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-Producing Lymphocytes Is a Manifestation of Immunologic Memory and Correlates with the Risk of Posttransplant Rejection Episodes
,§
Departments of
*
Medicine,
Pathology, and
Surgery and
§
Histocompatibility Laboratory, University Hospitals of Cleveland, Cleveland Veterans Affairs Medical Center and Case Western Reserve University, Cleveland, OH, 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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only when there is defined immunologic priming, thus
representing a measure of primed donor-specific immunity. Because the
environmental Ag exposure of the recipient is not a function of the HLA
mismatch between donor and potential recipient, the number of HLA
mismatches may not correlate with the frequency of pretransplant,
donor-specific IFN--producing PBLs. Studies of donor-specific
IFN--producing lymphocytes in a cohort of patients being evaluated
for renal transplantation corroborated this hypothesis. Moreover, for
recipients of both living and cadaver renal allografts, the
pretransplant frequency of donor-specific memory cells correlated with
the posttransplant risk of developing acute rejection episodes. This
improved ability to define the strength of the allospecific immune
response by enzyme-linked immunospot assay may allow improved pairing
of recipients with donors and identification of kidney allograft
donor-recipient pairs at high risk for acute rejection, thus permitting
targeted interventions aimed at prolonging graft
survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human immune repertoire is constantly being shaped through exposure to environmental Ags, resulting in generation of memory T cells primed to respond rapidly upon re-exposure to the inciting stimulus (5, 6). Some of these primed T cells cross-react with alloantigens, in part accounting for the high frequency of alloreactivity (7, 8). Such cross-reactive memory can result in presensitization to a potential donor despite lack of exposure to tissues from that donor (7, 8, 9, 10, 11, 12, 13). As primed memory cells have lower activation thresholds than their naive counterparts (14, 15, 16, 17), their presence before transplantation may increase the risk of a poor outcome after placement of the allograft.
If it were possible to identify a functional measure of primed, cross-reactive, donor-specific T cell reactivity, it might provide an improved assessment of the recipient’s pretransplant immune status, and thereby allow the selection of donors to which the recipient has little pre-established cellular immunity. Furthermore, such functional information may help to improve donor-recipient pairing, help to tailor immunosuppression for high vs low risk recipients, and potentially prevent graft failure in selected patients.
Primed memory T cells can be discriminated from naive T cells by their
ability to rapidly produce cytokines in short-term assays
(18). Naive cells produce low amounts of IL-2 alone upon
initial stimulation (although they will differentiate to produce other
cytokines over several days), while precommitted memory cells rapidly
produce IL-2 and other cytokines, including IFN-, IL-4, and/or IL-5
(18, 19, 20). It is also established that both
CD4+ and CD8+ T lymphocytes
can mediate allograft rejection through direct cytotoxicity and/or
through induction of cytokine-induced inflammation in the target organ
(21, 22, 23). In particular, IFN-, which can promote
macrophage and cytotoxic lymphocyte activation, has been linked
strongly to allograft rejection in both animal models and humans
(21, 22, 23, 24). These findings suggest that the ability to
detect IFN--producing alloreactive T cells in short-term culture may
be a useful functional measure of the primed strength of the
donor-specific alloresponse.
In this regard, our laboratory has developed a highly sensitive cytokine ELISPOT2 assay that is capable of characterizing the frequencies of alloantigen-specific T cells in short-term culture (18, 23, 25, 26), thus providing a reflection of their function in vivo. In this report we show that this assay can detect cytokines produced by individual human PBLs. Moreover, the data suggest that the assay provides a measure of the strength of pretransplant alloreactivity that is independent of the number of Ag mismatches at MHC loci, and that it provides information useful in predicting the risk of acute rejection following renal transplantation. This assay may be a useful immunologic tool for selecting allograft donors and merits further investigation as a means of assessing the risk of acute and chronic allograft rejection in human recipients of organ transplants.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples were obtained in heparinized tubes from patients being evaluated for renal transplantation at the Transplant Clinic of the University Hospitals of Cleveland (Cleveland OH). We studied 45 individuals from 17 families being evaluated for living related renal transplants, 11 patients being evaluated for living unrelated renal transplantation from 13 potential donors, and 10 patients who were recipients of cadaver renal allografts. All samples were obtained before transplantation. Blood samples also were obtained from eight normal healthy volunteers.
