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Division of Clinical Research, Fred Hutchinson Cancer Research Center, and
Department of Medicine, Division of Oncology, University of Washington, Seattle, WA 98105
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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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Adoptive transfer of cultured CD4 or CD8 T cell clones into allogenic recipients causes severe vascular leak, leading to death within 5 days (2, 3). Cultured T cell clones, however, cannot be used to recapitulate the pathogenesis of GVHD, which is initiated primarily by resting T cells in clinical marrow transplantation. T cells from TCR-transgenic strains have been used in adoptive transfer experiments to evaluate the events that occur after recognition of alloantigen in vivo (4, 5, 6, 7). The results of these studies have confirmed that alloactivated T cells show a burst of proliferation within several days after the adoptive transfer. After an initial expansion, however, the population declines sharply. Most remarkably, these immunologic events often appear not to be accompanied by any significant pathologic events resembling GVHD. The reasons for the absence of overt GVHD have not been apparent. We have now developed a system in which a TCR-transgenic T cell population initiates GVHD against recipients that express the specific alloantigen recognized by the TCR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6), (B6 x BALB/c)F1 (CB6),
and BALB/c H2-dm2 (dm2) mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). (dm2 x B6)F1 (dm2B6) mice
were bred at Fred Hutchinson Cancer Research Center (Seattle, WA).
Founders for 2C transgenic mice were provided by Dr. Dennis Y. Loh
(Nippon Roche Research Center, Kamakur-shi, Japan). These mice express
rearranged TCR 
and ß genes from a cytotoxic T cell clone that
recognizes the MHC class I Ag Ld. CD8 cells that
express the 2C TCR are positively selected in the thymus by H2
Kb (8), and they can be identified
by staining with the clonotypic mAb 1B2. CD8+
cells from the 2C TCR-transgenic strain have robust,
helper-independent, proliferative, and cytolytic responses against
Ld+ cells in vitro (9). Mice were
housed under specific pathogen-free conditions with twice weekly cage
changes and received sterilized chow and acidified water (pH 3.5) ad
libitum. Experiments were reviewed and approved by the institutional
animal care and use committee of the Fred Hutchinson Cancer Research
Center.
T cell purification
For purification of CD8+/1B2+ cells by positive selection, a magnetic cell separation system was used according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA). Lymph node cells were incubated with anti-CD8-conjugated MicroBeads for 15 min at 4°C. After washing, the cell suspension was passed through a VS+ separation column placed in a magnetic field. The VS+ column was washed two or three times with 0.5% BSA/PBS and then removed from the magnetic field. Retained cells were flushed from the VS+ column with 0.5% BSA/PBS. The isolated cells were passed over a second VS+ column to increase the purity of enriched cells. After separation, cells were stained with FITC-conjugated 1B2 mAb and PE-conjugated anti-CD8 mAb, and analyzed by flow cytometry. The purity of CD8+/1B2+ cells ranged from 94 to 98%.
Transplantation and LPS challenge
B6, dm2B6, or CB6 recipient mice were exposed to 750 cGy from dual opposed 60Co sources at 20 cGy/min as previously described (10). Purified CD8+/1B2+ lymph nodes cells from 2C donors were suspended in PBS and were injected via the tail vein into 8- to 10-wk-old irradiated recipients within 24 h after irradiation. The number of donor T cells injected ranged from 6 to 10 x 106/recipient, but in any given experiment, equal numbers of cells were transplanted into each recipient. Recipients and controls were monitored for weight loss and manifestations of GVHD. As a challenge test for the presence of GVHD, LPS (Escherichia coli, L-3024, Sigma, St. Louis, MO) dissolved in PBS and sterilized by 150 Gy of irradiation was injected i.v. at a dose of 50 µg/mouse 7 days after donor cell transfer.
