD8+ cells for further analysis. Most
blood NK1.1 CD8+ cells were also
CD44int/lo (Figure 1D, open profile, left panel).
TCR+CD8+ cells in the marrow accounted
for 1.1% of nucleated cells (Figure 1A), and 15.8% were
NK1.1+ (mean 0.9% ± 0.2% and 18% ± 1%,
respectively). In contrast to the blood NK1.1
CD8+ cells, most NK1.1 CD8+ cells
were CD44hi (Figure 1D, shaded profile, left
panel).
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| Figure 1.
Two-color flow cytometric analysis of
TCR+ T cells from PB and BM of C57BL/6 mice.
(A) Staining of TCR versus CD4 or CD8. (B) The analysis of the
gated TCR+CD4+ or
TCR+CD8+ cells from panel A for NK1.1
versus CD4 or CD8. (C) One-color analysis of the gated
NK1.1 CD4+TCR+ from PB
(open profiles) or BM (shaded profiles) for CD44, CD62L, and CD45RB.
(D) One-color analysis of the gated NK1.1
CD8+ TCR+ T cells. Percentages of gated
cells are shown above the boxes. One representative of 4 is
shown.
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Because CD44 is expressed at high levels in early T-cell development in
the thymus and on activated/memory T cells,16,17 the
expression of CD62L and CD45RB was studied also. The latter markers are
down-regulated on activated/memory cells and present at higher levels
on naive T cells.18,19 As shown in Figure 1D, both blood
and marrow NK1.1 CD8+ T cells expressed high
levels of CD62L and CD45RB. Thus, the blood NK1.1
CD8+ T cells showed a typical naive T-cell pattern
(CD44int/loCD62LhiCD45RBhi), but
the marrow NK1.1 CD8+ T cells showed an
unusual CD44hiCD62LhiCD45RBhi
pattern. Figure 1C shows that blood NK1.1
CD4+ T cells had a naive
CD44int/loCD62LhiCD45RBint T-cell
pattern. However, the marrow NK1.1 CD4+ T
cells had a down-regulated bimodal pattern of CD62L expression and a
bimodal down-regulated pattern of CD45RB expression. Most expressed
either low ( channel 1) or intermediate (channel 1-10) levels, and
the remainder expressed high ( channel 10) levels of CD45RB.
Two-color analysis of CD44 versus CD62L or CD44 versus CD45RB on the
latter cells revealed that 30% to 50% had an unusual CD44hiCD62LhiCD45RBint/hi phenotype
similar to the marrow NK1.1 CD8+ T cells
(data not shown). The remainder of the
CD44hiNK1.1 CD4+ T cells
expressed the CD62LloCD45RBlo activated/memory
cell phenotype.
Different cytokine secretion profiles of CD4+ and
CD8+ T cells from blood and BM
To study the cytokine secretion profiles of the blood and marrow
T-cell subsets, all T cells were first enriched with immunomagnetic beads, and desired subsets were thereafter isolated with flow cytometry. Sorted subsets (100 × 103) were stimulated
with phorbol myristate acetate and calcium ionophore in vitro for 48 hours. The culture supernatants were assayed with enzyme-linked
immunosorbent assay for the concentration of IL-2, IFN-, IL-4, and
IL-10. As shown in Table 1, sorted blood
CD4+ T cells (> 99% NK1.1) produced large
amounts of IL-2 (mean 2856 pg/mL) but little IFN- (mean 78 pg/mL),
IL-4 (mean 26 pg/mL), or IL-10 (mean 17 pg/mL). In contrast, sorted
marrow CD4+ T cells containing about 25%
NK1.1+ cells (Figure 1) produced about 5 times less IL-2
but 10 times more IFN- and 20 times more of the Th2 cytokines IL-4
and IL-10. The sorted marrow NK1.1 CD4+ T
cells still produced 7-fold less IL-2 but about 10 times more IFN-,
IL-4, and IL-10 as compared with the sorted blood CD4+ T
cells. Marrow CD4+ NK1.1+ T cells produced
small amounts of IL-2 (mean 355 pg/mL) and large amounts of IFN-
(mean 1051 pg/mL), IL-4 (mean 3072 pg/mL), and IL-10 (mean 380 pg/mL).
