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,
* Universität Leipzig, Leipzig, Germany;
Department of Medicine and
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44122; and
Department of Immunology and Urologic Institute, Cleveland Clinic Foundation, Cleveland, OH 44195
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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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In addition to direct recognition, alloreactive T cells can recognize and respond to donor-derived determinants that have been processed and presented by recipient APCs and are thus self-restricted (1, 2, 4). This indirect alloresponse also seems to derive primarily from naive T cells but comprises only a minority (<10%) of the total alloimmune T cell repertoire (4).
In an effort to further account for the strength of the alloresponse,
it has been hypothesized that a portion of the alloimmune repertoire
derives from a clonally expanded population of memory T lymphocytes
that have been primed to environmental Ags and exhibit chance
cross-reactivity to alloantigens (9, 10, 11). In support of
this view, in vitro studies showed that cultured T cell clones specific
for OVA react with a variety of allostimulator cells at a high
frequency (up to 30%; Ref. 12). In addition, Lechler and
colleagues (9) demonstrated 
10 years ago that a
significant portion of alloreactive T cells in humans have been
sensitized to alloantigens. This latter finding has been confirmed by a
number of investigators, including our laboratory (11),
using a variety of approaches. Although blood transfusions and
pregnancy can directly expose an individual to alloantigens in vivo and
could potentially account for the presence of alloreactive memory T
cells, the T cell repertoires of many individuals without any such
exposure often contain alloreactive T cells with a memory phenotype as
well. Circumstantial evidence suggests that environmental exposure,
including exposure to infectious agents and immunizations, may be
sufficient to prime T cells that cross-react with alloantigens, despite
a lack of exposure to the alloantigens themselves. This hypothesis has
been difficult to evaluate in humans or in animal models.
The presence of such memory/primed alloreactive T lymphocytes is not merely a matter of academic interest. Memory T cells have less stringent costimulatory requirements, lower activation thresholds, alterations in intracellular signaling that result in higher avidity, and broader trafficking patterns than naive T cells (13, 14, 15, 16, 17, 18, 19, 20, 21), thus permitting them to respond rapidly and efficiently to infectious agents to which the organism has been previously exposed. Although such features are clearly beneficial to the health of an individual when exposed to recurrent infections by the same pathogen, the presence of primed alloreactive T cells before a transplant may subject that individual to a higher risk of rejecting the transplanted organ. For example, experimental studies in animal models have shown that primed alloreactive T cells mediate accelerated, "second set" rejection in animals that previously rejected a skin graft from the same donor (22). Moreover, others and we have provided correlative evidence in humans that the presence of donor reactive T cells in an individual predisposes them to a higher risk of posttransplant acute rejection episodes (9, 11, 23). Previously primed or memory T cells may even be resistant to immunomodulation or tolerance induction, an issue that is extremely relevant to present clinical trials in transplantation in which tolerance is being attempted in the human transplant recipients.
In an effort to address these issues, we identified and characterized an animal model in which T cells primed in response to an infectious agent cross-react with one defined set of alloantigens. Our data provide novel in vivo evidence that such cross-reactivity can naturally develop as the protective antipathogen immune response evolves, and further provide new insight into the clinical effects of these cross-reactively primed T cells on the outcome of a transplant.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (B6, H-2b), BALB/c (H-2d), B10.D2 (H-2d), C3H (H-2k), B10.BR (H-2k), P/J (H-2p), DBA (H-2q), RIIIS (H-2r), and SJL (H-2s) mice aged 6–8 wk, were purchased from The Jackson Laboratory (Bar Harbor, ME) or the National Cancer Institute (Frederick, MD). All mice were maintained in the specific pathogen-free animal facility at the Louis Stokes Cleveland Department of Veterans Affairs Medical Center (Cleveland, OH) or at the Cleveland Clinic Foundation (Cleveland, OH). All animal protocols were approved by the Animal Care and Use Committees of the Louis Stokes Cleveland Veterans Affairs Medical Center, Case Western Reserve University (Cleveland, OH), and/or Cleveland Clinic Foundation.