Flow cytometry
Peripheral blood, spleen, lymph node cells, and thymocytes were stained by direct immunofluorescence with Abs specific for 2C TCR, CD8, CD4, or B220 in various two- or three-color combinations, as indicated. The 1B2 hybridoma, which produces a clonotypic mAb specific for the 2C TCR, was provided by Dr. Loh (8). Purified and FITC-conjugated mAb 1B2 was prepared in our laboratory. All other Abs were purchased from PharMingen (San Diego, CA). Flow cytometric analysis was performed with a FACScan and CellQuest software (Becton Dickinson, San Jose, CA). CD8+/1B2+ cells were considered of donor origin, because cells with this phenotype could not be detected in the control mice that were not transplanted. CD8+/1B2-, CD4+, and B220+ cells were considered CD8, CD4, and B cells of host origin, respectively.
Cell culture and proliferation
Cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 15 mM HEPES, 1 mM sodium pyruvate, 5 x 105 M 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. For measuring proliferative responses, splenocytes were collected and used as responder cells 6 days after transplantation, seeded at 105/well, and cocultured with irradiated (20 Gy) CB6 or dm2B6 splenocytes at 2 x 105/well in the presence or the absence of 50 U/ml human rIL-2 (Immunex, Seattle, WA) in triplicate at 0.2 ml/well. After 3 days of culture, cells were labeled with 1 µCi of [3H]TdR/well for 8 h. DNA was harvested onto glass-fiber filters and was quantitated as counts per minute in a Topcount liquid scintillation counter (Packard, Meridian, CT). The stimulation index was calculated by the counts per minute in cultures with Ld+ stimulators, IL-2, or both in combination divided by the counts per minute in cultures with Ld- stimulators without IL-2.
Anti-host cytotoxicity
Cytotoxic activity was measured directly, without in vitro restimulation, by testing spleen cells from the recipients as effectors against 51Cr-labeled P815 (H2d) or EL-4 (H2b) targets. Spleen cells were added to U-bottom 96-well plates to achieve E:T cell ratios of 6, 12, 25, 50, and 100:1 with 2.0 x 103 targets/well. The plates were centrifuged at 200 x g for 2–3 min and then incubated at 37°C for 4–5 h. Chromium release was measured with a Topcount (Packard, Meridian, CT), and the percent cytotoxicity was calculated as (experimental release - spontaneous release)/(maximal release - spontaneous release) x 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the fate of CD8+ T cells after
exposure to specific alloantigen in vivo, we transplanted 2C
CD8+ T cells into sublethally irradiated (750
cGy) nontransgenic B6 recipients or (BALB/c x
B6)F1 (CB6) recipients. At multiple time points
after transplantation, 2C cells were identified in the peripheral
blood, spleen, lymph nodes, and thymus of the recipients by two-color
staining with an mAb specific for the 2C TCR (1B2) and an mAb specific
for CD8. After adoptive transfer, transgenic 2C T cells engrafted in
the spleen and lymph nodes of nontransgenic B6 recipients (Fig. 1
, middle panels)
and were detected in the blood as late as 126 days after
transplantation, the latest time point analyzed in the experiment. In
these recipients, 2C cells maintained expression of CD8 and TCR at
pretransplant levels (Fig. 2A). Background staining of
CD8+/1B2+ cells in
irradiated CB6 controls was <0.2% at all time points tested (Fig. 1, left panels).
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, right panels).
CD8+/1B2+ cells could be
found in the recipient spleen on day 2, the earliest time point tested,
and the number of CD8+/1B2+
started to increase on day 3 after transplant (data not shown). Both
TCR and CD8 on 2C cells were expressed at lower levels after transfer
to allogenic CB6 recipients than after transfer to syngenic B6
recipients (Fig. 1). The modulation of TCR and CD8 in CB6 recipients
indicated that 2C T cells had interacted with specific Ag in vivo
(11). We followed the presence of 2C cells in the
peripheral blood of individual recipients for 4 mo after
transplantation. The number of
CD8+/1B2+ cells peaked on
day 13 and decreased dramatically on day 21 (Fig. 2A). A
second peak was consistently seen between days 35 and 42, and then the
number of 2C cells started to decline until becoming indistinguishable
from background staining by day 90. The kinetics of 2C T cell expansion
showed similar patterns in peripheral blood and lymphoid organs
(Fig. 2).