When the relative concentrations of IL-2, IFN-, IL-4, and IL-10 were
compared (Table 2), PB CD4+ T
cells had extremely high ratios of IL-2/IFN- (37:1), IL-2/IL-4 (110:1), and IL-2/IL-10 (168:1) as compared with those from BM CD4+, CD4+NK1.1+, and
CD4+NK1.1 T cells (all < 3:1). On the other
hand, ratios of IFN-/IL-4 or IFN-/IL-10 were low (all < 5:1)
for all CD4+ T cells from blood and marrow (Table 2). As
compared with blood CD4+ T cells, the blood
CD8+ T cells produced about 2-fold less IL-2 (mean 1505 pg/mL) and 20-fold more IFN- (mean 1403 pg/mL) but similar amounts
of IL-4 (mean 20 pg/mL) and IL-10 (mean 42 pg/mL) (Table 1). This
resulted in high ratios (> 33:1) of the Th1 (IL-2 or IFN-) to Th2
(IL-4 or IL-10) cytokines for blood CD8+ T cells but a low
ratio of IL-2/IFN- (Table 2). The latter ratio was markedly reduced
as compared with blood CD4+ T cells (Table 2).
Comparison of cytokine profiles from blood CD8+ T (99%
NK1.1) and marrow NK1.1 CD8+ T
cells showed both had high ratios of Th1 (IL-2 or IFN-) to Th2 (IL-4
or IL-10) cytokines (Table 2). The marrow
NK1.1+CD8+ T cells produced small amounts of
IL-2 (mean 700 pg/mL) but large amounts of IFN- (mean 2074 pg/mL),
IL-4 (mean 2248 pg/mL), and IL-10 (mean 224 pg/mL). Inclusion of the
NK1.1+ T cells among marrow CD8+ T cells
resulted in increased levels of IL-4 and IL-10 as compared with marrow
NK1.1 CD8+ T cells (Table 1).
Differences in the function of CD4+ and
CD8+ T cells from blood and BM in the GVH disease
assay
Because blood and marrow CD4+ and CD8+
T-cell subsets had markedly different surface receptor expression
and/or cytokine secretion profiles, we tested their function in a model
of GVH disease using major histocompatibility complex-mismatched
C57BL/6 (H-2b) donors and BALB/c (H-2d)
hosts.5 Graded numbers of sorted blood and marrow T-cell subsets from the donor mice were coinjected with
1.5 × 106 stringently TCD donor marrow cells into
lethally irradiated 8 Gy (800 rads) hosts. As shown in Figure
2A, all the recipients given TCD marrow
cells alone survived for more than 100 days without any signs of GVH
disease, and the recipients given no TCD marrow cells all died by day
14 (data not shown). Addition of graded numbers
(25 × 103-500 × 103) of sorted blood
CD4+ T cells induced severe diarrhea, weight loss, and
mortality in a dose-dependent manner but no hair loss. The survival was
significantly reduced as compared with that of the recipients given TCD
marrow alone (P < .001, log-rank test). In contrast,
addition of 500 × 103 sorted marrow CD4+
(NK1.1+ and NK1.1) T cells induced no
clinical signs of GVH disease, and all the recipients survived for more
than 100 days (Figure 2A). Furthermore, addition of
500 × 103 marrow NK1.1 CD4+ T
cells induced only transient and mild signs of GVH disease, such as
slight diarrhea and weight loss between days 40 to 60 after
transplantation (data not shown), and all hosts survived for more than
100 days.
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| Figure 2.
Marked difference in the ability of PB and BM
CD4+ and CD8+ T cells to induce lethal GVH
disease.
Graded numbers of sorted CD4+, CD8+, or
CD4+/CD8+ (CD4+ and
CD8+ together as a pool) T cells from C57BL/6 donors were
added to a constant number (1.5 × 106) of C57BL/6 TCD BM
cells and injected intravenously into lethally irradiated (8 Gy) BALB/c
hosts. Survival of hosts over a 100-day observation period is shown in
groups of 10 mice. Data are combined from 2 replicate experiments. (A)
Graded numbers of sorted PB and BM CD4+ T cells. (B) Graded
numbers of PB and BM CD8+ T cells. (C) Graded numbers of PB
and BM CD4+/CD8+ T cells.
