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Departments of
*
Immunology,
Renal Medicine, and
Histopathology, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Donor-specific cytotoxic T cell
responses were also attenuated. In contrast, the frequencies of
indirectly alloreactive cells were maintained, patients with CAN having
significantly higher frequencies of CD4+ T cells indirectly
activated by allogeneic peptides when compared with controls with good
allograft function. An extensive search for alloantibodies has revealed
significant titers in only a minority of patients, both with and
without CAN. In summary, this study demonstrates widespread
donor-specific hyporesponsiveness in directly activated
CD4+ T cells derived from longstanding recipients of renal
allografts, whether they have CAN or not. However, patients with CAN
have significantly higher frequencies of CD4+ T cells
activated by donor Ags in an indirect manner, a phenomenon resembling
split tolerance. These findings provide an insight into the
pathogenesis of CAN and also have implications for the development of a
clinical tolerance assay. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD4+ T cell plays a central role in the rejection response by directing the effector functions of not only fellow CD4+ T cells, but also CD8+ cytotoxic T cells and alloantibody-producing B cells. Alloreactive CD4+ T cells may be activated by either the direct or indirect pathways. In the former, CD4+ TCRs recognize foreign HLA molecules on allogeneic cells, while in the latter pathway, alloantigens are processed by the conventional Ag-processing pathways by autologous APCs and are then presented to CD4+ T cells as peptides, in the context of self MHC molecules (8). There is now a body of evidence from our group and others that the direct pathway is down-regulated in a donor-specific fashion in recipients of renal (9, 10, 11, 12, 13) and other solid organ allografts (14, 15, 16). As a result, interest has focused on the second, indirect pathway. Indeed, there is some evidence that this pathway may be active in CAN and other forms of chronic rejection. It has been demonstrated that T cells taken from the peripheral blood of patients with CAN are primed against donor-derived HLA allopeptides (17). Similar results have been described in recipients of cardiac allografts suffering from transplant vasculopathy (18, 19) and lung transplant recipients suffering from bronchiolitis obliterans (20). In a rodent model, one group has been able to transfer skin lesions similar to chronic rejection with an indirectly primed alloreactive T cell clone (21).
Although many clinical studies have been described, none has made a simultaneous comparison of both direct and indirect pathways in the same group of patients. Such an experiment has been performed in rodents in which both pathways were clearly demonstrated to be active in the early phase after transplantation, although the direct pathway was more vigorous (22). In this study, we have investigated the direct and indirect pathways concurrently in a cohort of longstanding renal transplants (n = 22), which divide into a subgroup with good graft function (n = 13) and another group with CAN (n = 9). The results of these studies are presented below.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-two recipients of live-related allografts were selected
by virtue of being longstanding recipients of allografts (median
duration, 157 mo; range, 96–333), and more importantly because it was
possible to isolate fresh cells from the original donor to set up
cellular assays. The median age of the patients at transplantation was
32 years (range, 19–47 years). This cohort of patients was divided
into two categories, those with CAN and those without. CAN was defined
as biopsy-proven changes of CAN, as assessed by a renal transplant
histopathologist (H. T. Cook) according to the 1997 Banff scheme
(23) (eight of nine patients), or at least two of the
following clinical criteria: serum creatinine >170 µmol/L;
proteinuria > ++ on dipstix; or hypertension requiring
medication. Patient characteristics are summarized in Table I
. Third party cells were derived from
volunteers around the laboratory who had all been previously tissue
typed.
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At the time of transplant, class I HLA-A, B, and class II HLA-DR were typed by microlymphocytotoxicity test based on the assay developed by Terasaki et al. (24). On the day of the functional assays described in this work, samples of 10 ml of blood in EDTA were taken from the donors and recipients for retrospective typing. Samples were then typed by sequence-specific amplification using the PCR with a unified PCR-sequence-specific amplification system allowing the detection of HLA-A, B, C, DRB1, DRB2, DRB4, DRB5, and DQB1 (25, 26)
LDAs
PBMC were obtained by density gradient centrifugation over Lymphoprep (Nycomed, Oslo, Norway). CD4+ T cells from transplant recipients were obtained by incubation with different mAbs: CD14, CD33 and CD19, CD16, and CD56 (BD Biosciences, San Jose, CA); CD8 (Serotec, Kidlington, U.K.), followed by negative selection on magnetic beads (Dynal Biotech, Oslo, Norway). Efficacy of depletion was measured by flow cytometry, and in all cases CD4+ fractions were >95% pure.
