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Diabetes Division, University of Massachusetts Medical School, Worcester, MA 01655
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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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CD154 (CD40 ligand) is rapidly up-regulated on Ag-activated CD4+ T cells (2, 3, 4, 5, 6). Blockade of the interaction of T cell-associated CD154 with CD40 on APCs inactivates the in vivo and in vitro CD4+ T cell response (4, 7, 8). We used these observations to develop a two-element transplantation protocol consisting of a single donor-specific transfusion (DST)3 plus a brief course of anti-CD154 mAb. We hypothesized that DST, acting as "signal 1," would initiate the activation of host CD4+ and CD8+ alloreactive T cells. Interference with the interaction of CD154 and CD40 would preclude costimulation ("signal 2") (1, 9, 10, 11).
Consistent with that hypothesis (1), our "two-element protocol" leads to prolonged survival of murine skin (12, 13, 14, 15) and heart (9) allografts. It also leads to permanent islet allograft survival in euthymic mice (1, 10, 16, 17) and to permanent skin allograft survival in the majority of thymectomized mice (14).
The mechanism by which this method prolongs graft survival is not fully
understood. It is known that CD4+ cells and
IFN-
are required (14). In addition, blockade of the
interaction of CTLA4 with B7-1 and B7-2 using either anti-CTLA4 mAb
(14) or CTLA4-Ig (11) abrogates the ability
of DST and anti-CD154 mAb to prolong graft survival. In vitro mixed
lymphocyte reactivity to donor alloantigen can be detected in tolerized
graft-bearing recipients treated with the two-element protocol
(15). However, donor-cell lymphohemopoietic chimerism is
undetectable (15). The mechanism by which the two-element
protocol inactivates CD8+ alloreactive cells has
remained perplexing because CD154 is preferentially expressed by
CD4+, not CD8+, T cell
populations (2, 8, 18, 19, 20). The reason for the dependence
of the protocol on CTLA4 is unknown.
To investigate these issues, we established an alloreactive transgenic T cell model system to analyze the fate of a tracer population of alloreactive CD8+ T cells in normal mice subsequently treated with DST and anti-CD154 mAb. We report that the DST component of our two-element protocol mediates rapid deletion of alloreactive transgenic CD8+ T cells, and that deletion of CD8+ T cells appears to be required for allograft survival. We also demonstrate that anti-CTLA4 mAb abrogates graft survival induced by DST and anti-CD154 mAb by preventing deletion of alloreactive CD8+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA/JCr (H2k), C57BL/6 (H2b), and BALB/c (H-2d) mice were obtained from the National Cancer Institute (Frederick, MD). C57BL/6 mice in which the CD8 lymphocyte surface Ag gene was disrupted by homologous recombination were obtained from The Jackson Laboratory (Bar Harbor, ME). To investigate the fate of specific alloreactive T cells, we established in our animal colony the KB5 TCR transgenic mouse, which has specificity to native H2b alloantigen (21, 22). This TCR transgenic mouse was the generous gift of Dr. John Iacomini (Harvard Medical School, Boston, MA), who obtained it from the original developer, Dr. Andrew Mellor (Medical College of Georgia, Augusta, GA). The TCR transgene is expressed by CD8+ cells in CBA (H2k) mice and has specificity for H2-Kb.
All animals were certified to be free of Sendai virus, pneumonia virus of mice, murine hepatitis virus, minute virus of mice, ectromelia, latic dehydrogenase elevating virus, GD7 virus, Reo-3 virus, mouse adenovirus, lymphocytic choriomeningitis virus, polyoma, Mycoplasma pulmonis, and Encephalitozoon cuniculi. All animals were housed in microisolator cages, given ad libitum access to autoclaved food, and maintained in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996) and the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts Medical School.
PCR typing of KB5 TCR transgenic mice
KB5 TCR transgenic mice were typed by PCR using DNA from ear
punches (23). The PCR primers specific for the transgenic
-chain were designed using Oligo primer analysis software (National
Biosciences, Plymouth, MN) and the published sequence
(24). The forward primer, 5'-GCAGCAGGTGAGACAAA-3'
(specific for the V region), and the reverse primer,
5'-ATACCGTGGTTCCTGTTC-3' (specific for the J segment), produce a
323-bp product from the rearranged gene. PCR was performed in the
presence of 2 mM MgCl2 at an annealing
temperature of 55°C.