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The colon and skin tissues were harvested from moribund recipients
given blood CD4+ T cells between days 45 to 60. For
comparison, tissues from recipients given marrow CD4+ or
NK1.1 CD4+ T cells were harvested on day 45 as well, and both sources of tissues were studied for histopathology
(Figure 3A-F). As shown in Figure 3A, the
blood CD4+ T cells induced severe intestinal lesions
associated with GVH disease. The lamina propria was expanded and
markedly infiltrated with lymphocytes (asterisk). The atrophic
intestinal crypts were depleted of plump mucin-containing glandular
cells, and intraepithelial lymphocytic infiltration (black arrows) was
observed along with apoptotic bodies (white arrows), indicating crypt
cell death. Consistent with the lack of hair loss, recipients
had no skin lesions (Figure 3B). The marrow CD4+ T cells
induced no clinical GVH disease, and their large intestine and skin
tissues looked normal (Figure 3C,D). The marrow NK1.1
CD4+ T cells induced characteristic microscopic GVH disease
lesions in the colon (Figure 3E), although they were less severe than those induced by blood CD4+ T cells. The marrow
NK1.1 CD4+ T cells induced no microscopic
skin lesions (Figure 3F). There was no noticeable difference in body
weight and tissue histology among the recipients given TCD marrow
alone, TCD marrow with marrow CD4+ T cells, or
NK1.1 CD4+ T cells 100 days after
transplantation (data not shown). These results indicate that marrow
NK1.1 CD4+ T cells can induce typical but
mild histologic changes of GVH disease in the colon with mild transient
clinical signs of GVH disease.
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| Figure 3.
Ability of CD4+ and
CD8+ T-cell subsets from PB and BM to induce
histopathologic lesions of GVH disease in the colon and skin.
Sections are stained with hematoxylin and eosin and the original
magnification is × 400. Each panel is representative of 4 recipients.
(A,B) Intestine and skin of a BALB/c recipient with severe clinical GVH
disease injected 50 days earlier with TCD BM and sorted PB
CD4+ T cells. There is an expanded lamina propria with
marked lymphocytic infiltration (asterisk) and infiltration into the
glandular epithelium (black arrow). There are apoptotic bodies in the
glandular epithelium also (white arrow). The skin appears to be normal.
(C,D) The colon and skin sections of a healthy recipient injected 45 days earlier with TCD BM and BM CD4+ T cells. Plump
mucin-containing glandular cells are seen lining the crypts with little
or no inflammation. The skin appears to be normal. (E,F) The tissue
sections of a recipient with slight diarrhea injected 45 days earlier
with BM NK1.1 CD4+ T cells. The colon shows
lesions of GVH disease, but the skin appears to be normal. (G,H) The
sections of a recipient with severe clinical GVH disease injected 50 days earlier with TCD BM and PB CD8+ T cells. The colon
shows lesions of GVH disease with lymphocytic infiltration of the
lamina propria (asterisk) and crypts (black arrows) and apoptotic crypt
cells (white arrow). The skin shows hyperplasia (black arrow) and
microabscesses (white arrow) in the epidermis and lymphocyte
infiltration in the dermis (asterisk). (I,J) The sections of a healthy
recipient injected 45 days earlier with TCD BM and BM CD8+
T cells. No abnormalities are seen. (K,L) The sections of a recipient
with slight diarrhea and hair loss injected 45 days earlier with TCD BM
and BM NK1.1 CD8+ T cells. There are lesions
of GVH disease in both tissues, including inflammation of intestine
crypts, epidermal hyperplasia, and dermal
infiltration.