Preparation of PBL and stimulator cells
PBLs were isolated from 12 to 20 ml of peripheral blood by standard Isoprep (Robbins Scientific, Sunnyvale CA) centrifugation (26). Stimulator cells (either PBLs from live donors or spleen cells from cadaver donors) were prepared by standard Isoprep centrifugation followed by treatment with mitomycin C (Boehringer Mannheim, Indianapolis, IN; 50 µg/ml) for 20 min and then three washes in PBS. In some experiments the stimulator cells were irradiated with 4000 rad followed by a single wash in HBSS before use. Viable cells were counted using an immunofluorescence microscope in the presence of acridine orange/ethidium bromide (26).
Determination of MHC I and MHC II phenotypes
HLA phenotypes were determined by standard, clinically applicable techniques. Ags encoded by HLA class I loci (A, B, and C) were identified by the basic microlymphocytotoxicity assay (27), using local antisera. Briefly, donor cells to be typed for HLA class I Ags were incubated with antisera at room temperature for 30 min and then incubated with complement at 20°C for another 60 min. The presence of Ags recognized by particular antisera was inferred from the extent of cell death in the corresponding wells, as gauged by two-color immunofluorescence using acridine orange and ethidium bromide. Class II alleles were determined by sequence-specific priming and PCR (28). Donor and recipient cells to be typed for HLA-DR ß1 and DQ ß loci were disrupted for isolation of genomic DNA using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Sets of primers (One Lambda, Canoga Park, CA) specific for one or a few alleles were then used to amplify segments of the corresponding genes using a Gene Amp 9600 Thermal Cycler (Perkin-Elmer, Norwalk, CT) and PCR. The amplified products were subjected to electrophoresis in 2% agarose gels and stained with ethidium bromide. The resulting patterns of observable bands were used to assign DR and DQ alleles.
Isolation and maintenance of the NVH1 cell line
NVH1 is a CD4+ alloreactive human T cell line developed in our laboratory. To produce this cell line, 3 x 106 PBLs from donor NG (A33, B14, DR1, DR5) were mixed with 3 x 106 stimulator PBLs from donor PH (A2, A28, B13, B14, DR1, DR7) in complete RPMI medium (90%RPMI,10% newborn calf serum, and 1% L-glutamine) and placed at 37°C in 7% CO2 for 5 days. The cells were washed, and their number was adjusted to 3 x 105/ml in complete RPMI plus 10 ng/ml (2 U/ml) recombinant human IL-2 (a gift from Sandoz, Basel, Switzerland). The feeding cycle was repeated every 5 days. One month after the cell line was started, it was restimulated with donor stimulator cells and placed in a 30-day alternating cycle of stimulation followed by feeding with IL-2. Surface staining for CD4 and CD8 by flow cytometry (25) revealed a pure population of CD4+ cells (data not shown). Alloantigen specificity was determined by ELISPOT assay and intracytoplasmic staining/flow cytometry as outlined below.