Transplantation procedures
Male C57BL/6 or CBA/JCr recipient mice 6–8 wk of age were
tolerized and transplanted with skin allografts using previously
published techniques (14, 15). As described in the table
and figure legends, certain groups of experimental mice received a
single DST. Briefly, 107 C57BL/6 or BALB/c
splenocytes from female donors were injected i.v. in a volume of 0.5 ml
into CBA/JCr or C57BL/6 mice, respectively. In the case of otherwise
unmanipulated C57BL/6 skin graft recipients, DST was given 7 days
before skin transplantation. CBA/JCr mice were all TCR chimeras (see
below) and were given DST 2 days after establishing chimerism and 4
days before any skin transplantation. The MR1 hamster anti-mouse
CD154 mAb was produced as ascites in scid mice (7, 25). Ab concentration in ascites was determined by ELISA.
Anti-CD154 mAb was administered i.p. at a dose of 0.5 mg per mouse on
varying schedules that are described in detail in the table and figure
legends. Nontransgenic mice given anti-CD154 mAb (Table I
; see Fig. 4) received either 4 doses on
days -7, -4, 0, and +4 relative to transplantation or doses twice
weekly for a total of 14 doses, with the first 4 doses on days -7,
-4, 0, and +4. Transgenic TCR chimeric mice that were given
anti-CD154 mAb received 1–4 doses at various intervals (Table II and see
Figs. 1–3, 5, and
6).
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Anti-CD8 and anti-CTLA4 mAb administration
Hybridoma cells secreting a depleting rat mAb directed against mouse CD8 (clone 2.43) were obtained from the American Type Culture Collection (Manassas, VA) (27). To deplete CD8+ cells, mice were given 0.5 mg of mAb i.p. daily for 3 days. This protocol was documented by flow microfluo-rometry to deplete >95% of CD8+ T cells 48 h after the last dose of mAb. A hybridoma cell line secreting hamster anti-mouse CTLA4 mAb (clone 9H10) was the gift of Dr. James Allison (University of California, Berkeley, CA). Anti-CTLA4 mAb was injected i.p. at a dose of 0.075 mg per mouse daily for 3 days. Both Abs were produced as ascites in scid mice. For the anti-CD8 mAb, rat Ig concentration was measured by radial immunodiffusion (The Binding Site, San Diego, CA); for the anti-CTLA4 mAb, hamster Ig concentration was measured by ELISA.
Analysis of cell number
In preliminary experiments, the lymph nodes that drain skin grafts placed in the standard flank location were identified. To do so, Evans blue dye (0.01 ml of 2.5% w/v) was injected directly either into the bed prepared for skin grafts or directly into successful skin grafts on mice that had been treated with DST and anti-CD154 mAb. In both cases, dye was subsequently observed only in the axillary and lateral axillary nodes (28). For cell counts, lymph nodes or intact spleens were dissected free of other tissues and extruded through a cell sieve. The total number of viable mononuclear cells present was determined by the method of trypan blue exclusion using a hemocytometer. Cell viability was >95% in all cases.
Flow microfluorometry
PE-conjugated anti-mouse mAbs directed against CD4 (clone
GK1.5) and the activation markers CD44 (clone IM7) and CD62L (Mel-14)
were obtained from PharMingen (San Diego, CA). Cy-Chrome-conjugated
anti-mouse CD8 mAb (clone 53-6.7) was also obtained from
PharMingen. A hybridoma cell line secreting the clonotypic DES Ab
(21) was the gift of Dr. John Iacomini. Isotype control
mAbs including the PE-conjugated rat IgG2a 
(clone R35-95) for CD44
and CD4, PE-conjugated rat IgG2b (clone A95-1) for CD62L, rat
Cy-Chrome-conjugated IgG2a (clone R35-95) for CD8, and mouse IgG2a
anti-TNP (clone G155-178) for DES were purchased from
PharMingen.
Two- and three-color flow cytometric analyses were performed as previously described (29). Briefly, 1 x 106 viable lymph node or spleen T cells were reacted with a mixture of conjugated mAbs for 20 min at 4°C. Cells were then washed and fixed with 2% paraformaldehyde. KB5 cells were incubated in the presence of anti-DES Ab, washed, and then reacted with FITC-conjugated anti-mouse IgG2a (PharMingen). Cells were washed and fixed with 2% paraformaldehyde. Labeled cells were analyzed using a FACScan instrument (Becton Dickinson, Sunnyvale, CA). Lymphoid cells were gated according their light-scattering properties. Levels of background fluorescence were subtracted.