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The capacity of blood and marrow CD8+ T-cell subsets to
induce GVH disease were compared also. As shown in Figure 2B, TCD
marrow cells alone induced no GVH disease, but addition of
500 × 103 blood CD8+ T cells induced severe
GVH disease, and 70% of the recipients died within 100 days. Addition
of 100 × 103 blood CD8+ T cells still
induced typical clinical signs of GVH disease, but only 10% of the
recipients died (Figure 2B). In contrast, addition of
500 × 103 marrow CD8+ (NK1.1+
and NK1.1) T cells induced no clinical signs of GVH
disease or death. The addition of 500 × 103 marrow
NK1.1 CD8+ T cells induced only transient
mild signs of GVH disease, such as slight diarrhea, weight loss, and
hair loss between days 40 to 60, and all the latter hosts survived for
more than 100 days (Figure 2B). Colon and skin tissues from the
moribund recipients given blood CD8+ T cells were harvested
between days 45 to 60, as were the tissues from recipients given marrow
CD8+ or NK1.1 CD8+ T cells
(Figure 3G-L). The blood CD8+ T cells induced
characteristic GVH disease lesions in the colon with a marked
lymphocytic infiltration in the lamina propria (asterisk) and between
glandular epithelium (black arrow) (Figure 3G). Crypt cell death with
appototic bodies (white arrow) was observed as well as depletion of
plump mucin-containing glandular cells. In the skin (Figure 3H), there
was hyperplasia (black arrow), neutrophilic microabscesses (white
arrow) in the epidermis, and lymphocytic infiltration in the dermis
(Figure 3H). The marrow CD8+ T cells induced no microscopic
evidence of GVH disease, and the intestine and skin tissues looked
normal (Figure 3I,J). However, the marrow NK1.1
CD8+ T cells induced characteristic GVH disease lesions in
the colon and skin tissues between days 40 to 60 (Figure 3K,L). There
were also no clear differences among the recipients given TCD marrow alone, TCD marrow with marrow CD8+ T cells, or
NK1.1 CD8+ T cells 100 days after
transplantation with regard to appearance, body weight, and histology,
indicating that any mild transient GVH disease had resolved by this
time point (data not shown). Although both blood CD4+ and
CD8+ T cells induced severe lethal GVH disease, the former
were still 20 times more potent than the latter (Figure 2A,B). Blood
CD4+ and CD8+ T cells also had a synergistic
effect, because as few as 6.25 × 103 blood
CD4+ and CD8+ T cells, sorted as a single pool
(CD4+/CD8+ T), induced a similar pattern of
mortality (Figure 2C) as that of 25 × 103 blood
CD4+ T cells or 500 × 103 blood
CD8+ T cells (Figure 2A,B, P > .1, log-rank
test). In contrast, addition of 500 × 103 marrow
CD4+/CD8+ T cells induced no clinical signs of
GVH disease or mortality (Figure 2C).
Different tissue distribution of blood and marrow CD4+
and CD8+ T cells in irradiated allogeneic hosts
To distinguish donor cells derived from the injected blood or
marrow T cells from those derived from the TCD marrow cells, blood
CD4+/CD8+ or marrow
CD4+/CD8+ T cells (500 × 103)
from C57BL/6 (H-2b, CD45.2) mice were coinjected with TCD
marrow cells (1.5 × 106) from congenic C57BL/6 mice
(H-2b, CD45.1) into lethally irradiated BALB/c recipients
(H-2d, CD45.2). Seven days later, the mononuclear cells
from blood, gut, spleen, liver, and marrow were harvested and stained
with anti-TCR, anti-H-2b, and anti-CD45.2 mAbs.
Figure 4 shows the percentage of injected
donor T cells and/or their progeny among the mononuclear cells from
different tissues of the BALB/c recipients. About 20% of the blood
mononuclear cells from the recipients given sorted blood
CD4+/CD8+ T cells and TCD marrow cells were
donor-type T cells (TCR+H-2b+, enclosed
in box in Figure 4A). Gated donor-type T cells were all
CD45.2+ (Figure 4A,B), derived from the injected donor
blood T cells. In contrast, only 0.5% of the blood mononuclear cells
from the recipients given sorted marrow
CD4+/CD8+ T cells and TCD marrow cells were
donor-type T cells, and insufficient numbers of latter cells were
available for analysis of CD45.2 expression (Figure 4C,D). Similarly,
88% of the mononuclear cells from the gut of the recipients given
blood CD4+/CD8+ T cells and TCD marrow cells
were donor-type T cells (Figure 4E), but only 7% of the mononuclear
cells from the gut of the recipients given marrow
CD4+/CD8+ T cells and TCD marrow cells were
donor-type T cells (Figure 4G). Again, all donor-type T cells were
derived from the injected sorted T cells (Figure 4 F,H). There were
high levels (86% and 74%) of donor-type T cells in the liver tissues
of the recipients given blood CD4+/CD8+ T cells
or marrow CD4+/CD8+ T cells (Figure 4I,K),
respectively, and all were derived from injected T cells (Figure 4J,L).