ELISPOT assay
Ninety-six-well ELISPOT plates (Polyfiltronics, Rockland MA)
were coated with capture Abs for either IL-5 (TRFK5, isolated from
hybridoma in our laboratory; 5 µg/ml), IL-4 (8D4-8, PharMingen, San
Diego CA; 5 µg/ml), IL-2 (5334.21, R & D Systems, Minneapolis MN; 2
µg/ml), or IFN- (2G1, Endogen, Woburn, MA; 4 µg/ml) in PBS
overnight at 4°C. The plates were then blocked with PBS/0.1% BSA and
washed with PBS. Three hundred thousand responder PBLs were added to
each well in 100 µl of complete RPMI medium. Newborn calf serum was
obtained from HyClone (Logan, UT); it was tested in multiple assays and
heat inactivated at 56°C for 30 min before use (comparison of newborn
calf serum to autologous human serum revealed no difference in
results). The PBLs were activated in vitro with donor stimulator PBLs,
tetanus toxoid (1/100; Connaught Laboratories, Willowdale, Canada),
purified protein derivative (PPD; 1/200; Evans Medical, Langurst,
U.K.), cat pelt Ag (500–1000 U/ml; Berkeley Biologicals, Berkeley,
CA), or PHA (10 µg/ml final concentration; Sigma, St. Louis, MO) in a
total volume of 200 µl. Control wells contained responder PBLs plus
medium alone. After 24 h for IFN- and IL-2 or 48 h for
IL-4 and IL-5, the plates were washed, and biotinylated detection Abs
(IL-5: JES1-5A10, PharMingen, 2 µg/ml; IL-4: MP4-25D2, PharMingen, 2
µg/ml; IL-2: BG5, Endogen, 3 µg/ml; IFN-: B133.5, Endogen, 4
µg/ml) were added to the wells overnight at 4°C. Streptavidin-HRP
(Dako, Carpenteria, CA) was then added for 2 h at room
temperature. The spots were developed using 3-amino-9-ethylcarbazole
(Pierce, Rockford, IL; 10 mg/ml in N,N-dimethyl
formamide) freshly diluted 1 ml into 30 ml of 0.1 M sodium acetate, pH
5.0, filtered, and mixed with 15 µl of
H2O2 (200 µl/well). The
resulting spots were counted on a computer-assisted ELISPOT image
analyzer Immunospot (Cellular Technology, Cleveland, OH).
Intracytoplasmic staining and flow cytometric analysis of cytokine production
Intracellular staining was performed as previously described
(29). Briefly, 3 x 106/ml PBLs
or 3 x 105/ml NVH1 cells were stimulated
with medium, PPD, PHA, or 1 x 106/ml
stimulator cells and cultured for 2 h. Ten microliters per
milliliter of brefeldin A (diluted to 1 mg/ml; Sigma) was added for
2 h. The cells were harvested, washed, fixed with 4%
formaldehyde, and then resuspended at 1 x
106/culture tube and pelleted. Permeabilization
was performed with 150 µl of permeabilization buffer containing PBS,
0.5% BSA, azide, and 0.5% Saponin (Sigma) for 10 min at room
temperature. After washing, 20 µl of PE-conjugated anti-human
IFN- Ab or control Ab (Becton Dickinson, San Jose, CA) was added and
incubated for 30 min. at room temperature with frequent shaking. The
cells were then washed once in permeabilization buffer followed by two
washes with PBS, azide, and BSA without saponin. Surface staining with
PerCP-conjugated anti-CD3 (Becton Dickinson) was performed,
followed by three washes in PBS. The cells were analyzed on a FACScan
flow cytometer (Becton Dickinson). Three thousand to five thousand
cells were acquired and analyzed for each experiment.
Ethics
All studies were performed under the approved guidelines set forth by the internal review board for human studies at University Hospitals of Cleveland and the Cleveland Veterans Affairs Medical Center.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether our cytokine ELISPOT assay accurately measures
frequencies of responding memory T cells at single cell resolution, we
initially tested cytokine production by an alloreactive T cell line,
NVH1 (NG anti-PH). Under these conditions, the numbers of T cells
plated in each well are known, and the frequency of the cells that
actually produce cytokine can be independently determined using
intracytoplasmic staining and flow cytometric analysis. As shown in
Fig. 1
A, NVH1 cells produced
predominantly IFN- in response to PH stimulator cells, but did not
respond to the control Ags tetanus toxoid or PPD. Unstimulated cells
did not produce cytokines. Fig. 1B reveals that 38.7% of
the NVH1 cell line produced IFN- by ELISPOT assay at all cell
numbers plated when tested over a wide range of cell concentrations.