In some experiments, the forward light-scatter characteristics of lymphoid cells were quantified and used as an index of cell size and, by extension, of cell activation. In these experiments, the forward scatter parameters (in arbitrary units) obtained from all untreated controls analyzed at the same time were averaged. For analysis, we calculated the ratio of each control and experimental value (obtained during the same run) to that mean control value. This procedure was adopted to account for day-to-day variability in machine settings, which caused variation in the forward scatter parameter associated with control cells. In all instances, a minimum of 50,000 events was acquired for each analysis.
T cell transgenic chimeras
To examine the fate of alloreactive CD8+ T cells under conditions that are "normal," we used the adoptive transfer chimera system originally described by Jenkins and colleagues (22, 30). In this system, small numbers of transgenic T cells are injected into syngeneic nontransgenic hosts to permit the engraftment of low levels of tracer transgenic cells in a host cellular microenvironment that is essentially unaltered by transgene expression. Transgenic T cells were enriched from spleen and lymph nodes of CBA-derived transgenic KB5 mice (H2k). These transgenic T cells express an anti-H2-Kb-specific TCR that is recognized by the anticlonotypic mAb, DES (21). Before their transfusion into adoptive recipients, spleen and lymph node cells from KB5 mice were enriched for the CD8+DES+ population using a CD8 Cellect column purification kit (Biotex Laboratories, Edmonton, Canada). Briefly, spleens and lymph nodes were removed and gently extruded through nylon sieves in cold PBS containing 2% FBS. Erythrocytes were lysed with hypotonic NH4Cl, and the samples were reacted with rat anti-mouse CD4 mAb. The cell suspension was then loaded onto the column (which is provided with bound goat anti-mouse Ig) and eluted with PBS-2% FBS. Cells not retained by the column were analyzed by flow microfluorometry and found to contain <0.1% CD4+ cells and 60–80% CD8+DES+ cells. CBA/JCr (H2k) skin allograft recipients and controls were transfused with 1–3 x 106 purified transgenic T cells. The number of CD8+DES+ cells present in adoptive recipients at various times after transfusion was analyzed by flow microfluorometry.
Statistics
Average duration of graft survival is presented as the median. Graft survival among groups was compared using the method of Kaplan and Meier (31). The equality of allograft survival distributions for animals in different treatment groups was tested using the log rank statistic (31). The p values <0.05 were considered statistically significant. Comparisons of three or more arithmetic means used one-way analyses of variance (32) and either the least significant difference procedure or Bonferroni-adjusted unpaired t tests (33) for a posteriori contrasts.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first tested the hypothesis that events predictive of skin
allograft survival would be detectable in graft-draining lymph nodes.
To do so, we first measured the total number of cells present in the
axillary and lateral axillary lymph nodes in five groups of
thymectomized, nontransgenic mice treated in accordance with published
protocols (14). The control group was untreated; the
remaining groups were comprised of skin graft recipients that were
otherwise untreated or that were treated with DST and anti-CD154
mAb alone or in combination. The total number of cells present in
untreated control lymph nodes ranged between 2.7 and 3.1 x
106 and remained constant over time (Table I
). In
contrast, the number of cells present in the draining axillary nodes of
otherwise untreated skin graft recipients was more than three times
greater on day 7 (by which time grafts had all been rejected). It was
eight times greater on day 9 (p < 0.001 vs
untreated, ungrafted controls). Lymph node cell numbers in skin graft
recipients that had received either anti-CD154 mAb or DST as
monotherapy were also much higher than in ungrafted controls on days 9
(p < 0.025) and 13 (p
< 0.001) after grafting (Table I). As expected (15), mice
in these groups uniformly rejected their grafts by day 13. In contrast,
the number of cells present in draining axillary nodes of graft
recipients treated with both anti-CD154 mAb and DST was
statistically similar to the number of cells present in the ungrafted,
untreated controls at each time point. Mice in this last group also had
healthy grafts on day 13, the final day of observation. To exclude any
influence of thymectomy on these results, comparable data were also
obtained in a similar experiment conducted using five groups of
nontransgenic, euthymic mice treated in the same way (data not
shown).