Placement and evaluation of skin grafts
Full thickness trunk skin allografts were placed using standard techniques (24, 25). Skin was harvested from euthanized donor mice, cut into 0.5-cm2 pieces, and placed in sterile PBS until used for transplantation (<30 min). Recipient mice were anesthetized with pentobarbital (50 µg/g body weight) and shaved around the chest and abdomen. The skin graft was placed in a slightly larger graft bed prepared over the chest of the recipient and secured using Vaseline gauze and a bandage. Bandages were removed on day 7, and the grafts were then visually scored daily for evidence of rejection. Donor-specific transfusion and costimulatory blockade were performed as described (26, 27). A total of 20 x 106 donor spleen cells were administered i.v. on day -7 with respect to graft placement. Repeat i.p. injections of anti-CD40 ligand (CD40L)3 Ab MR1 (purchased from BioExpress, West Lebanon, NH) were given on days -7, -4, 0, and +4 with respect to graft placement (250 µg per injection). Grafts were considered fully rejected when they were >90% necrotic. Selected grafts were harvested, embedded in paraffin, stained with H&E, and examined by light microscopy.
Leishmania infections
L. major promastigotes were grown in vitro as previously described (28). Two million stationary phase promastigotes were injected into the hind footpads of B6 mice. Maximal footpad swelling occurred at 2–3 wk following infection and was fully resolved by wk 4–5 after injection.
Antigens
Soluble Leishmania Ag (SLA) was prepared from Leishmania promastigotes as described (29). Renal tubular Ag (RTA) was prepared from rabbit renal cortex as previously described (30). Hen eggwhite lysozyme (HEL) and chicken OVA were purchased from Sigma-Aldrich (St. Louis, MO). Peptides HY1pb (MHC Ib-restricted, immune dominant determinant derived from the Uty gene of the male Ag (31), WMHHNMDLI) and HY2pb (MHC II I-Ab-restricted, immune dominant determinant derived from the Dby gene of the male Ag (32), NAGFNSNRANSSRSS) were synthesized by Research Genetics (Huntsville, AL).
T cell subset isolation
Splenic and lymph node-purified CD3+ and CD4+ T cells were purified using commercially available murine T cell isolation columns from R&D Systems (Minneapolis, MN) following the instructions supplied by the manufacturer. Resultant cells were washed in HBSS, assessed for viability by trypan blue exclusion, and resuspended at appropriate concentrations for use in the various assays. All T cell subpopulations were >92% pure by flow cytometry (data not shown).
Ab determination by flow cytometry
Blood samples were collected from the tails of experimental animals and serum was isolated by centrifugation. Single-cell suspensions of P/J or third party SJL thymocytes were prepared, live cells were counted by trypan blue exclusion, and the cells were divided into aliquots of 1 x 106 cells. The thymocytes were pelleted by centrifugation and resuspended in 100 µl of diluted serum (serial dilutions were made from 1/10 to 1/2430 in PBS with 5% FBS and 0.02% NaN3) followed by a 1-h incubation on ice and three washes with PBS with 5% FBS and 0.02% NaN3. Detecting FITC-conjugated goat anti-mouse IgG Ab (BD PharMingen, San Diego, CA) was diluted 1/100 in PBS plus 5% goat serum, and 0.02% NaN3 and 100 µl were added to the samples. Thymocytes were incubated for 1 h on ice in the dark, washed three times, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry using a BD Biosciences FACScan and CellQuest software (BD Biosciences, Mountain View, CA) using 10,000 ungated events.
ELISPOT assays
Assays were performed as outlined previously in detail (4, 30, 33, 34). Briefly, ImmunoSpot M200 plates (Cellular Technology, Cleveland, OH) were coated overnight with the capture Abs in sterile PBS. The plates were blocked for 1 h with sterile 1% BSA in PBS and washed three times with sterile PBS. Spleen cells (106/well) or purified T cells were plated in HL-1 medium (BioWhittaker, Walkersville, MD) with or without mitomycin C-treated stimulator cells (300,000/well) and/or soluble Ags (i.e., SLA, RTA, OVA at 10 µg/ml). The plates were then incubated at 37°C, 5% CO2 for 24 h. After washing with PBS followed by PBS 0.025% Tween (PBST), detection Abs were added overnight. After washing with PBST, alkaline phosphatase-conjugated anti-biotin Ab (Vector Laboratories, Burlingame, CA) diluted 1/1000 in PBST was added for 2 h at room temperature. The plates were developed using a nitroblue tetrazolium chloride (Bio-Rad Laboratories, Hercules, CA) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich) substrate. Sixty-six microliters of 60-mM nitroblue tetrazolium chloride in 70% dimethyl formamide plus 33 µl of 250 mM 5-bromo-4-chloro-3-indolyl phosphate in 100% dimethyl formamide were dissolved in 10 ml of 0.1 M Trizma base, 0.1 M NaCl, 0.1 M MgCl2 (pH 9.5), 200 µl were placed in each ELISPOT well. The plates were then washed with water and allowed to air dry. The resulting spots were counted on an ImmunoSpot Series 1 Analyzer (Cellular Technology). Digitized images were analyzed for the presence of areas in which color density exceeds the background by a factor set on the basis of the comparison of control (containing T cells and APC without Ag) and experimental wells (containing Ag). After separating spots that touch or partially overlap, additional criteria of spot size and circularity were applied to gate out speckles and noise caused by spontaneous substrate precipitation and nonspecific Ab binding. Objects that do not meet these criteria were ignored and areas that meet them were recognized as spots, counted, and highlighted.