All assays were performed in RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with penicillin/streptomycin (Life Technologies), 2 mM L-glutamine (Life Technologies), and 10% human AB serum (Harlan, Loughborough, U.K.). Each responder was tested against donor and third party allogeneic cells (PBMC). Assays against donor and third party were always performed at the same time. As donor and recipient shared one HLA haplotype, third party cells were chosen on the basis of sharing the matched DR type between donor and recipient.
Replicates of 24 wells at seven doubling dilutions of responder cells
(from 1 x 105 per well) in 100 µl of
medium were aliquoted, and medium alone was added to the 24 control
wells. Irradiated (30 Gy) stimulator PBMC (5 x
104 in 100 µl) were added to all wells. Two
plates were prepared in duplicate. From the first set, IL-2, IFN-,
and proliferation were measured; from the second set, the remaining
cytokines were measured.
Proliferation assays
After 6 days, 50 µl 1 µCi/ml [3H]thymidine was added. Plates were harvested after a further 12 h. Radioactivity was measured in a scintillation counter (Wallac, Turku, Finland). Wells were scored positive when counts were higher than the mean + 3 SDs of the control wells (irradiated stimulator cells only).
IL-2 estimation
After 72 h, 50 µl of supernatant was harvested into another plate, and 5 x 103 CTLL-2 in 75 µl of medium was added to each well. The frequency of IL-2-producing cells was obtained by bioassay with the IL-2-dependent CTLL-2 line, as described previously (27, 28).
IFN-, IL-5, IL-10, IL-13, and TGF-
ELISAs
After 6 days of culture, supernatants were harvested from each
well and frozen (-20°C) until further use. Paired Abs for each
cytokine were used (IL-5 and IFN- from Immunokontact, Frankfurt,
Germany; TGF- from R&D Systems, Abingdon, U.K.; IL-13 from BD
Biosciences; IL-10-coating Ab (clone 9D7); and biotinylated Ab from
Biosource, Nivelles, Belgium), and the ELISAs were performed using a
standard protocol. Wells were scored positive when greater than the
limit of detection of the assay as determined by the standard
curve. The limit of detection varied between 1 and 5 pg/ml for IFN-
and IL-10 and between 5 and 20 pg/ml for IL-5, IL-13, and TGF-.
51Cr release cytotoxic assays
Replicates of 24 wells at seven doubling dilutions of responder PBMC (from 1 x 105 per well) in 100 µl of medium were aliquoted into wells; medium alone was added to the 24 control wells. Irradiated (30 Gy) stimulator PBMC (from donor and third party; 5 x 104 in 100 µl) were added to all wells. Wells were supplemented with 5 U/ml IL-2 after 3 and 6 days of culture. At day 9–10, 2 x 104 51Cr-labeled PHA-blasted target PBMC were added to each well and, after 4-h incubation, 51Cr release in the supernatant was measured in a Top Count (Packard Instrument, Meriden, CT). Cells were scored positive when counts per minute were higher than the mean + 3 SDs of the control wells, in which only target cells were added.