Analysis of the spleen and marrow cells showed that donor-type T cells
accounted for less than 10% of mononuclear cells (data not shown).
Further analysis of the recovered donor-type T cells from different
tissues showed that they were all CD4+ or CD8+
NK1.1 TCR+ T cells (data not shown).
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| Figure 4.
Different tissue distributions of PB and BM T cells in
the allogeneic recipients at 7 days after BM transplantation.
Lethally irradiated BALB/c recipients (H-2Kd) were injected
with 1.5 × 106 donor C57BL/6 (H-2Kb, CD45.1)
TCD BM and 500 × 103 PB
CD4+/CD8+ or BM
CD4+/CD8+ T cells from congenic C57BL/6
(H-2Kb, CD45.2) mice. The mononuclear cells from blood,
gut, and liver of the recipients were harvested and stained with
anti-H-2Kb, TCR, and CD45.2 mAbs. In the first
column, panels A, E, and I show the tissues from the recipients given
PB CD4+/CD8+ T cells, and panels C, G, and K
show the tissues from the recipients given marrow
CD4+/CD8+ T cells. Analyses in panels A, C, E,
G, I, and K show TCR versus H-2Kb with
TCR+H-2Kb+ cells enclosed in boxes. In
the second column, the gated TCR+H2kb+
cells were analyzed further for TCR versus CD45.2 in panels B, D,
F, H, J, and L. Each panel is representative of 4 recipients.
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The mean percentages and yields of donor-type T cells from different
tissues are shown in Table 3. The mean
absolute numbers of the donor-type T cells in blood, gut, and spleen
from the recipients given blood CD4+/CD8+ T
cells were found to be respectively 40-fold (P < .001),
20-fold (P < .001), and 2-fold (P < .05)
higher than that in the recipients given marrow
CD4+/CD8+ T cells. However, there was no
significant difference (P > .1) in the livers and marrow
of the 2 kinds of recipients. These results indicate that the marked
difference in the capacity of blood and marrow
CD4+/CD8+ T cells to induce GVH disease is
associated with their markedly different early expansion in the blood
and gut tissues of the recipients.
Function of BM CD4+ and CD8+ T cells in
graft versus tumor and facilitation of engraftment
assays
To test the antitumor activity of marrow CD4+ and
CD8+ T cells, graded numbers of sorted T cells were
coinjected with 1.5 × 106 C57BL/6 Rag-2/
marrow cells and 60 BCL1 B cell lymphoma cells into
lethally irradiated (8 Gy [800 rads]) BALB/c recipients. As shown in
Figure 5A, all the irradiated recipients
given 1.5 × 106 Rag-2/ marrow cells
alone survived for more than 100 days, but all the recipients given the
same dose of marrow cells in combination with 60 BCL1 cells
died of tumor growth by day 43. In contrast, addition of sorted marrow
CD8+ T cells to the RAG-2/ marrow cells
increased the survival in a dose-dependent manner. Addition of as few
as 25 × 103 marrow CD8+ T cells allowed 10%
of the recipients to survive for more than 100 days
(P > .05, log-rank test). Addition of
100 × 103 and 500 × 103 marrow
CD8+ T cells, respectively, allowed 40% and 100% of the
recipients to survive for more than 100 days (P < .0001).