Statistical analysis
Statistical analysis to determine differences between groups for immune responses was performed using the Student t test for equal or unequal variances. A value of p < 0.05 was considered statistically significant. Kaplan Meier survival analysis was used to determine the difference in median graft survival between groups.
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Results |
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by ELISPOT
in a <24-h assay is a marker of a primed T cell and directly
correlates with down-regulation of L-selectin on the T cell surface
(3).
Spleen cells from BALB/c (H-2d), C3H
(H-2k), and B6 (H-2b) mice
immunized with either HEL, OVA, or RTA mixed in IFA or CFA did not
respond stronger than naive controls to any of the tested alloantigenic
stimulator cells (data not shown). However, spleen cells obtained from
B6 mice 6 wk following infection with L. major (a time at
which the animals had clinically recovered from the infection)
exhibited an intriguing pattern suggesting cross-reactivity. Spleen
cells from the L. major-infected animals responded by
producing IFN- at a significantly higher frequency to P/J
(H-2p) stimulator cells compared with spleen
cells from naive mice (90/million spleen cells vs <5/million,
respectively). In contrast, the spleen cells from naive mice and from
L. major-infected mice responded equally, and at low
frequency, to SJL (H-2s) stimulator cells (Fig. 1
, <20 per million IFN- producers for
each group), and at a similar low frequency to a variety of other
stimulator cells (H-2d, k, q, r haplotypes, data
not shown).
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). The T cells isolated from
naive mice produced predominantly IL-2 in response to P/J and SJL
stimulators (at a frequency of 30–40 per 50,000 T cells) with few
IFN-, IL-4, or IL-5 producers (<10 per 50,000). This low frequency
response is consistent with our previous studies demonstrating that
naive animals have largely naive T cell repertoires that do not contain
primed IFN--producing T cells specific for alloantigens
(3). As anticipated for a naive T cell repertoire, T cells
isolated from naive B6 mice did not respond to SLA or to a control RTA
(Fig. 2B). In contrast, T cells obtained from animals that
resolved L. major infections responded vigorously to SLA
(250 IFN- ELISPOTs per 50,000 T cells) showing that specific
anti-L. major immunity was induced by the infection.
Consistent with the spleen cell results shown in Fig. 1
, the T cells
from L. major-primed mice also produced IFN- in response
to P/J stimulator cells (40–70 IFN- ELISPOTs per
50,000 T cells), confirming cross-reactive T cell priming. This
cross-reactive immunity was type 1 in character, as no IL-4 or IL-5 was
produced in response to P/J alloantigens (Fig. 2A).
Importantly, T cells isolated from L. major-infected mice
did not produce IFN- in response to SJL stimulators, showing that
the effect was not simply unspecific activation of a large proportion
of the T cell repertoire. As one specificity control to determine
whether T cell priming to any Ag would induce a cross-reactive immune
response, we placed B6 male skin grafts on B6 female recipients, and 8
wk later tested splenic T cells for responses to P/J stimulators. As
shown in Fig. 2B, T cells from these male Ag-primed mice did
not cross-react with P/J stimulators, but did respond at high frequency
to donor male stimulator cells and to both MHC I- and MHC
II-restricted, HY-derived peptides. The relative specificity of the
L. major:P/J cross-reactive T cell immune response was
confirmed by additional experiments in which spleen cells obtained from
B6 mice 2 mo following pulmonary infection with Mycobacterium
bovis (Bacillus of Calmette and Guerin) responded at
high frequency (>400 per 50,000 T cells) to self APCs, as well as to a
panel of stimulator cells (H-2d, k, q, r, s, p
haplotypes, data not shown). Similar results were noted using spleen
cells isolated from B6 mice 4 wk following oral infection with
Salmonella typhimurium. Thus, some chronic intracellular
infections, such as Mycobacterium and Salmonella,
seem to nonspecifically activate a wide repertoire of alloreactive T
cells, while infection with L. major seems to result in a
relatively specific cross-reactive T cell alloimmune response.