Note that the detected response titrates linearly, suggesting no
significant contribution from the stimulator cells (which remain
constant at all T cell line titrations). As shown in Fig. 1, C and D, intracytoplasmic staining and flow
cytometric analysis revealed that IFN- was produced by a remarkably
similar 36.3% of the cell line. These data suggest that the ELISPOT
assay can, in fact, detect IFN- produced by individual responder T
cells, and therefore can accurately measure frequencies of
IFN--producing cells.
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production by primed PBLs in bulk culture
We have previously shown that IFN- produced in short-term
culture by murine T cells is a marker of Ag-specific memory
(18). To similarly establish whether IFN- production by
freshly isolated human PBLs is also a manifestation of Ag-specific
memory, we studied the PPD recall response in Bacillus
Calmette-Guerin-vaccinated and unvaccinated individuals. As shown in
Table I, minimal cytokine production
occurred in the absence of stimulation (PBLs cultured in medium alone),
while PPD-specific immune responses were detectable in PPD-positive
(Bacillus Calmette-Guerin-vaccinated), but not in PPD-negative,
individuals (Fig. 2 and Table I).
Notably, this PPD-specific immunity was dominated by IFN- (with
minimal IL-4 or IL-5 activity), consistent with a primed,
proinflammatory response, presumably capable of mediating immunity to
Mycobacterium tuberculosis. As a control to demonstrate that
not all immune responses were IFN- dominated, we also tested for
primed responses to allergens. As shown in Table I, an individual with
type I allergy to cat pelt Ag exhibited an IL-4- and IL-5-dominated
recall response to the allergen (Table I). Nonallergic individuals did
not respond when exposed to the same allergen. Mitogen stimulation with
PHA resulted in production of IFN-, IL-4, and IL-5 (Table I and
Fig. 2).
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spots
titrated linearly with the number of responder cells plated (Fig. 2
;
for mitogen responses this was 
0.5% responding cells). Strikingly,
although we could readily detect IFN- production by ELISPOT assay in
freshly isolated PBLs, the frequencies were too low to be routinely
detectable by intracytoplasmic staining and flow cytometry (Fig. 2).
These findings reveal that the ELISPOT assay 1) accurately reflects the
cytokine profiles of the responding cells; 2) detects IFN- produced
in response to some, but not all, Ags; 3) detects IFN- production
only when there is defined immunologic priming; and 4) is significantly
more sensitive than intracellular staining and flow cytometric
analysis.
IFN- producing cross-reactive, memory responses against
alloantigens
Having established that we can determine the frequency of
IFN--producing cells in freshly isolated PBLs as a measure of
Ag-specific memory, we next studied the immune response to alloantigens
by PBLs from normal volunteers. Also shown in Table I, incubation with
allogeneic stimulators (from an unrelated individual who shared a
single MHC I allele, but no MHC II alleles, with the responder), but
not syngeneic (self) stimulators, led the responder PBLs to produce
IFN-. The presence of IFN--producing cells in the responder PBLs
suggested that the alloreactive immune repertoire contained some
previously environmentally primed T cells that cross-react with the
donor’s alloantigens. Once again, the detectable number of IFN-
spots titrated linearly with respect to the number of responder cells
present in the assay (Fig. 2), suggesting 1) that only the responder
cells produced the IFN-, and 2) that the true frequencies of
responding cells can be assessed by this technique. Consistent with the
low frequency of responding cells as detected by ELISPOT assay (Fig. 3), we were unable to detect allospecific
responses by intracytoplasmic staining and flow cytometry (Fig. 2).