Alloantigen-specific CD8+ T cells in allografted mice treated with DST and anti-CD154 mAb are depleted within 7 days of skin transplantation
The first experiment demonstrated that the presence of a skin
allograft was associated with an increase in the overall cellularity of
draining lymph nodes unless the grafted mice had been treated with DST
and anti-CD154 mAb. The result suggested that alloreactive T cell
numbers in draining nodes might be reduced in this last group.
Therefore, we specifically sought to determine the fate of alloreactive
cells in graft-draining lymph nodes using the KB5 TCR transgenic
system. Five groups of euthymic CBA/JCr (H2k)
mice were transfused with a tracer population of syngeneic KB5
DES+ transgenic T cells (specific for
H-2Kb). One group received no further treatment.
The other four groups received a C57BL/6 (H2b)
skin graft plus no additional treatment, DST alone, anti-CD154 mAb
alone, or both anti-CD154 mAb and DST. The interval between the DST
or the first injection of anti-CD154 mAb and transplantation on day
0 was 
84 h. The number of
CD8+DES+ cells
present in the draining lymph nodes in all groups was measured on day
+7 relative to transplantation (Fig. 1).
The number of CD8+DES+ T
cells in the draining lymph nodes of ungrafted, untreated euthymic
controls was 7.6 ± 5.1 x 103,
representing 0.9–1.5% of the total lymph node cell population. The
number of CD8+DES+ T cells
in the axillary lymph nodes of otherwise untreated graft recipients
(69.0 ± 21.8 x 103) was much greater
than in the ungrafted controls (Fig. 1; p < 0.001).
The number of CD8+DES+ T
cells in graft recipients treated with DST as monotherapy (74.0 ±
48.7 x 103) was similar to the number in
the otherwise untreated graft recipients. The number of
CD8+DES+ T cells in graft
recipients treated with anti-CD154 mAb monotherapy (27.8 ±
16.5 x 103, p < 0.001) was
also significantly greater than in the ungrafted controls, but less
than in the untreated graft recipients (p <
0.05) or in the grafted recipients treated with DST as monotherapy
(p < 0.05). In striking contrast, the number
of CD8+DES+ T cells in the
draining lymph nodes of skin allograft recipients treated with both DST
and anti-CD154 mAb (1.6 ± 1.3 x
103; Fig. 1) was not only less than that in the
nodes of each of the other graft recipient groups, but was also
significantly less than in the ungrafted controls
(p < 0.025; Fig. 1).
Depletion of CD8+ alloreactive T cells occurs rapidly after combined treatment with DST and anti-CD154 mAb
Treatment with DST and anti-CD154 mAb appeared to reduce the
number of CD8+ alloreactive transgenic
CD8+ T cells in draining lymph nodes of skin
allograft recipients by day 7 after transplantation. This observation
prompted us to investigate the kinetics of deletion in both lymph nodes
and (to help exclude alteration homing) spleen. Four groups of euthymic
CBA/JCr (H2k) mice were transfused with syngeneic
KB5 DES+ transgenic T cells. None of the animals
received grafts. The groups received no further treatment, DST alone,
anti-CD154 mAb alone, or both anti-CD154 mAb and DST. Cell
number was measured 84 h later (the time point at which the grafts
had been transplanted in the previous experiment). The number of
CD8+DES+ cells present in
the spleens of mice treated with DST and anti-CD154 mAb was
statistically significantly less than that in any other treatment group
(p < 0.03; Fig. 2A). The number of
CD8+DES+ cells present in a
pooled preparation of axillary and inguinal lymph nodes was also
significantly lower in mice treated with DST and anti-CD154 mAb
than in any other treatment group (p < 0.002;
Fig. 2B). In contrast, the number of
CD8+DES+ cells present in
the lymph nodes of mice treated with DST alone was significantly higher
than in any other treatment group (p < 0.005;
Fig. 2B). For mice treated with both DST and anti-CD154
mAb, the number of CD8+DES+
cells present in both spleen and lymph nodes was <10% of the number
present in untreated controls.
CD8+ alloreactive T cells rapidly acquire an activated phenotype after combined treatment with DST and anti-CD154 mAb
Having demonstrated that CD8+DES+ cells appeared to have been deleted in mice treated 84 h earlier with DST and anti-CD154 mAb, we next attempted to determine whether the DES+ cells were activated before their disappearance. Four groups of euthymic CBA/JCr (H2k) mice were transfused with a tracer population of syngeneic KB5 DES+ transgenic T cells. None of the animals received grafts. The groups received no further treatment, DST alone, anti-CD154 mAb alone, or both anti-CD154 mAb and DST. Spleens and lymph nodes were recovered 48 h later and were counted. KB5 CD8+DES+ cells were analyzed by flow microfluo-rometry for forward and side light scatter (as an index of size) and for surface expression of CD44 and CD62L, markers indicative of an activated state (34).