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). CD4+ T cells
from L. major-primed mice, but not from naive B6 mice,
responded specifically to P/J (but not SJL) stimulator cells (200
per 100,000 CD4 T cells) by producing IFN-. No IL-4 or IL-5 was
detected (data not shown). Consistent with the development of activated
or memory T cells as suggested by IFN- ELISPOT, the percentage of
CD4+ T cells from L. major-infected
mice expressing cell surface markers associated with memory phenotype
(either CD44high or CD62 L-selectin
(CD62L)low) increased 2- to 3-fold compared with
naive mice (Table I).
CD8+ T cells isolated from naive or L.
major-primed mice did not respond strongly to either P/J or SJL
stimulators (<10 per million IFN- ELISPOT), and L. major
infection had only minimal effects on altering the cell surface
expression of memory cell markers in this population of cells (Table I). The data demonstrate that the induced cross-reactivity was found
within the CD4+ T cell population, consistent
with the fact that CD4+ T cells are the dominant
immune mediators in response to L. major in this mouse
strain (35, 36).
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100 IFN- ELISPOTs per 50,000 T cells,
Fig. 2). However, these CD4+ T cells did not
react with SLA, suggesting that the anti-P/J cross-reactivity
primed by L. major infection was directed toward
Leishmania Ags not found in the SLA preparation (which only
contains a portion of the total antigenic repertoire of the L.
major organism; Ref. 29). To determine whether the induced anti-L. major-immune response also exhibited B cell cross-reactivity to P/J alloantigens, we tested serum from naive mice, L. major-primed mice, and P/J skin graft-primed B6 mice for binding to P/J (or control SJL) thymocytes by flow cytometry. No anti-P/J Abs were detected in naive mice or in mice that cleared L. major infections (n = 6, data not shown), suggesting that the anti-P/J cross-reactivity induced by L. major infection was restricted to T cells and did not involve the humoral immune response (as a positive control, anti-P/J-specific alloantibodies were detected in B6 mice that rejected P/J skin grafts).
To determine the in vivo relevance of the detected cross-reactivity, we
next placed P/J or control SJL skin grafts on L.
major-infected B6 mice. We reasoned that if the L.
major infection-primed T cells with cross-reactivity to P/J
alloantigens, then these animals might specifically reject P/J skin
grafts with accelerated kinetics compared with naive animals. As shown
in Fig. 4, mice previously infected with
L. major rejected P/J skin grafts 2 days faster than naive
mice, an acceleration that was both statistically and biologically
significant (p < 0.05 by Kaplan Meier survival
analysis). In addition, the accelerated rejection rate was not
statistically different from second set rejection of P/J skin (median
survival time of 11.5 days, n = 4, data not shown),
which is considered the maximum rate of rejection in this model. In
control experiments, naive mice and mice that previously cleared
L. major infections both rejected third party SJL skin
grafts with similar kinetics (median survival time of 14.8 days per
group, Fig. 4), confirming that the L. major-induced
cross-reactive immune response was specific for P/J.
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We chose to study the effects of DST plus anti-CD40L Ab MR1
treatment (DST/MR1) on graft survival using a previously published
protocol in which animals are treated with DST (20 million spleen
cells, i.v.) combined with repeat i.p. injections of MR1 (250 µg) on
days -7, -4, 0, and +4 with respect to graft placement (26, 27, 38). Used in this manner, P/J DST/MR1 treatment of naive mice
induced prolonged skin graft survival in naive mice (Fig. 5, median survival time >45 days,
vs 14 days in untreated mice). Despite the prolongation of graft
survival, the animals were not truly tolerant to the skin grafts, as
the grafts were all eventually rejected and second P/J skin grafts were
not accepted (data not shown).
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All six of the P/J grafts were rejected within 15–20 days of graft
placement.