Assays of the indirect alloresponse
Responder PBMC were set up at a single dilution (29), with 48 replicates using as stimulator a cytoplasmic membrane protein preparation from donor PBMC, in a method developed from previous work (18, 30). A total of 6–8 x 107 PBMC was lysed by three cycles of freezing to -80°C and thawing at 37°C in a Tris-EDTA-based buffer containing 1/5000 Nonidet P-40 (BDH Laboratory Supplies, Poole, U.K.), 0.1 mM PMSF (Sigma, Dorset, U.K.), 1/200 protease inhibitor mixture (Sigma), and 5 ng/ml soybean trypsin inhibitor (Sigma). The suspension was centrifuged at 1,000 x g for 2 min, and the supernatant was collected and further centrifuged for 45 min at 14,000 x g. The pellet was then collected and resuspended in assay medium and added to the responders. As no whole cells were present in the preparation (confirmed by microscopy), the stimulation of recipient CD4+ T cells could only occur through the indirect pathway. The presence of HLA molecules in the pellet was confirmed by Western blot probing with anti-class I (W6/32 hybridoma) and anti-class II (L243 hybridoma) Abs (data not shown). Proliferation was measured by [3H]thymidine uptake at day 5 for the last 12 h of culture. A single dilution of 48 wells with PBMC without donor Ag was set up as a control. Wells were scored positive when counts per minute were higher than the mean + 3 SDs of the six lowest wells.
Calculation of precursor frequencies
For all the assays, the frequencies with confidence intervals
were calculated with the modified score function, which is based on a
maximum likelihood method (31), using Excel 7.0 and
software written with Visual Basic (Redmond, WA). For each
assay, experimental data are fitted to the function, and a
2 value of goodness of fit with the
corresponding p value is obtained with the program.
Frequencies were only recorded if they adjusted to the function with a
p > 0.05. All frequencies are given as 1 in
n number of cells.
Definition of hyporesponsiveness
An antidonor frequency was considered to reflect donor-specific hyporesponsiveness when it was lower than anti-third party frequency with nonoverlapping confidence intervals.
Flow cytometric cross-matching
A total of 1 x 105 donor PBMC/well was incubated with 30 µl of a mix 1/1 of FCS and mouse serum for 30 min at 4°C, washed, and then incubated for 30 min at 37°C with 30 µl of serial dilutions of the recipient’s serum (neat, 1/8). After three washes, 1 µl of goat F(ab')2 anti-human IgG (Fc specific) (Caltag Laboratories, Burlingame, CA), 2.5 µl of anti-CD19 PE (Cymbus, Hants, U.K.), and 1 µl of anti-CD3 Quantum Red (Sigma) were added and incubated for 10 min at 4°C. Samples were washed, fixed (FACS lysing solution; BD Biosciences), and analyzed in a FACSCalibur (BD Biosciences). A biparametric gate in the size-granularity dot plot was drawn around the lymphocyte population, and events were acquired until there were at least 1500 events in the CD19+ population. The dilution of serum that gave the highest mean fluorescence intensity (MFI) in the green (FL1) detector was considered to establish positivity. A serum was considered positive when the ratio of fluorescence means (RFM = MFI of sample/MFI of pooled AB serum) was higher than the mean + 3 SDs of control sera. As a control, sera from six nontransfused males were assayed in the same manner, and the mean of all the RFMs was calculated and then a threshold value for T cells and another one for B cells was established. Sera that bound exclusively to B cells were classified as anti-class II. Sera that bound to T were considered as certain anti-class I Abs; the presence or absence of anti-class II Abs could not be determined in these patients.
ELISA-based screening of allospecific Abs
Blood was collected on the day of the assay in Vacutainers (Becton Dickinson, Plymouth, U.K.) and serum was stored at -30°C. When all the samples were collected, solid-phase ELISA for the detection of IgG Abs to HLA class I (QuickScreen; GTI, Brookfield, WI) and to HLA class II (B-Screen; GTI) were performed following manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This was conducted by performing
limiting dilution assays (LDAs) that permitted the enumeration of
donor- and third party-specific T cells (32). To ensure
consistency in these assays, a number of controls were performed.
First, in all the assays, the immunogenicity of the irradiated donor
and third party cells was ensured by stimulation of a completely
mismatched fourth party responder. Second, to control for the
possibility that 1-haplotype-matched relatives would provoke lower
frequencies due to closer matching than similarly DR-mismatched third
parties, assays were performed using CD4+ T cells
from four volunteers against both their respective parents and
similarly DR-mismatched third parties. No significant differences in
frequencies were observed against parents or third parties, when
measured by proliferation or the secretion of IL-2, IL-5, and IFN-
(data not shown).