We observed first that the numbers of
CD8+DES+ spleen cells and
lymph node cells at the 48-h time point were statistically similar in
all four groups (Table II). However, for each of the three parameters
used as an index of activation, we observed that the results for both
of the groups that had been treated with DST were significantly
different from those obtained in either the untreated group or the mAb
monotherapy group (Table II). In all cases, both spleen and lymph node
CD8+DES+ cells from the
groups treated with DST (alone or with anti-CD154 mAb) were larger
and evidenced up-regulation of CD44 and down-regulation of CD62L. There
were no statistically significant differences between the untreated
controls and the anti-CD154 mAb monotherapy group. In addition,
there were no statistically significant differences between the DST
monotherapy and DST plus anti-CD154 mAb groups for any of the three
parameters, indicating that the Ab had not prevented T cell activation.
Representative histograms illustrating the expression of CD44 and CD62L
on CD8+DES+ cells for each
of the four experimental groups are shown in Fig. 3.
We also examined CD8+DES+
cells from identical groups of animals obtained 84 h after
treatment. CD8+DES+ cells
from mice treated with either DST or anti-CD154 mAb as monotherapy
also exhibited activated and nonactivated phenotypes, respectively
(data not shown). The phenotype of
CD8+DES+ cells from mice
treated with both DST and anti-CD154 mAb could not be determined
because, consistent with the results in Fig. 2, almost no
CD8+DES+ cells were
present.
Skin allograft survival in mice treated with a combination of anti-CD8 and anti-CD154 mAbs is comparable to that achieved using DST and anti-CD154 mAb
The data generated using tracer populations of KB5 DES+ alloreactive T cells suggest that the mechanism by which DST plus anti-CD154 mAb promotes allograft survival is at least in part dependent on the depletion of host alloreactive CD8+ T cells. Because anti-CD154 mAb is known to react primarily with CD4+ T cells, we hypothesized that the role of DST in our two-element protocol was to facilitate the deletion of CD8+ alloreactive cells. We tested this hypothesis by substituting the depleting 2.43 anti-CD8 mAb for DST in our two-element protocol.
A cohort of euthymic nontransgenic C57BL/6 mice was randomized to
receive anti-CD8 mAb alone, anti-CD154 mAb alone, combined
therapy with both mAbs, or combined therapy with DST and anti-CD154
mAb. All mice also received a BALB/c skin graft. As shown in Fig. 4A, a course of anti-CD154
mAb plus a depleting anti-CD8 mAb prolonged skin allograft survival
(median survival time (MST) = 76 days) to an extent comparable to
that achieved with DST and anti-CD154 mAb (MST = 95 days).
Monotherapy with either anti-CD154 mAb (MST = 12 days) or
anti-CD8 mAb alone (MST = 10 days) did not prolong skin
allograft survival.
Anti-CD154 mAb monotherapy prolongs skin allograft survival in CD8 knockout mice
As an additional test of our hypothesis that DST in the
two-element protocol serves to facilitate the deletion of
CD8+ alloreactive cells, we analyzed the survival
of BALB/c skin allografts on C57BL/6 CD8 knockout mice. Three groups of
graft-recipient mice were randomized to receive either no additional
treatment or anti-CD154 mAb monotherapy on one of two dosing
schedules. Median graft survival in untreated mice was 14 days. At the
lower dose of mAb, median graft survival was prolonged to 23 days
(p < 0.03), and at the higher dose it was
prolonged to 57 days (p < 0.01; Fig. 4B).
Treatment with anti-CTLA4 mAb prevents the deletion of KB5 DES+CD8+ alloreactive T cells in recipient mice given DST and anti-CD154 mAb
We have previously demonstrated that coadministration of anti-CTLA4 mAb prevents the prolongation of allograft survival induced by DST and anti-CD154 mAb (14). The mechanism was not identified. Given the above data, we hypothesized that anti-CTLA4 mAb abrogates the process of prolonging graft survival by interfering with the deletion of CD8+ T cells. To test this hypothesis, a cohort of euthymic CBA/JCr mice was injected with a tracer population of alloreactive CD8+DES+ transgenic T cells and then randomized into three groups 2 days later. Group 1 received no further treatment. Group 2 received standard treatment with DST and anti-CD154 mAb. Group 3 received DST and anti-CD154 mAb plus three daily injections of anti-CTLA4 mAb, beginning on the day of DST.