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) revealed a dominance of IFN-
(range of 5–150 spots/300,000 responder PBLs) over the other cytokines
in each case tested, although significant numbers of IL-2 and IL-5
spots were noted as well. Moreover, the frequency of allo-induced
IFN- spots varied minimally when responders were retested serially
with the same stimulator over a 2- to 4-mo time span (Fig. 4), revealing that the results this assay
are relatively stable. These data implicate IFN- as the primary
memory cytokine in the pretransplant alloresponse. Furthermore, the
frequency of PBLs induced to secrete IFN- in response to allogeneic
stimulators reflects a reproducible measure of the number of memory
cells directed against that donor.
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-producing PBLs does not
correlate with HLA Ag mismatch
We next sought to test whether the frequency of allospecific
IFN--producing cells would provide functional information above and
beyond that determined by HLA analysis. Previous studies have
demonstrated that the risk of graft rejection and the overall frequency
of potentially alloreactive T cells in an individual correlate with the
number of HLA mismatches between donor and recipient (9, 11). However, as the pretransplant frequency of donor-specific,
IFN--producing cells (as determined by this ELISPOT assay) is a
manifestation of cross-reactive immunologic memory (and not a measure
of the total alloreactive T cell precursor frequency), it may bear no
relationship to the number of HLA mismatches between donor and
recipient. To test whether this is indeed the case, we compared the
frequency of allostimulator-induced IFN--producing cells with the
number of HLA mismatches between donor and recipient, using a panel of
individuals being evaluated for living related or living unrelated
renal transplantation.
Fig. 5 shows results from four different
assays in which the responder and the stimulator were either fully
matched or fully mismatched at the A, B, and DR loci (by the typing
methods used for renal transplantation). Interestingly, one
well-matched pair had no detectable IFN- spots, while the other had
a strong response (Fig. 5). Similarly, responder cells from one of the
poorly matched pairs produced a high frequency of IFN- spots, while
responders from another pair produced only a low frequency response
(Fig. 5). Mitogen stimulation induced equally strong responses of >300
spots/well in all four situations, confirming the viability of the
cells (not shown).
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. Two male individuals that differed at
only a single MHC I allele were studied. A2 was expressed by one
person, and A30 by the other. The frequency of IFN--producing cells
was much stronger when the A2-expressing individual, as opposed to the
A30-expressing individual, was the responder. Interestingly, specific
donor:recipient HLA mismatches have been associated with an increased
rejection rate (30), providing evidence that the results
of this assay may be relevant to the clinical outcome of graft
function. The findings clearly illustrate that donor-specific memory
cells can be present in the responder regardless of the number of
matches at HLA loci.
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spots was then correlated with
the number of HLA mismatches for a population of patients being
evaluated for living related (Fig. 7)
renal transplants. The data are expressed as a function of mismatches
at all tested loci (A, B, C, DR, DQ), loci generally studied for
matching of renal allografts (A, B, and DR alone), MHC I loci alone (A,
B, C), or MHC II loci alone (DR, DQ). As shown, the frequency of
detectable spots ranged from 0–103/300,000 PBLs plated. Notably,
although fully matched pairs tended to have lower responses than all
other groups, there was no direct correlation between the number of
mismatches and the number of spots detected under any of the studied
circumstances (r < 0.3 in all situations). There were
many poorly matched pairs with allospecific IFN- frequencies in the
same range as the fully matched pairs.
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). The majority of the pairs tested (30
of 34) exhibited allospecific IFN- frequencies of <105/300,000
cells, fully overlapping with the detected responses found in the
living related pairs, despite a greater degree of mismatching. Only
four of the 34 responders tested produced donor-specific IFN- spots
at higher frequencies than the living related pairs, ranging from
160–570 spots/300,000 cells. As was noted for the living related
pairs, there was no direct correlation between the number of mismatches
at any locus (or loci) and the number of spots detected (Fig. 8). In
sum, the findings are consistent with our hypothesis that the frequency
of primed, donor-specific, pretransplant IFN--producing cells may
not be predictable based on the number of HLA mismatches alone.