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A),
donor-specific hyporesponsiveness was present in 6 of 9 patients with
CAN, as opposed to 11 of 13 patients with good graft function. This
difference was not statistically significant. These results were in
accordance with descriptions of donor-specific hyporesponsiveness
measured by simple mixed lymphocyte reactions, although previous
reports have concentrated on patients with good graft function alone
(9, 10, 11, 13).
Fig. 1B, showing IL-2-secreting frequencies of
CD4+ T cells, demonstrates donor-specific
hyporesponsiveness in all 22 patients studied. The most striking aspect
of these findings was the similarity between those patients with CAN
and those without. This result implies that the direct pathway is not
involved in the process of CAN. We have previously described a similar
phenomenon in some patients suffering from late renal allograft failure
(12) and also in a group of recipients of cardiac
allografts who had developed transplant vasculopathy (16).
The frequencies of alloreactive T cells, as measured by IFN- and
IL-5 secretion, are shown in Fig. 1, C and D,
respectively. These cytokines were chosen as surrogate markers for Th1
and Th2 polarization, as originally described by Mosmann
(33). In particular, IL-5 was chosen in preference to IL-4
because previous work (27) from our laboratory has shown
that levels are higher and more consistent than IL-4. In addition,
levels of IL-5 follow similar kinetics to IFN-, enabling the
measurement of both cytokines at the same time point
(62). Evidence for this form of polarization of
CD4+ responses has been demonstrated in
autoimmune, infectious, and allergic-type diseases, but the relevance
of this phenomenon to transplantation remains at best controversial.
While it is true that a number of animal models of tolerance display
some features of Th2-type responses and acute rejection is associated
with a number of facets of Th1-type responses, attempts to produce
tolerance by immune deviation have been uniformly unsuccessful across
fully allogeneic MHC barriers (34, 35). The relevance of
cell polarization to the outcome of clinical transplantation is not
clear, although IL-10, traditionally a Th2-type cytokine in murine
systems, is one of the best markers of acute rejection
(36). Our results show that longstanding recipients of
renal allografts show significant donor-specific hyporesponsiveness,
according to the secretion of both cytokines, whether they have CAN or
not. In Fig. 2, the donor-specific
frequencies for both cytokines are superimposed, revealing that
IFN--secreting frequencies were significantly higher than IL-5
frequencies in three of nine patients with CAN, as opposed to 3 of 12
patients without, a result that is not significantly different.
Interestingly, a similar analysis of responses to third party controls
shows the opposite trend, namely, 
75% of patients exhibited
predominantly Th1-type responses in both groups of patients (Fig. 2B). This suggests that there may be an overall decline in
donor-specific Th1 responses with preservation of Th2 cells in
transplant patients. The role of Th2 polarization in the indirect
pathway is discussed below.
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). The reduced CTL
frequencies in the CAN patients highlight one of the fundamental
distinctions between the acute and chronic rejection processes. During
acute allograft rejection, the finding of tubulitis and the prominence
of CD8+ T cells in inflammatory infiltrates
suggest an important pathogenic role for these cells. This contention
is further supported by the increase in donor-specific CTLs
demonstrated during episodes of acute rejection (37).
However, it should be noted that the third party cells expressed a
larger number of HLA class I mismatches than the living related donors,
and this may have contributed to the lower antidonor frequencies (data
not shown).
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, we have
used a modification of a freeze-thawing technique described by Yamada
(30) to prepare a mixture of donor membrane proteins,
which were then used to pulse autologous APCs. Because no whole cells
were present, only CD4+ T cells previously
stimulated through the indirect pathway in vivo would respond in
measurable numbers to such antigenic stimulation. It can be seen that
frequencies of indirectly activated T cells were generally higher in
patients with CAN, and the median frequency was significantly higher
than in the group with good allograft function
(p = 0.021; Mann-Whitney U test). In
this experiment, we did not measure anti-third party frequencies
because previous experiments from our laboratory had shown them to be
beneath the limit of detection for the system used
(18).