Consistent with the results shown in Fig. 2, the number of
CD8+DES+ T cells present in
the spleens of mice treated with DST and anti-CD154 mAb was
statistically significantly less than in untreated controls
(p < 0.005; Fig. 5A). In contrast, the number
of CD8+DES+ T cells present
in mice treated with DST, anti-CD154 mAb, and anti-CTLA4 mAb
not only failed to decline, but actually expanded by an order of
magnitude (p < 0.003 vs other groups; Fig. 5A). Similarly, the number of
CD8+DES+ T cells present in
a pooled preparation of axillary and inguinal lymph nodes from mice
treated with DST and anti-CD154 mAb was statistically significantly
less than in untreated controls (p < 0.005;
Fig. 5B). Again, the addition of anti-CTLA4 mAb to DST
and anti-CD154 mAb was associated with a dramatic expansion of the
tracer population of
CD8+DES+ T cells
(p < 0.003 vs other groups; Fig. 5B). Representative histograms illustrating the deletion of
the CD8+DES+ population in
the presence of DST and anti-CD154 mAb, and its expansion in the
presence of anti-CTLA4 mAb are shown in Fig. 6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using tracer populations of KB5 transgenic T cells, we have shown that treatment with DST plus anti-CD154 mAb is associated not only with graft survival but also with rapid activation and subsequent deletion of alloreactive CD8+ T cells. Neither DST nor anti-CD154 mAb as monotherapy prolonged graft survival or deleted alloreactive KB5 DES+CD8+ T cells. The data led us to hypothesize that prolongation of graft survival by our two-element protocol is dependent on the deletion of CD8+ alloreactive T cells. This hypothesis was strengthened by our observation that skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus the depleting 2.43 anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. The effect of DST on alloreactive CD4+ T cells cannot be determined from our data, but we believe that any such effect is likely to be small. This belief is based on previous data showing that skin graft survival is comparable in mice treated with both anti-CD154 mAb and anti-CD8 mAb or the combination of both mAbs plus DST (Ref. 14 and our unpublished observations).
Given these data, we then predicted that the addition of anti-CTLA4 mAb to our two-element protocol, which is known to render the protocol ineffective in prolonging skin allograft survival (14), would interfere with the deletion of alloreactive CD8+ T cells. We observed that anti-CTLA4 mAb not only prevented the deletion of tracer populations of KB5 DES+CD8+ alloreactive T cells, but actually led to an increase in the number of these cells.
We interpret our observations as evidence of alloantigen-specific peripheral deletion of CD8+ cells in the presence of costimulatory blockade. We recognize that this interpretation is based on observations made with a tracer population of alloreactive transgenic KB5 CD8+ T cells. We have not directly measured the behavior of the various alloreactive host T cell populations after treatment with DST and anti-CD154 mAb. The KB5 cell population is clonal and expresses a single alloreactive TCR. Unlike host alloreactive cell populations, the tracer population tests an alloreactive TCR of only one affinity. However, both the tracer and nontracer data we present are internally consistent and suggest that the behavior of the KB5 transgenic T cell is indeed representative of the behavior of the native host T cell population that mediates graft rejection. The data that are not dependent on the KB5 system show 1) a rapid decline in draining lymph node cell number in animals treated with DST and anti-CD154 mAb, 2) prolonged allograft survival in animals treated with anti-CD154 mAb and a depleting anti-CD8 mAb in lieu of DST, and 3) prolonged graft survival in CD8 knockout allograft recipients treated only with anti-CD154 mAb.
Based on the sequence of events that leads to T cell activation and the recognition that T cell activation in the absence of costimulation leads to inactivation (35, 36), we previously hypothesized that prolongation of graft survival by DST and anti-CD154 mAb could be viewed as a four step process (16): 1) nonprofessional APCs like small resting donor-specific B cells with low basal levels of B7 expression would begin the process by presenting alloantigen; 2) intercurrent anti-CD154 mAb therapy would prevent the CD40-CD154 interaction that would normally up-regulate B7 costimulatory molecules on APCs; which would 3) lead to Ag recognition in the absence of costimulation; and 4) result in T cell inactivation and a state of Ag-specific nonresponsiveness. However, accrual of additional data suggests that this hypothesis requires modification.