In an effort to evaluate how the DST/MR1 treatment affected the induced
T cell immune response in these mice following graft placement, an
additional set of experiments was performed. Naive or L.
major-infected B6 mice were treated with DST/MR1 and a P/J skin
graft was placed as above. As positive controls, P/J skin grafts were
placed onto naive B6 mice without any treatment. All animals were
sacrificed on day 13 following placement of the skin graft. At this
time point, the P/J skin grafts placed on naive, untreated recipients
were fully rejected, the grafts placed on the L.
major-infected mice treated with DST/MR1 were undergoing acute
rejection (50% necrotic by visual inspection), and the grafts
placed on uninfected recipients treated with DST/MR1 did not
demonstrate any visual evidence of rejection. Immune cells were
isolated from animals in each group and were tested in recall cytokine
ELISPOT assays (Fig. 6). Immune cells
from uninfected, untreated recipients that rejected P/J skin grafts
contained a high frequency of IFN- and IL-2 producers specific for
P/J stimulators (1200 IFN- producers per million lymph
node cells), consistent with the results in Fig. 2. Immune cells
from uninfected mice treated with DST/MR1 associated with prolonged
skin graft survival responded only weakly to donor stimulator cells at
frequencies no different from those detected in naive mice (Fig. 6, 50 per million IFN- producers). Furthermore, there was no
evidence for type 2 immune deviation as no donor-reactive IL-4 (or
IL-5, data not shown) was detected. In contrast, immune cells from the
L. major-infected mice treated with DST/MR1 that were
undergoing acute rejection of P/J skin responded to donor P/J
stimulator cells (700 per million IFN- producers), albeit at
lower frequencies than that found in untreated mice (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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at increased
frequency in comparison to naive animals (
Figs. 1–3). The induced T
cell immunity exhibited a type 1 cytokine phenotype (IFN-, IL-2
without IL-4 or IL-5) consistent with what is known about the
anti-L. major immune response in B6 mice (35, 36, 39) and further consistent with the dominant cytokine phenotype
induced following transplantation (3, 24). Moreover, the
cross-reactive immune response was confined to the cellular arm of the
immune repertoire, as no alloantibodies were detected in L.
major-primed mice. The detection of cross-reactive immunity between an infectious agent and alloantigens is reminiscent of the concept of "molecular mimicry" between infectious agents and autoantigens. This phenomenon is relevant to initiation of certain autoimmune diseases both in mice and in humans (40, 41, 42). Our findings in a model of transplant rejection provide the first experimental evidence that an analogous molecular mimicry phenomenon can alter the course of transplant rejection.
The results have important implications for human recipients of organ transplants. It has been noted for many years that peripheral blood of normal human volunteers contains alloreactive T cells from within both the primed and the naive T cell populations (9, 10). Although some of these human alloreactive T cells were likely primed directly to alloantigens through exposure to blood transfusion and/or pregnancies, it has been hypothesized that infections and/or vaccinations could prime Ag-specific T cells that happen to cross-react with alloantigens. In vitro studies using human CTL clones have in fact documented cross-reactivity between immunodominant epitopes of the EBV and specific HLA molecules (43). Our data provide proof of principal that immunity to an infectious agent can cross-react with some alloantigens with relative specificity, and provide the first animal model with which to carefully study this phenomenon and its consequences on transplantation.
Although we have presented strong evidence for cross-reactivity, we do not know the specific L. major-derived determinants that cross-react with P/J alloantigens. T cells from B6 mice sensitized to P/J skin grafts did not respond to SLA, an Ag preparation produced from the promastigote stage of the L. major life cycle. The Ags found in this soluble preparation contain well-defined immunodominant Ags, such as LACK (44), and it is known that soluble Ag is comparable to irradiated whole parasite for induction of in vivo resistance or susceptibility to subsequent infection (29, 45). Nonetheless, the SLA preparation excludes insoluble promastigote Ags as well as some determinants that are unique to the tissue-phase, amastigote form of L. major (29, 45). Thus, it seems likely that the cross-reactive epitopes derive from one of these latter sources. Alternatively, it is possible that Leishmania infection triggers anti-P/J Ag T cells specificities not typically induced by P/J allograft rejection, such as a normally sequestered Ag.