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A shows the results of both
flow cytometric T cell cross-matches and anti-class I HLA Abs
measured by ELISA in all of the patients studied. In two of nine
patients with CAN, anti-class I HLA Abs were detected by ELISA
compared with none of the patients with good graft function. In
contrast, only one of nine patients with CAN had a positive T cell
cross-match, compared with 4 of 13 patients with good function. It can
readily be seen that there is no concordance between the results of
these two assays, as the positives were all in entirely different
patients. Furthermore, there were no significant differences between
patients with or without CAN. In Fig. 5B, similar results
are shown for B cell cross-matches and anti-HLA class II Abs
detected by ELISA. In this case, one of nine patients with CAN had both
a positive B cell cross-match and anti-class II Abs detected by
ELISA. In the group of patients with good graft function, 1 of 13
patients had anti-class II Abs by ELISA, whereas 2 of 13
(different) patients had mildly positive B cell cross-matches. Although
results are inconclusive, there is little to suggest that
circulating alloantibodies are currently contributing to CAN in the
majority of these patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism whereby directly activated CD4+ T
cell donor-specific hyporesponsiveness is brought about remains a
subject of speculation. We have performed experiments specifically
designed to investigate the possibility of immune deviation as a
contributory mechanism, as there is some evidence that allograft
acceptance is associated with a Th2-type CD4+ T
cell response in animal models (34). There is surprisingly
little data available in human transplantation, although evidence from
protocol renal biopsy series suggests that up-regulation of cytokines
such as IL-10 and IL-4 is not associated with a good histological
appearance (41). We cannot conclude that direct
antidonor-polarized Th2 responses avoid the appearance of chronic
rejection, since in our data there was no difference between the two
groups of patients. A general antidonor Th2 bias might be a feature of
longstanding renal transplants and additional experiments after a
shorter interval since transplantation may help to clarify this point.
Evidence was sought in these assays for the secretion of other
immunosuppressive cytokines that might be maintaining
hyporesponsiveness. However, analysis of the same LDA plates by ELISAs
revealed no evidence of IL-10, IL-13, or TGF- secretion in the
patients that had reduced frequencies of IL-2-secreting T cells (data
not shown).
Two alternative mechanisms for the observed hyporesponsiveness are the
deletion, or the induction of anergy, in donor-specific T cells. Based
on the rodent experiments referred to above, we would argue that
alloantigen presentation by the parenchymal cells of the graft plays a
key role, since rat kidney allografts that were depleted of donor bone
marrow-derived cells were spontaneously accepted, without
immunosuppression, in some strain combinations (38). This
led to the proposal that the graft parenchymal cells were antigenic,
but not immunogenic. More importantly, in vitro studies with primary
cultures of IFN--treated thyroid and renal epithelial cells
demonstrated the capacity of these cells to induce allospecific
unresponsiveness, due to T cell anergy, in
CD45RO+CD4+ T cells
(42).
Immune responsiveness can be recovered in anergic CD4+ T cells by the addition of exogenous IL-2 (43), and data from our laboratory have shown that in some patients donor-specific CD4+ T cell frequencies can be restored upon incubation with IL-2, suggesting that a form of anergy may be operating (44). In keeping with the proposition that anergy is induced by encounter with renal epithelial cells, we have shown recently in patients receiving renal allografts that donor-specific hyporesponsiveness in the direct pathway emerges preferentially in the subset of CD4+ T cells capable of trafficking through the allograft, thereby interacting with parenchymal cells (CD4+CD45RO+) (45). One recent study investigated a live-related recipient who had stopped all immunosuppression. T cells were analyzed by the extremely sensitive method of RT-PCR, looking for clonotypic alloreactive T cells (46). This patient had previously been shown to exhibit donor-specific hyporesponsiveness in functional assays (47). Despite this fact, a high level of donor HLA-specific T cell clonotypic mRNA was detected in the patient’s circulation, implying that the T cells were still circulating, but had either become anergic or were in some way being suppressed.