To exclude the possibility that 2C T cells recognized alloantigens
other than Ld through a distinct TCR with a
second rearranged TCR -chain, we tested dm2B6 recipients. Because
dm2 is a BALB/c Ld-loss mutant, the only H2
difference between the two strains is that CB6 mice express
Ld, while dm2B6 mice do not. In dm2B6 recipients,
the number of 2C T cells showed little variation across time, and these
2C T cells did not modulate their CD8 or TCR, while in CB6 recipients,
the 2C T cell population expanded, and 2C cells modulated both CD8 and
TCR (Fig. 2B and data not shown). Thus,
Ld is the only alloantigen recognized by 2C T
cells in CB6 mice. We found that 2C T cells were less numerous in the
blood of Ld+ recipients than in the blood of
Ld- recipients, but 2C T cells were more
numerous in the spleen of Ld+ recipients than in
Ld- recipients (Fig. 2 and not shown).
Function of 2C T cells in vivo
To demonstrate that 2C T cells had been activated in vivo and were
functional, we examined the ability of 2C cells to proliferate after Ag
restimulation ex vivo. Six days after transplant, splenocytes isolated
from either dm2B6 or CB6 recipients were cultured with irradiated
splenocytes from dm2B6 or CB6 mice. In the absence of exogenous IL-2,
no proliferation could be detected with splenocytes from either dm2B6
or CB6 recipients. When naive 2C T cells were mixed with dm2B6
splenocytes at frequencies <2.5% in the absence of IL-2, stimulation
with Ld did not induce incorporation of
[3H]TdR over background (Fig. 3A, inset), suggesting that
proliferation of 2C cells in response to Ld is
helper dependent when 2C cells are present at low frequency. Therefore,
the lack of any detectable response with 2C T cells from the spleen of
dm2B6 mice after restimulation with Ld in the
absence of IL-2 could be explained by the low frequency of
2C T cells (<2.5%; Fig. 3C). In CB6 recipients, however,
the absence of a proliferative response cannot be explained by a low
frequency of 2C cells (Fig. 3, A and C).
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A). On days 13, 21, 42, and 70 after transplantation, the
proliferative responses to Ld by splenocytes from
CB6 mice transplanted with 2C cells were similar to the response on day
6 (data not shown).
We examined the ability of 2C T cells to generate cytotoxic activity in
vivo. Splenocytes from dm2B6 or CB6 mice transplanted with 2C T cells
were tested on days 6, 13, 21, 42, and 70 after transplantation in a
direct cytotoxicity assay against Ld+ (P815) or
Ld- (EL-4) targets in a 4- to 5-h
51Cr release assay without prior restimulation in
vitro (Fig. 3B). Splenocytes from CB6 recipients
demonstrated cytotoxicity against Ld+ P815
targets at all time points tested, with the highest activity between 13
and 21 days after transplantation. In contrast, splenocytes from dm2B6
recipients showed no activity against P815 targets at any time point.
Splenocytes from CB6 and dm2B6 recipients did not have detectable
cytotoxic activity against Ld- EL-4 targets
(data not shown).
To test the sensitivity of the cytotoxicity assay and to estimate the
cytolytic activity of CTL effectors in the recipient spleen, we mixed
in vitro primed 2C effectors with splenocytes from either dm2B6 or CB6
mice and tested their cytolytic activity against P815 or EL-4 targets.
As few as 0.4% 2C effectors mixed with splenocytes from either strain
were detected by the CTL assay, and the percentage of 2C effectors
correlated closely with percent specific lysis (Fig. 3B,
inset).