Regardless of the initial antigenic stimulus, it is important to note that primed T cells exhibit immunologically relevant properties that differ from naive T cells. Recent studies have revealed that priming of naive T cells leads to differentiation into effector cells, massive expansion, and alteration of cell surface marker expression, thus enabling the primed cells to traffic into peripheral organs (16, 20). Following resolution of the immune response, there is widespread apoptosis of this Ag-reactive T cell repertoire leaving a small number of residual, specific memory cells (46, 47, 48). In comparison to naive T cells that are largely confined to the primary lymphoid organs, these memory T cells maintain their ability to circulate into peripheral organs (16, 20). In addition, compared with naive cells, memory T cells are resistant to apoptotic signals (49, 50, 51), and have lower costimulatory requirements (17, 18), lower activation thresholds (19), higher functional avidities (14), and more rapid onset of effector functions upon re-encounter of their Ag (16, 20, 21). These are key features for protection from reinfection as they permit effective immune surveillance and consequent rapid control of infectious agents previously encountered by the host.
However, for T cells that recognize alloantigens, these same features
could theoretically result in deleterious effects with regard to the
outcome of a transplanted organ. In support of this concept, the data
presented in this manuscript show that the pretransplant presence of
primed alloreactive T cells induced through a cross-reactive infection
were specifically associated with accelerated rejection of a skin graft
(Fig. 4). Mice that recovered from L. major infections and
developed cross-reactive immune responses to P/J alloantigens rejected
P/J skin grafts at rates commensurate with second set rejection (Fig. 4). The L. major-infected animals rejected third party SJL
skin grafts at the same rate as did naive mice, consistent with the
lack of detectable cross-reactivity to SJL stimulators in vitro (
Figs. 1–4). Overall, the data demonstrate that the induced anti-P/J
immune response following L. major infection was not simply
an in vitro observation, but had specific, clinically significant in
vivo consequences. It should be noted that live L. major
parasites can be isolated from B6 mice for many months after they have
clinically resolved their infections (52), thus making it
possible that both effector and/or memory CD4+ T
cells contributed to the L. major-specific (and
cross-reactive P/J-specific) responses detected in our experiments.
As the T cell repertoire of previously nontransplanted humans often contains alloreactive memory T cells (9, 11, 23), some of which were presumably primed through cross-reactivity to infectious stimuli, the findings from our studies may have important implications for transplant patients. Indeed, others and we have provided evidence that the frequency of donor-specific memory T cells pretransplant may predict the posttransplant risk of acute rejection episodes (9, 11, 23).
Our data additionally highlight a heretofore under-appreciated
observation, that an immune manipulation effective in prolonging
allograft survival in naive mice (DST/MR1) is largely ineffective in
animals with preexisting alloreactive T cells primed through a
cross-reactive infectious stimulus. Although DST plus anti-CD40L
mAb significantly prolonged P/J skin graft survival in naive animals,
the same therapy had essentially no effect in mice that clinically
controlled an L. major infection (Fig. 5). Moreover,
previous L. major infection prevented donor-specific T cell
hyporesponsiveness induced by DST/MR1 treatment (Fig. 6). It should be
noted that recently published studies by Rossini and colleagues
(53, 54), in which acute LCMV infection was shown to
abrogate the effects of DST/MR1, addressed a related but clearly
different question. Although certainly quite relevant to the transplant
patient who develops an acute viral infection, such data do not address
the question evaluated by our studies: how does the presence of primed
T cells that cross-react with alloantigens affect the course of graft
rejection?
CD40 binding to CD40L is known to be an important costimulatory signal for activation of naive T cells, and Ag recognition by naive T cells in the absence of such costimulation can actively induce a tolerant state (26, 27, 55, 56). However, in contrast to naive T cells, the lower costimulatory requirements of effector or memory T cells can allow activation in the total absence of costimulation (even in the absence of CD4 or CD8 interactions), particularly if the TCR:MHC:peptide interaction is of high affinity. In addition, the up-regulated expression of antiapoptotic genes such as bcl-2 renders memory/effector cells resistant to activation-induced cell death (51, 57). Therefore, it might be anticipated that costimulatory blockade would not have the same effects on primed vs naive T cells—a hypothesis supported by the present data. It remains to be determined whether higher doses of costimulatory blockade and/or more prolonged treatment would be effective in mice with primed T cell immune responses in this system. Alternate protocols of costimulatory blockade have indeed been effective in selected animals that were presensitized to alloantigens directly before graft placement (58). Nonetheless, our data strongly suggest that cross-reactive T cell immunity is sufficient to render standard DST/MR1 therapy ineffective—a finding that in itself has important implications for human transplantation.