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-producing cells
correlates with posttransplant acute rejection episodes
As it is well established that secondary stimulation of memory T
cells elicits a more vigorous and rapid response than primary
stimulation of naive cells (5, 6), an increased frequency
of pretransplant, donor-specific, IFN--producing PBLs in the
recipient may increase the risk of posttransplant rejection episodes.
To test this hypothesis, we followed a cohort of patients that received
living or cadaver renal allografts and compared their clinical outcome
with the pretransplant frequency of donor-specific IFN--producing
cells. Our initial findings suggest that the pretransplant
donor-specific IFN- spot frequency may indeed have important
predictive value (Tables II and III).
Table II shows the outcome of nine
recipients of allografts from living donors, six of whom were related
and three of whom were unrelated. Five of these recipients had
uncomplicated postoperative courses with no episodes of rejection 3–15
mo after transplant, and all five had good renal function (serum
creatinine, 1.0–1.9 mg/dl) at their most recent evaluation.
Pretransplant allospecific IFN- ELISPOT frequencies were <10, <10,
<10, 15, and 40/300,000 PBLs in these individuals. Notably, patient 4
(donor-specific IFN- spot frequency of 15/300,000 cells), the
recipient of an unrelated (nonspouse) allograft matched only at one DR
locus (1/6 match), has done extremely well. Four other recipients of
allografts from living donors had episodes of biopsy-proven graft
rejection with complete resolution to baseline renal function after
therapy with OKT3. Three of the patients had somewhat elevated
pretransplant allospecific IFN- spot frequencies of 34, 53, and
68/300,000 PBLs. The final patient was a male recipient of a spouse
graft matched at one DR and one A locus (2/6 Ag match). Six weeks after
transplantation, this patient developed severe biopsy-proven acute
allograft rejection that resulted in a marked deterioration in renal
function (serum creatinine stabilized at 2.5 mg/dl, well above his
lowest posttransplant creatinine of 1.0 mg/dl). Strikingly, this
patient produced the highest pretransplant frequency of donor-specific
IFN- spots of this study population: 570/300,000 cells.
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). For these patients,
pretransplant, donor-specific, IFN--producing cell frequencies were
defined using recipient PBLs obtained at the time of transplantation
(before surgery and before instituting immunosuppression) tested in
response to donor spleen cells. In this cohort, seven recipients with
donor-specific responses of <20/300,000 have not had a rejection
episodes, while the other three recipients (frequencies of 34, 50, and
450/300, 000) developed biopsy-proven acute rejection. Remarkably,
patient 19, with the highest pretransplant donor-specific response in
this subgroup, had primary nonfunction of his allograft, with two
biopsies confirming severe histologic rejection.