We have shown, for example, that prolongation of allograft survival is
dependent on the presence of CD4+ cells, IFN-,
and CTLA4. Depletion of CD4+ cells, removal of
IFN- using either mAb or IFN- knockout mice, and, as noted above,
blockade of CTLA4 by either anti-CTLA4 mAb or CTLA4-Ig abrogates
the ability of DST and anti-CD154 mAb to prolong skin allograft
survival (11, 14). These observations cannot be explained
by or predicted from our original hypothesis. In particular, it has
been difficult to explain how blockade of CD154, which is expressed
primarily on activated CD4+ T cells (2, 8, 18, 19, 20), leads to inactivation of alloreactive
CD8+ T cells.
Based on the new data we have obtained, we now propose a mechanism by
which DST and anti-CD154 mAb prolong allograft survival. We propose
that DST initiates the process by activating both
CD4+ and CD8+ alloreactive
T cells. The alloantigen-activated CD4+ T cells
produce helper factors generated in a CD40-CD154-independent manner. In
the absence of CD40-CD154 interaction, reciprocal APC activation and
costimulation do not occur. In the absence of costimulation, the
alloantigen-activated CD8+ cell population that
is receiving CD4 help undergoes apoptosis. This concept is consistent
with the observation that costimulation prevents Ag-induced apoptosis
in vitro (37, 38, 39). Our data suggest that one of the
CD4-derived helper factors is very likely to be IFN-
(14). Conversely, we can exclude IL-4 and IL-10 as helper
factors because neither is required for prolongation of graft survival
by the two-element protocol (14).
It is important to point out that the generation of CD4-derived soluble
helper factors is known to be at least partially independent of APC
activation (via CD40-CD154 costimulation) and factors derived from
activated APCs. For example, after in vivo Ag-specific priming, in
vitro production of IL-2 and IFN- by lymph node cells is relatively
unimpaired in CD40 knockout compared with that in wild-type mice
(40).
Our hypothesis does not require alloactivated
CD4+ cells to be present in close physical
proximity to alloantigen-activated CD8+ cells. An
intermediary high-frequency host population activated by CD4-derived
soluble factors could amplify the activity of partially activated
TCR-ligated CD4+ cells. We hypothesize that one
such intermediary cell population may be comprised of NK
(NK1.1+) cells. NK cells are known to be
activated by IL-2 and to secrete IFN- (41). This
argument predicts that NK cells should participate in prolongation of
graft survival, and we have observed that, in the absence of
NK1.1+ cells, DST and anti-CD154 mAb
treatment does not prolong allograft survival (T. G. Markees,
unpublished observations). We postulate that in the absence of
APC-derived costimulation, TCR-ligated alloreactive
CD8+ T cells are, in effect, short-circuited and
driven to undergo activation-induced apoptosis.
Our hypothesis also predicts that APC activation by a CD40-CD154-independent process will interfere with alloreactive CD8+ T cell deletion and prevent graft prolongation in animals treated with DST and anti-CD154 mAb. One process that activates APCs in a costimulation-independent manner is viral infection (42). Consistent with our prediction, we have observed in preliminary studies that infection of mice with lymphocytic choriomeningitis virus at the time of allotransplantation abrogates the ability of DST and anti-CD154 mAb to prolong skin graft survival (A. A. Rossini, unpublished observations).
Our proposed mechanism of graft survival prolongation is consistent with traditional views of clonal deletion of peripheral T cells. That view is based on studies of CD4+ cells in mice exposed to minor lymphocyte-stimulating superantigens (43, 44, 45, 46, 47, 48). Deletion of CD4+ cells in these systems is more extensive than deletion of CD8+ cells, although deletion of the latter has also been observed (43, 49, 50, 51). Deletion of CD8+ cells has also been documented in systems of clonal exhaustion. Naive H-Y-specific CD8+ T cells transfused into nude male hosts largely disappear after a period of extensive proliferation, leaving behind a small proportion of anergic cells (52). Similar observations have been made in mice expressing a transgenic TCR specific for H2-Kb (53) or Ld (54). Additional support for our proposed mechanism has been obtained in an OVA-specific TCR transgenic system. The data demonstrate that CD4 T cell help is required to prevent the deletion of Ag-activated CD8+ T cells (55). Additional data have shown that the CD4 T cell help needed for the generation of CTL in this kind of TCR transgenic system is provided by APCs activated by a CD40-dependent signaling mechanism (56).