It is further interesting to note that preliminary work in nonhuman primates suggests that some animals are relatively resistant to the effects of costimulatory blockade, particularly in the absence of concomitant immunosuppression (59, 60). Although there are many differences between rodent models and primate or human transplant recipients, one speculative explanation for the difference in efficacy is that the immune repertoires of outbred primates maintained under standard living conditions (but not naive laboratory rodents) may contain donor-reactive memory T cells that are resistant to costimulatory blockade.
In conclusion, these studies definitively demonstrate that cross-reactive priming of alloreactive T cells can occur in vivo and furthermore provide evidence that such primed T cells can have a significant impact on the outcome of a subsequent transplant. The results have important implications for human transplant recipients whose immune repertoires may contain cross-reactively primed allospecific T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anna Valujskikh, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, NB-30, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: valujsa{at}ccf.org
3 Abbreviations used in this paper: CD40L, CD40 ligand; SLA, soluble Leishmania Ag; RTA, renal tubular Ag; HEL, hen eggwhite lysosome; DST, donor-specific transfusion; CD62L, CD62 L-selectin.
Received for publication May 28, 2002. Accepted for publication July 29, 2002.
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References |
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  H. Zheng, C. Matte-Martone, D. Jain, J. McNiff, and W. D. Shlomchik Central Memory CD8+ T Cells Induce Graft-versus-Host Disease and Mediate Graft-versus-Leukemia J. Immunol., May 15, 2009; 182(10): 5938 - 5948. [Abstract] [Full Text] [PDF]
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Z. Wu, Y. Wang, F. Gao, X. Shen, Y. Zhai, and J. W. Kupiec-Weglinski Critical Role of CD4 Help in CD154 Blockade-Resistant Memory CD8 T Cell Activation and Allograft Rejection in Sensitized Recipients J. Immunol., July 15, 2008; 181(2): 1096 - 1102. [Abstract] [Full Text] [PDF]
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D. Stapler, E. D. Lee, S. A. Selvaraj, A. G. Evans, L. S. Kean, S. H. Speck, C. P. Larsen, and S. Gangappa Expansion of Effector Memory TCR V{beta}4+CD8+ T Cells Is Associated with Latent Infection-Mediated Resistance to Transplantation Tolerance J. Immunol., March 1, 2008; 180(5): 3190 - 3200. [Abstract] [Full Text] [PDF]
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 J. Yang, M. O. Brook, M. Carvalho-Gaspar, J. Zhang, H. E. Ramon, M. H. Sayegh, K. J. Wood, L. A. Turka, and N. D. Jones Allograft rejection mediated by memory T cells is resistant to regulation PNAS, December 11, 2007; 104(50): 19954 - 19959. [Abstract] [Full Text] [PDF]
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Y. Zhai, Y. Wang, Z. Wu, and J. W. Kupiec-Weglinski Defective Alloreactive CD8 T Cell Function and Memory Response in Allograft Recipients in the Absence of CD4 Help J. Immunol., October 1, 2007; 179(7): 4529 - 4534. [Abstract] [Full Text] [PDF]
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 A. Valujskikh and X. C. Li Frontiers in Nephrology: T Cell Memory as a Barrier to Transplant Tolerance J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2252 - 2261. [Full Text] [PDF]
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L. K. Selin and M. A. Brehm Frontiers in Nephrology: Heterologous Immunity, T Cell Cross-Reactivity, and Alloreactivity J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2268 - 2277. [Abstract] [Full Text] [PDF]
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 M. A. Brehm, J. Mangada, T. G. Markees, T. Pearson, K. A. Daniels, T. B. Thornley, R. M. Welsh, A. A. Rossini, and D. L. Greiner Rapid quantification of naive alloreactive T cells by TNF-{alpha} production and correlation with allograft rejection in mice Blood, January 15, 2007; 109(2): 819 - 826. [Abstract] [Full Text] [PDF]
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B. Koehn, S. Gangappa, J. D. Miller, R. Ahmed, and C. P. Larsen Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation. J. Immunol., March 1, 2006; 176(5): 2691 - 2696. [Abstract] [Full Text] [PDF]