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ELISPOT frequencies are independently
predictive of posttransplant rejection when other known risk factors
are taken into account. Notably, however, neither the percent reactive
Ab (PRA) values (Tables II and III), the transfusion histories (data
not shown), nor the pregnancy histories (data not shown) directly
correlated with the IFN- spot frequencies. Four of the patients were
recipients of second or third organ transplants (patient 9 in Table II
and patients 13, 17, and 19 in Table III), a known high risk group. Of
these patients, the individual with the lowest donor-specific response
(patient 13), remains free of a rejection episode, while the other
three patients developed acute rejection, suggesting that the ELISPOT
assay may indeed have some independent predictive value. In summary,
for recipients of both living and cadaver renal allografts, those with
the lowest frequencies of donor-specific IFN- spots did not
experience clinical acute rejection over the follow-up period, while
the highest responders experienced rejection episodes. Thus, in this
cohort of transplant recipients, the frequency of allospecific IFN-
spots correlated with short-term clinical outcome, consistent with our
hypothesis that the existence of memory cells specific for the donor
portends an increased risk of posttransplant acute rejection. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies, using a highly sensitive ELISPOT assay, revealed that the
frequency of donor-specific IFN--producing PBLs is a measure of the
primed alloimmune response that seems to be independent of the degree
of HLA match. We first demonstrated that the ELISPOT assay detects
cytokines produced by single cells (Fig. 1), that it is more sensitive
than intracellular staining and flow cytometric analysis (Fig. 2), that
it provides a measure of Ag-specific memory, and that it detects
anticipated patterns of cytokine production in appropriate situations
(i.e., IFN- in response to PPD, and IL-4/IL-5 in response to
allergens; Table I). When alloimmune responses were tested in normal
healthy volunteers, we found that the cytokine pattern was dominated by
IFN- (Fig. 3), and that single samples were representative of that
individual’s allospecific response for at least a 2- to 4-mo period
(Fig. 4). These findings are consistent with the presence of
environmentally primed immunity in the recipient directed toward the
donor. In a cohort of individuals being evaluated for either living
related or living unrelated renal allografts, we next demonstrated that
the pretransplant frequency of donor-specific IFN--producing cells
in the peripheral blood did not directly correlate with the number of
mismatches at HLA A, B, DR, and DQ (
Figs. 5–8).
The data suggest that factors other than the number of HLA mismatches
at A, B, DR, and DQ loci between donor and recipient may influence the
strength of the primed, donor-specific, IFN--producing cells before
a transplant. There are a number of potential explanations for these
findings. T lymphocytes, the central mediators of allograft rejection,
recognize peptide:MHC complexes on donor (direct recognition) and/or
recipient (indirect recognition) APCs (34). The ability of
T cells to respond to an alloantigen thus depends on the nature of the
peptide(s) presented by donor and recipient MHC molecules and by the
TCRs available to recognize them. An allograft may express some
MHC:peptide complexes that are not immunogenic and therefore ignored by
the recipient’s T cells or may express potential alloantigens to which
the recipient is tolerant. In contrast, even a small number of
immunodominant peptide:MHC complexes could induce strong T cell immune
responses and may overwhelmingly influence the T cell repertoire that
ultimately leads to rejection (35, 36). Secondly, the
alloimmune response to minor Ags expressed by the donor graft are not
accounted for by standard HLA matching. Although it is generally
accepted that minor Ags are less immunogenic than allo-MHC Ags, there
are a number of situations in which immunity to minor Ags is
detrimental to the graft (37, 38, 39). Thirdly, although our
data suggest that allelic differences at A, B, DR, and DQ do not
account for the detected ELISPOT frequencies, it remains possible that
HLA differences at other loci (i.e., DP) or differences as determined
by high resolution typing may explain some of our findings. Strong
mixed lymphocyte responses have been noted, for example, in
donor:recipient pairs matched at HLA loci by standard techniques, but
mismatched at the nucleotide level using high resolution techniques
(40, 41). High resolution HLA typing is not routinely used
clinically for renal transplantation; however, and perhaps more
importantly, the functional significance of any noted nucleotide
sequence differences at HLA loci is not readily predictable. Thus, the
results of this ELISPOT analysis may provide a previously unavailable
functional measure of the significance of such allelic differences.
Finally, it is well established that T cells primed to environmental
Ags (i.e., through previous blood transfusions, pregnancy, viral
infections, etc.) can cross-react with both autoantigens and
alloantigens (7, 8, 12, 13). As we have shown that the
ability to detect IFN--producing cells by ELISPOT is a measure of
primed immunity (Table I), we favor the hypothesis that donor-specific
IFN- production is likely to be the result of such cross-reactive
priming (10, 11). As shown by our results in Fig. 7D, even selected well-matched patients, i.e., living
related donors matched at DR loci, may develop primed immunity specific
for their donor, in part due to cross-reactive memory.