The recovery of donor-specific alloresponsiveness in recipient CD4+ T cells suggests that deletion of alloreactive T cells has not taken place; however, it is possible that the magnitude of the antidonor response is diminished in some way by clonal deletion, and indeed recent evidence from murine models suggests that tolerance induction is dependent upon significant shrinkage in the alloreactive repertoire due to activation-induced cell death (48, 49). Therefore, it may be desirable to reduce the repertoire size by creating circumstances that favor a wave of deletion and then allow encounter with graft cells to promote anergy over ensuing months. Previous work from animal models has suggested that deletion may be required to reduce the alloreactive T cell repertoire down to levels that are then controllable by regulatory mechanisms (35). This complex subject clearly requires further study.
With such prominent donor-specific hyporesponsiveness in the direct pathway, suspicion falls upon the indirect pathway to provide the ongoing CD4+ T cell stimulation that may contribute to the development of CAN. Significantly increased frequencies of indirectly activated donor-specific CD4+ T cells were recorded in our patients with CAN. This is in marked contrast to the pretransplant situation in which the direct response is extremely vigorous in 1-haplotype-mismatched pairs, while the indirect response is usually unrecordable. Our findings are consistent with other groups that have described sensitization to donor-derived allogeneic HLA peptides in patients with CAN, when compared with controls with good graft function (17). In addition, studies in patients with chronic rejection of other solid organs such as hearts and lungs have also shown evidence of activation of indirect pathway T cells (18, 19, 20). An intriguing recent finding has been the suggestion that the indirect responses in CAN are polarized toward a Th1 type of response, and this warrants further investigation (50).
Elegant rodent experiments, performed with class II knockout mice, have suggested that the generation of IgG donor-specific alloantibodies requires processing of donor MHC Ag through the indirect pathway to bring about isotype switching (51). This finding has also been confirmed by other groups (52, 53). If this mechanism is also operating in human transplantation, then conversely, the presence of donor-specific IgG alloantibodies represents circumstantial evidence of ongoing T cell activation through the indirect pathway. In addition, some recent studies have suggested that alloantibodies are significantly more prevalent in patients with CAN, particularly those directed against allogeneic class II molecules (54). In our patients, we were unable to consistently detect circulating alloantibodies. However, a possible role of anti-HLA alloantibodies in the pathogenesis of CAN cannot be dismissed without a more extensive search for Ab-mediated disease. Novel techniques, such as the staining of peritubular capillaries for the C4d component of complement or ultrastructural analysis of the peritubular capillaries for multilayering of the basement membrane, might prove more sensitive (55, 56, 57, 58).
There has recently been a great deal of interest in the idea of developing a clinical tolerance assay. Ideally, such an assay would form the experimental basis for measured drug withdrawal. This has become increasingly important with the evolution of strategies for the induction of donor-specific tolerance. From this work, we think it unlikely that assays of direct alloreactivity will prove useful in this respect. Likewise, assays for circulating alloantibodies would not appear to be discriminatory. We would suggest that such an assay would require monitoring of the indirect pathway of allorecognition. Our own assays have limitations. They are time consuming and require large numbers of both donor and recipient cells. They are somewhat insensitive at low responder frequencies (1/500,000), despite the fact that such responses may still be biologically relevant. However, they give a very accurate comparison between two different stimuli. Given all these circumstances, it is also possible that the final tolerance assay will involve a panel of different tests used in combination with other promising candidates such as rapid ELISPOT assays (22, 59) and trans vivo analysis in nude mice (60, 61).
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Acknowledgments |
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Footnotes |
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2 R.J.B. and M.P.H.-F. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Robert I. Lechler, Department of Immunology, Imperial College, Hammersmith Hospital, Du Cane Road, London, W12 0NN, U.K. E-mail address: r.lechler{at}ic.ac.uk
4 Abbreviations used in this paper: CAN, chronic allograft nephropathy; LDA, limiting dilution assay; LRD, living related donor; MFI, mean fluorescence intensity; RFM, ratio of fluorescence means.