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M. D. Vu, M. R. Clarkson, H. Yagita, L. A. Turka, M. H. Sayegh, and X. C. Li Critical, but Conditional, Role of OX40 in Memory T Cell-Mediated Rejection J. Immunol., February 1, 2006; 176(3): 1394 - 1401. [Abstract] [Full Text] [PDF]
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Q. Zhang, Y. Chen, R. L. Fairchild, P. S. Heeger, and A. Valujskikh Lymphoid Sequestration of Alloreactive Memory CD4 T Cells Promotes Cardiac Allograft Survival J. Immunol., January 15, 2006; 176(2): 770 - 777. [Abstract] [Full Text] [PDF]
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D. Baeten, S. Louis, C. Braud, C. Braudeau, C. Ballet, F. Moizant, A. Pallier, M. Giral, S. Brouard, and J.-P. Soulillou Phenotypically and Functionally Distinct CD8+ Lymphocyte Populations in Long-Term Drug-Free Tolerance and Chronic Rejection in Human Kidney Graft Recipients J. Am. Soc. Nephrol., January 1, 2006; 17(1): 294 - 304. [Abstract] [Full Text] [PDF]
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M. A. Brehm, K. A. Daniels, J. R. Ortaldo, and R. M. Welsh Rapid Conversion of Effector Mechanisms from NK to T Cells during Virus-Induced Lysis of Allogeneic Implants In Vivo J. Immunol., June 1, 2005; 174(11): 6663 - 6671. [Abstract] [Full Text] [PDF]
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 T. El-Sawy, M. Miura, and R. Fairchild Early T Cell Response to Allografts Occuring Prior to Alloantigen Priming Up-Regulates Innate-Mediated Inflammation and Graft Necrosis Am. J. Pathol., July 1, 2004; 165(1): 147 - 157. [Abstract] [Full Text] [PDF]
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Y. Chen, P. S. Heeger, and A. Valujskikh In Vivo Helper Functions of Alloreactive Memory CD4+ T Cells Remain Intact Despite Donor-Specific Transfusion and Anti-CD40 Ligand Therapy J. Immunol., May 1, 2004; 172(9): 5456 - 5466. [Abstract] [Full Text] [PDF]
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G. Demirci, F. Amanullah, R. Kewalaramani, H. Yagita, T. B. Strom, M. H. Sayegh, and X. C. Li Critical Role of OX40 in CD28 and CD154-Independent Rejection J. Immunol., February 1, 2004; 172(3): 1691 - 1698. [Abstract] [Full Text] [PDF]
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C. He, S. Schenk, Q. Zhang, A. Valujskikh, J. Bayer, R. L. Fairchild, and P. S. Heeger Effects of T Cell Frequency and Graft Size on Transplant Outcome in Mice J. Immunol., January 1, 2004; 172(1): 240 - 247. [Abstract] [Full Text] [PDF]
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F. G. Lakkis and M. H. Sayegh Memory T Cells: A Hurdle to Immunologic Tolerance J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2402 - 2410. [Full Text] [PDF]
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M. A. Brehm, T. G. Markees, K. A. Daniels, D. L. Greiner, A. A. Rossini, and R. M. Welsh Direct Visualization of Cross-Reactive Effector and Memory Allo-Specific CD8 T Cells Generated in Response to Viral Infections J. Immunol., April 15, 2003; 170(8): 4077 - 4086. [Abstract] [Full Text] [PDF]
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) or from B6 mice 8 wk following infection with L.
major (
) and tested in IFN-
, statistically significant
prolongation in graft survival vs nontreated naive mice,
p < 0.05). Eight to 12 wk following infection with
L. major, B6 mice were treated with P/J DST plus MR1 and
engrafted with P/J skin (•). n = 4–8 per group.
All of the skin grafts in the naive mice treated with DST plus MR1
eventually rejected (days 50–60) and second P/J skin grafts were
rejected by these mice by day 15 demonstrating that true tolerance was
not achieved in any animal.
) and tested in recall cytokine ELISPOT assays against P/J and
third party SJL stimulator cells. n = 3 per group.
The depicted results represent the mean values of duplicate wells
(<10% variability between wells) as counted by the computer-assisted
image analyzer. The results are representative of two independently
performed experiments with similar results. *, Reached statistical
significance vs the DST/MR1-treated group, p <
0.05.