In vivo elimination of Ld+ recipient thymocytes and B cells by donor 2C T cells
To investigate whether 2C T cells cause immunopathology in
Ld+ recipients after activation in vivo, we
examined central and peripheral lymphoid organs in the mice
transplanted with 2C T cells. At each time point, two mice per group
were sacrificed, and thymocyte, splenocyte, and lymph node populations
were enumerated. In B6 and dm2B6 recipients and in irradiated CB6
controls, the thymus was smaller than in unirradiated normal mice, but
the distribution of thymocyte subsets was normal, with 80–90%
CD4+CD8+ double-positive
cells at all time points tested (Fig. 4A and data not shown). In CB6
recipients, however, the thymus was reduced to a small remnant between
35 and 56 days after transplant. During this period, the number of
double-positive thymocytes in CB6 recipients was 4–5 logs lower than
in the other groups. By day 70, coincident with the reduction of the
numbers of 2C cells, CB6 recipients recovered double-positive
thymocytes at numbers comparable to those in mice in other groups (Fig. 4B).
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), reaching a nadir between 13 and 42
days after transplantation (Fig. 6,
top panels). By day 70, the number of B cells in CB6
recipients had begun to recover. After transplantation with 2C T cells,
the numbers of recipient-derived CD8 and CD4 T cells were also lower in
CB6 recipients than in dm2B6 or B6 recipients (Fig. 6, middle and bottom panels), possibly as a
consequence of decreased thymic production or direct killing by 2C T
cells.
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Nonlymphoid changes in allogenic recipients of 2C T cells and GHVD lethality induced by LPS
We examined whether adoptively transferred 2C T cells affected
nonlymphoid tissues and caused clinical disease. All recipients had
5–8% weight loss after irradiation. CB6 recipients recovered their
original weight at 5 wk after transplant, while B6 recipients and
irradiation controls recovered at 2 wk (data not shown). Between 2 and
4 wk after transplant, the hematocrit was markedly lower in CB6
recipients than in B6 recipients or irradiation controls (Fig. 7A). Between 13 and 56 days
after transplantation, white blood counts were lower in CB6 recipients
than in B6 recipients or irradiation controls (Fig. 7B). No
appreciable pathology was seen in lung, liver, kidney, or skin in any
recipient (data not shown). Nearly all CB6 recipients survived until
sacrifice at up to 140 days after transplant.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We could not demonstrate "cold target" inhibition of cytolytic activity when in vitro primed 2C effectors were mixed with resting CB6 splenocytes compared with dm2B6 splenocytes. Although resting splenocytes were not able to mediate cold target inhibition, we suspect that activated splenocytes in recipients with GVHD might have caused some degree of cold target inhibition in assays with P815 targets, because CTL activity was lower than might have been expected from the number of 2C cells in the spleen. G0 cells are resistant to CTL-induced apoptosis, but cells become susceptible to CTL lysis when growth factors, cellular transformation, or infection have induced entry into G1 or a G1-like state (16). We speculate that cells can cause cold target inhibition only when they are susceptible to lysis.
In contrast to our observations, Zhang et al. (6, 7)
reported that 2C T cells isolated from Ld+ SCID
recipients were completely unresponsive to Ag restimulation in
both proliferative and cytolytic assays 4–18 days after
transplantation. A major difference between the two studies is that we
employed irradiated immunocompetent CB6 mice, while Zhang et al.
(6, 7) employed unirradiated BALB/c-SCID mice as
recipients. Irradiation enhances the production of inflammatory
cytokines such as TNF- and IL-1ß and also permits increased
translocation of LPS from the gastrointestinal lumen into the systemic
circulation (17, 18). Exposure to LPS can interfere with
the induction of peripheral tolerance and can activate
CD8+ T cells to cause immunopathology (19, 20). In our experiments, recipient-derived B cells might have
sustained antigenic stimulation and cytolytic function of 2C T cells,
while the absence of B cells in SCID mice might have promoted the
development of unresponsiveness. B cells are instrumental in processes
that amplify and sustain T cell activation initiated by professional
APCs (21, 22), and donor T cells that induce GVHD in
immunocompetent allogeneic recipients are more likely to become
tolerant in immunodeficient allogenic recipients (23).