The spleen cells of the recipients were stained with
anti-BCL1-Id mAb and analyzed by flow cytometry for the
presence of BCL1 cells. The spleens of moribund recipients
given only RAG-2/ marrow cells contained 50% to 80%
of BCL1 cells (data not shown). The spleen cells of 2 of
the 4 recipients given 100 × 103 marrow CD8+
T cells and RAG-2/ marrow cells that survived for more
than 100 days had 5% to 10% BCL1 cells, and the spleens
of recipients given 500 × 103 marrow CD8+ T
cells had no detectable BCL1 cells (data not shown). The
results indicate that high doses of marrow CD8+ T cells are
able to eliminate the BCL1 cells, but low doses induce "tumor dormancy."
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| Figure 5.
Ability of BM CD4+ and CD8+ T
cells to mediate anti-BCL1 tumor activity.
Graded numbers of sorted C57BL/6 BM CD4+, CD8+,
or CD4+/CD8+ T cells were added to a constant
number of C57BL/6 Rag-2/ BM cells
(1.5 × 106) and 60 BALB/c BCL1
cells and injected intravenously into lethally irradiated
BALB/c recipients. Survival over a 100-day observation period is shown
for groups of 10 mice. Data are combined from 2 replicate experiments.
(A) Graded numbers of BM CD8+ T cells. (B) BM
NK1.1+ or NK1.1 CD8+ T cells. (C)
C57BL/6 BM CD4+ T cells or BM CD8+ T cells from
C57BL/6xBALB/c F1 mice. (D) Graded numbers of BM
CD4+/CD8+ T cells.
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As shown in Figure 5B, addition of 100 × 103 marrow
NK1.1+CD8+ T cells showed little antitumor
activity. In contrast, addition of marrow NK1.1
CD8+ T cells showed dose-dependent antitumor activity, and
100 × 103 cells were able to rescue 60% of the
recipients for more than 100 days. The surviving recipients did not
have observable clinical signs of GVH disease, and their spleens did
not have detectable BCL1 cells as judged by flow cytometry
analysis (data not shown). C57BL/6xBALB/c F1 marrow CD8+ T
cells (500 × 103) and C57BL/6 marrow CD4+ T
cells (500 × 103) had little antitumor activity in the
BALB/c recipients, and all died by 65 days (Figure 5C).
Although marrow CD4+ T cells alone were not able to mediate
antitumor activity, they enhanced the antitumor activity mediated by
marrow CD8+ T cells. As shown in Figure 5D,
100 × 103 combined marrow
CD4+/CD8+ T cells were able to effectively
treat hosts given BCL1 cells, and all the recipients
survived for more than 100 days. In contrast, 100 × 103
sorted marrow CD8+ T cells alone rescued only 40% of the
BCL1-bearing recipients (Figure 5A,D,
P < .01, log-rank test). In addition, the survival of the
recipients given 25 × 103 or 6.25 × 103
marrow CD4+/CD8+ T cells was similar to that of
the recipients given 100 × 103 or
25 × 103 marrow CD8+ T cells, respectively
(Figure 5A,D, P > .05, log-rank test). We did not test
the antitumor activity of either blood CD4+ or
CD8+ T cells, because these cells induced lethal GVH disease.
The sorted marrow CD4+ and CD8+ T cells were
also tested for facilitation of engaftment of hematopoietic
progenitors. Graded numbers of marrow CD4+ or
CD8+ T-cell subsets were coinjected with a suboptimal dose
(750 × 103) of C57BL/6 donor TCD marrow cells into
lethally irradiated BALB/c recipients without tumor cells. As shown in
Figure 6A, only 10% of the recipients
given TCD marrow cells alone had long-term survival of more than 100 days. Addition of marrow CD8+ T cells to the TCD marrow
cells resulted in a dose-dependent increase in long-term survival.
Injection of as few as 6.25 × 103 marrow
CD8+ T cells increased the long-term survival from 10% to
30% (P < .05, log-rank test), and
25 × 103 marrow CD8+ T cells increased the
long-term survival to 70% (P < .001, log-rank test).
Further increase of the marrow CD8+ T-cell dose to
100 × 103 or 500 × 103 failed to further
increase the survival beyond this plateau (Figure 6A).
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| Figure 6.
Ability of donor BM CD4+ and CD8+ T cells to
facilitate engraftment.