Received for publication June 4, 2001. Accepted for publication October 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes. J. Immunol. 163:2267.This article has been cited by other articles:
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V. Mirenda, I. Berton, J. Read, T. Cook, J. Smith, A. Dorling, and R. I. Lechler Modified Dendritic Cells Coexpressing Self and Allogeneic Major Histocompatability Complex Molecules: An Efficient Way to Induce Indirect Pathway Regulation J. Am. Soc. Nephrol., April 1, 2004; 15(4): 987 - 997. [Abstract] [Full Text] [PDF]
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Y. Chen, Y. Demir, A. Valujskikh, and P. S. Heeger Antigen Location Contributes to the Pathological Features of a Transplanted Heart Graft Am. J. Pathol., April 1, 2004; 164(4): 1407 - 1415. [Abstract] [Full Text] [PDF]
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D. M. Richards, S. L. Dalheimer, B. D. Ehst, T. L. Vanasek, M. K. Jenkins, M. I. Hertz, and D. L. Mueller Indirect Minor Histocompatibility Antigen Presentation by Allograft Recipient Cells in the Draining Lymph Node Leads to the Activation and Clonal Expansion of CD4+ T Cells That Cause Obliterative Airways Disease J. Immunol., March 15, 2004; 172(6): 3469 - 3479. [Abstract] [Full Text] [PDF]
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A. D. Salama, N. Najafian, M. R. Clarkson, W. E. Harmon, and M. H. Sayegh Regulatory CD25+ T Cells in Human Kidney Transplant Recipients J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1643 - 1651. [Abstract] [Full Text] [PDF]
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D. S. Game, M. P. Hernandez-Fuentes, A. N. Chaudhry, and R. I. Lechler CD4+CD25+ Regulatory T Cells Do Not Significantly Contribute to Direct Pathway Hyporesponsiveness in Stable Renal Transplant Patients J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1652 - 1661. [Abstract] [Full Text] [PDF]
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W. F. Ng, M. Hernandez-Fuentes, R. Baker, A. Chaudhry, and R. I. Lechler Reversibility with Interleukin-2 Suggests that T Cell Anergy Contributes to Donor-Specific Hyporesponsiveness in Renal Transplant Patients J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2983 - 2989. [Abstract] [Full Text] [PDF]
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H. Regele, G. A. Bohmig, A. Habicht, D. Gollowitzer, M. Schillinger, S. Rockenschaub, B. Watschinger, D. Kerjaschki, and M. Exner Capillary Deposition of Complement Split Product C4d in Renal Allografts is Associated with Basement Membrane Injury in Peritubular and Glomerular Capillaries: A Contribution of Humoral Immunity to Chronic Allograft Rejection J. Am. Soc. Nephrol., September 1, 2002; 13(9): 2371 - 2380. [Abstract] [Full Text] [PDF]
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B. Watschinger and M. Pascual Capillary C4d Deposition as a Marker of Humoral Immunity in Renal Allograft Rejection J. Am. Soc. Nephrol., September 1, 2002; 13(9): 2420 - 2423. [Full Text] [PDF]
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), or equally mismatched third party cells
(
), in a LDA. For all the figures, the nine patients on the
left had CAN. Frequencies of alloreactive cells were
then calculated using either cytokine secretion or proliferation as a
readout. Reciprocal frequencies are plotted on a logarithmic scale.
Ninety-five percent confidence intervals for the calculated frequencies
are shown. A, Proliferation was measured after 6 days of
culture by 12 h of [3H]thymidine uptake and
radioactivity measurement in a beta counter. B, IL-2
production was measured with a CTLL bioassay after 72 h of
culture. IFN-
). The median and percentiles 25–75
for each group are shown as well (p < 0.05;
Mann-Whitney U test).
) (T cell cross-match for class I and B cell
cross-match for class II). Individual data are represented for each
patient. ELISA data are illustrated by OD readings on the left
axis, and cross-match data are shown on the right
axis as RFM (RFM = MFI obtained with recipient
serum and donor cells/MFI obtained with control serum and donor cells).
The nine patients on the left are the ones with CAN. The
threshold value for both assays is shown as a solid horizontal line in
both graphs. Right and left axes have
been adjusted so that the threshold coincides for both scales. Positive
control sera values for class I: OD2046, RFM range 2–40,
mean 15; for class II: OD1987, RFM range 3–21, mean
6.6.