Studies by Rocha et al. (4, 5) showed that CD8+ T cells from anti-HY TCR-transgenic donors can eliminate male B cells when female mice reconstituted with 10% male bone marrow cells were used as recipients. In contrast, male B cells persisted, and donor CD8+ T cells underwent exhaustion when female mice reconstituted with 50% male bone marrow cells were used as recipients, and anergy was induced when female mice reconstituted with 90% male bone marrow cells were used as recipients. We found that 2C cells induced B lymphopenia but were unable to eliminate host B cells in recipients that express Ld on all nucleated cells. By 70 days after transplantation, the donor T cells became tolerant, allowing recipient B cells to recover. Thus, T cell tolerance is the ultimate consequence if T cells confront an abundance of persistent Ag in vivo, as shown in other TCR transgenic systems (4, 5, 6, 7, 24, 25, 26).
After transplantation into allogeneic CB6 recipients, 2C transgenic T cells caused pathology primarily in lympho-hemopoietic organs, including spleen, lymph nodes, thymus, and bone marrow. Several explanations might account for the inability of 2C cells to cause epithelial damage in nonlymphoid organs and induce lethal GVHD. GVHD is ordinarily caused by a polyclonal population of T cells expressing TCRs that recognize multiple recipient alloantigens with a wide range of avidities. In comparison, 2C cells recognize a much more limited diversity of peptide-MHC complexes with a correspondingly limited diversity of avidities for triggering activation. Hence, the tissue distribution of Ags recognized by 2C cells might not be permissive for the development of epithelial injury. The high avidity of the interaction between the 2C TCR and Ld and the abundant expression of peptide-Ld complexes (11) might cause clonal exhaustion or rapid down-regulation of effector functions needed to cause epithelial injury. Alternatively, the repertoire of functional responses activated among 2C cells might lack one or more components needed to cause epithelial injury.
We favor the hypothesis that 2C cells do not induce lethal GVHD because they lack help from CD4+ cells. CD4+ cells are typically the first T cell subset to be activated after adoptive transfer in allogeneic recipients, and they are the predominant subset to produce cytokines (27). Activated CD4+ cells can also provide signals through CD40 ligand:CD40 interactions to activate host APCs, and these activated APCs can then directly stimulate CD8+ T killer cells (28, 29, 30). We are currently investigating whether host-reactive CD4+ T cells can indeed help CD8+ 2C T cells to mount a stronger immune response against Ld and induce lethal GVHD. The inability of recipients to withstand LPS challenge after adoptive transfer of 2C cells suggests that macrophages (12, 31) and possibly dendritic cells (32, 33) were primed for amplified production of TNF in vivo. This effect did not appear to require the participation of CD4 cells.
Taken together, our findings suggest that many interacting components contribute to the pathogenesis of GVHD. As argued by several investigators, the processes leading to clinical disease may begin with epithelial damage caused by the pretransplant conditioning regimen well before donor T cells are activated by alloantigens of the recipient. The use of TCR-transgenic T cell populations in adoptive transfer experiments will facilitate future studies of the immunopathologic mechanisms that result in GVHD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claudio Anasetti, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., D2-100, Seattle, WA 98109. E-mail address:
3 Abbreviations used in this paper: GVHD, graft-vs-host disease; B6, C57BL/6 mice; CB6, (B6 x BALB/c)F1 mice; dm2, BALB/c H2-dm2 mice.
Received for publication May 28, 1999. Accepted for publication August 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during graft-versus-host disease. J. Exp. Med. 175:405.This article has been cited by other articles:
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