Our hypothesis accounts not only for the basic functional success of the two-element protocol, but also for the ability of CTLA4-Ig under different conditions of administration to either prolong or shorten graft survival (11, 14, 57). CTLA4 can deliver a negative signal to T cells that have been stimulated by TCR engagement. Interaction of B7 with CTLA4 is hypothesized to lead to down-regulation of activated T cells and is reportedly required for the induction of unresponsiveness (58, 59). Blockade of CTLA4 precludes this inhibitory signal and permits very rapid expansion of T cells in weakly stimulatory situations. This may be due to the lowering of the threshold for expansion or to the blockade of CTLA4-mediated apoptotic signals (60, 61). In the context of the two-element protocol, we have documented that activation of tracer populations of CD8+ alloreactive T cells precedes their subsequent deletion. We have also documented that in the presence of anti-CTLA4 mAb, the number of these alloreactive cells increases rather than decreases. We interpret this observation to suggest that interference with basal CTLA4 signals causes Ag-activated CD8+ alloreactive T cells to proceed directly to a state of costimulation-independent activation and proliferation. This interpretation is also supported by in vitro analyses (11).
In contrast, it has also been reported that the administration of DST at the time of transplantation and of CTLA4-Ig 2 days later leads to permanent survival of murine cardiac allografts (62). However, if anti-CTLA4 mAb is given at the time of grafting, graft survival is significantly reduced (57). Based on the kinetics, we suggest that CTLA4 blockade at the time of allostimulation leads to uncontrolled expansion of the alloreactive T cells. Conversely, treatment with CTLA4-Ig two days after priming with DST and grafting blocks CD28-dependent costimulation of Ag-activated alloreactive T cells.
It has been reported that the combination of anti-CD154 mAb and CTLA4-Ig can prolong skin and heart allografts in mice (63) and renal allografts in primates (64). Our hypothesis would predict that prolongation of graft survival by that procedure may not involve deletion of alloreactive CD8+ T cells. We are currently investigating this possibility.
The hypothesis we propose has two important implications. On a general level, it provides testable predictions about how functional tolerance induction in the presence or absence of specific soluble factors, costimulatory molecules, and cell subsets should act at different time points relative to transplantation. On a more specific and practical level, it suggests that the behavior of reagents like CTLA4-Ig may be critically dependent on the kinetics of the tolerization process relative to Ag exposure and subsequent CD8+ T cell activation. We suggest that the use of CTLA4 blockade in clinical "tolerance-based" transplantation will require cautious attention to kinetics (1). The available data also suggest that the use of CTLA4 blockade in spontaneous, progressive, or chronic autoimmune disease will require some knowledge of the timing of the initial activation of autoreactive effector cell populations.
We do not know the fate of alloantigen-activated CD4+ cells that fail to receive costimulation after exposure to DST and anti-CD154 mAb. It has been shown in a CD4+ TCR transgenic system that Ag-activated CD4+ T cells can enter a functionally unresponsive state (22, 59). It has been speculated that such anergized cells may be mediators of tolerance (65, 66). Based on our observation that CD4+ cells are required for long-term graft maintenance (14), we hypothesize that a fraction of Ag-activated CD4+ cells that fail to receive costimulation become anergized and function as suppressor cells. It should be emphasized that the present data address the early events involved in the prolongation of allograft survival. The mechanism of CD4-dependent maintenance of allograft survival (14) is not known.
In conclusion, we propose that DST and anti-CD154 mAb induce "functional allotransplantation tolerance" by activation and deletion of CD8+ alloreactive T cells in the absence of costimulation but in the presence of CD4+ cells activated via TCR ligation. We hypothesize that the role of DST in this process is to prime the system for CD8+ alloreactive T cell deletion in advance of allograft placement.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Aldo A. Rossini, Diabetes Division, University of Massachusetts Medical School, 373 Plantation Street, Biotech 2, Suite 218, Worcester, MA 01605. E-mail address:
3 Abbreviations used in this paper: DST, donor-specific transfusion; MST, median survival time.
Received for publication June 30, 1999. Accepted for publication October 14, 1999.
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