Graded numbers of sorted C57BL/6 BM CD4+ and
CD8+ T cells were added to constant numbers
(750 × 103) of C57BL/6 TCD BM cells and injected into
lethally irradiated BALB/c recipients. Survival over a 100-day
observation period is shown for groups of 10 to 20 mice. Data are
combined from at least 2 replicate experiments. (A) Graded numbers of
BM CD8+ T cells. (B) C57BL/6 BM CD4+ T cells or
BM CD8+ T cells from C57BL/6xBALB/c F1 mice. (C) Chimerism
of the BALB/c recipients given C57BL/6 TCD-BM cells alone or C57BL/6
TCD-BM and C57BL/6 BM CD8+ T cells 100 day after BM
transplantation. The spleen cells were stained for
anti-H-2Kb versus TCR, B220, or MAC-1 and Gr-1
markers. The donor-type cells are shown in the upper box and host-type
cells in the lower box in each panel.
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In contrast, addition of 500 × 103 marrow
CD4+ T cells or C57BL/6xBALB/c F1 marrow CD8+ T
cells did not increase the survival rate as compared with TCD marrow
alone (Figure 6B). As shown in Figure 6C, the spleen cells of the
recipients were stained with anti-H-2b versus TCR,
B220, and MAC-1/Gr-1 mAbs after 100 days. The recipients given TCD
marrow only showed mixed chimerism in which about 80% of T cells were
donor type and 20% host type, but all the B cells, macrophages, and
granulocytes were donor type. In contrast, addition of donor marrow
CD8+ T cells resulted in a complete chimerism of T cells as
well as B cells, macrophages, and granulocytes. (Figure 6C). Marrow
CD4+ T cells were not able to induce complete chimerism
(data not shown) and facilitate engraftment.
| |
Discussion |
In the current study, the NK1.1 CD4+ and
CD8+ T cells in the marrow and blood were investigated with
regard to surface phenotype, cytokine secretion, and function. Most of
the blood NK1.1 CD8+ T cells were
CD44int/loCD62LhiCD45RBhi, but most
of the marrow NK1.1 CD8+ T cells were
CD44hiCD62Lhi CD45RBhi. The
phenotype of the blood T cells is representative of the typical naive
T-cell phenotype found in the lymph nodes and spleen.7 The
unusual phenotype of the marrow NK1.1 CD8+ T
cells has been reported previously in extrathymically derived CD8+ T cells and in those that have expanded in
T-cell-deficient adoptive hosts presumably stimulated by endogenous
self ligands.20-22 About 30% to 50% of the marrow
NK1.1 CD4+ T cells also showed the
CD44hiCD62LhiCD45RBhi phenotype,
and the remainder of the marrow NK1.1 CD4+ T
cells were mainly
CD44hiCD62loCD45RBlo. The latter
phenotype has been reported previously on activated/memory CD4+ T cells,7,17 extrathymically derived
CD4+ T cells,20 and CD4+ T cells
in the normal marrow.7 The former phenotype has been reported on extrathymically derived CD4+ T cells as
well.20 The unusual phenotype of T cells in the marrow may
reflect their derivation from an alternative extrathymic developmental
pathway that is present in the BM itself,8,9 activation by
endogenous ligands in the marrow, and migration to the marrow after
activation in the periphery.
The unusual surface receptor phenotype of marrow CD4+ T
cells was associated with an atypical cytokine secretion profile. Blood CD4+ T cells produced large amounts of IL-2 with little
IFN-, IL-4, or IL-10, as reported for freshly isolated naive
CD4+ T cells from the spleen after in vitro
stimulation.23 Marrow NK1.1 CD4+
T cells secreted 5- to 10-fold higher levels of IFN-, IL-4, and
IL-10 as compared with blood CD4+ T cells and a 7-fold
lower levels of IL-2. Thus, the marrow NK1.1
CD4+ T cells expressed a cytokine pattern that was unique
and similar to the previously described Th0 pattern.23,24
The cytokine profile of the blood NK1.1 CD8+
T cells was quite similar to that of the marrow NK1.1
CD8+ T cells, and both produced high levels of IL-2 and
IFN- and very low levels of IL-4 and IL-10, as described previously
as the Tc1 pattern.25
We |
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Abstract
Introduction