Based on these assertions, it is not be surprising that the number of
HLA mismatches at the tested loci does not correlate with the frequency
of IFN--producing T cells directed toward a prospective donor (Figs. 7 and 8). Moreover, as memory T cells respond more vigorously than
naive T cells upon secondary stimulation (5, 6, 14, 15, 16),
these findings raised the possibility that donor-specific immunity in a
graft recipient before transplantation might predict posttransplant
outcome. Clinical follow-up of a cohort of kidney transplant recipients
from both living and cadaver donors, in fact, revealed that those
patients with the highest pretransplant responses developed clinical
and histologic graft rejection, while those with low responses did not
(Tables II and III). Thus, our results not only provide further insight
into the human alloimmune response, but additionally may provide the
basis for a clinically useful test. Determination of high
donor-specific responses both pre- and posttransplant could guide the
use of alternative donors (particularly for recipients of living
transplants) and identify high risk recipients for prophylactic,
aggressive immune suppression. The feasibility of donor-specific
immunologic monitoring has furthermore been recently confirmed by
others using a flow cytometric approach (42). Whether such
approaches will be embraced by the transplant community or, in fact,
whether our promising data will hold true in a large scale prospective
trial still remains to be determined.
Our data additionally demonstrate directly that pretransplant
alloreactivity in freshly isolated normal human PBLs is predominantly
characterized by IFN- production, which occurs at a frequency of
1/7,000 to 1/20,000 (Fig. 3). The detection of IFN- in short-term
assays is consistent with the presence of primed memory cells as
indicated by others (20). Our detected frequencies were
similar to those detected by other methods (43, 44, 45) and
are significantly greater than the frequency of T cells responding to
defined protein Ags (which are as low as 1/million). In fact, our data
demonstrate that the alloresponse in an individual not previously
exposed to the alloantigen is approximately equal in frequency to the
recall response specific for cat pelt or mycobacterial proteins in
individuals primed to these Ags (Table I). This markedly increased
frequency of alloreactivity compared with that for nominal protein
determinants is most likely due to the large number of novel
peptide:MHC complexes expressed by the donor to which the recipient can
potentially respond as well as to cross-reactivity to environmental
Ags. Thus, it is not surprising that the alloresponse is 10- to 50-fold
stronger than the response to individual proteins (in which only a
limited number of antigenic determinants are available to the T cell
repertoire).
In conclusion, our data show that the pretransplant, donor-specific
memory T cell immune response, as determined by the frequency of
recipient alloreactive IFN--producing PBLs, does not directly
correlate with the number of HLA A, B, and DR mismatches between donor
and recipient, but instead seems to provide an independent assessment
of the pretransplant immune status of the individual in response to the
potential donor. The improved ability to define the strength of the
preexisting allospecific immune response may provide improved selection
of potential graft donors and furthermore could help to predict the
risks of acute and/or chronic rejection in human allograft recipients.
Large scale prospective studies correlating graft outcome
with the results of ELISPOT analysis are warranted to determine whether
this measure of alloreactivity provides additional prognostic
information above and beyond matching for HLA Ags in recipients of
renal allografts.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ELISPOT, enzyme-linked immunospot; PRA, percent reactive Ab; PPD, purified protein derivative.
Received for publication March 24, 1999. Accepted for publication June 1, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin 5 producing cells as a predictive marker for renal allograft failure. Transplantation 66:219.[Medline]
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, Responder cells were obtained on
12/96 and 1/97 and tested against 3/6 Ag-matched, living related
stimulator cells. 
, Responder cells were obtained on 12/97, 1/98,
2/98, and 3/98 and tested against stimulator cells from an unrelated
individual matched at one A locus and one DR locus. •, Responder
cells were obtained on 12/97, 1/98, 2/98, and 3/98 and tested against
fully mismatched unrelated stimulator cells (error bars fall within the
data points). Each data point represents the mean value of triplicate
wells counted by image analysis.
