Heart Institute (InCor) and
1 Division of Renal Transplantation, Hospital of Clinics, University of São Paulo Medical School, Av. Dr Enéas de Carvalho Aguiar 500, 3a, São Paulo 05403-000, Brazil
2 Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
3 Mental and Computational Biology, National Cancer Institute Frederick, NIH, MD 21702-1201, USA
Correspondence to: V. Coelho
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Abstract |
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Keywords: autologous limiting dilution assay, autoreactivity, multi-hit limiting dilution assay, regulatory cells, renal transplantation
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Introduction |
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Current knowledge regarding the molecular basis for T cell antigen recognition and MHCpeptideTCR interactions and mechanisms of thymic selection has shed new insights onto the biological meaning of autoreactivity. Data from several laboratories suggest that despite the massive elimination of strongly self-reactive thymocytes in the thymus, there remain positively selected T cells which have intermediate affinity for self-MHCpeptide complexes (12). It is, therefore, reasonable to assume that the mature peripheral T cell repertoire is potentially autoreactive. Recently, auto-MLR has been used by several groups to study the immune response in different models of autoimmune diseases (1316) and the molecular mechanisms of the suppressor effect generated in auto-MLR has also been investigated (17,18). In this scenario, autoreactivity may presently be revisited and, thus, the use of auto-MLR as an instrument to study the functions of autoreactive T cells.
In spite of the potentially autoreactive peripheral T cell repertoire, autoimmune disease only occurs infrequently. Several mechanisms of peripheral tolerance such as apoptosis, anergy and suppression seem to be important in regulating immune responses and, thus, limiting the potential risk of autoimmune disease (19). Some groups have suggested that peripheral self-tolerance is a homeostatic equilibrium achieved through a balance between the population of autoreactive cells and regulatory cells (2023). Accordingly, maintenance of immune homeostasis would not represent the lack of autoreactivity but, more likely, a dynamic equilibrium resulting in a predominant state of non-aggression to self-tissues. In addition, autoreactive T cells have also been implicated in the regulation of the immune response, and some investigators support the view that homeostasis is maintained because of autoimmunity and not despite it (24).
Considering that T cell repertoires to alloantigens, autoantigens and nominal antigens probably overlap (2529), it is conceivable that the expansion of one repertoire due to a particular stimulus may interfere with, and overcome peripheral regulation, expand T cells reactive to several different antigens and eventually induce alteration of self-tolerance. Indeed, it has been proposed that several immunological stimuli such as viral, parasitic and bacterial infections which activate and expand a particular repertoire of T cells may also expand autoreactive T cells and eventually surmount peripheral regulatory mechanisms, leading to breakdown of peripheral tolerance to self-antigens (3032).
Taking into account two important characteristics of the allogeneic response, (i) its intensity owing to the high frequency of alloreactive precursor T cells (3334) and (ii) the existence of a high homology of donor MHC alloantigens with recipient autoantigens, we hypothesize that a significant modification in the repertoire of autoreactive T cells may take place after allostimulation. In animal models, it has been demonstrated that the repertoire of autoreactive T cells may change post-allo-transplantation (Tx), leading to breakdown of tolerance to some self-antigens (3536). Breakdown, to some, may suggest reversal of tolerance on the cellular level. It should be pointed, however, that tolerance does not exist on this level for much of the self-reactive population, as shown by some investigators (37).
In the context of human Tx, positive auto-MLR and T cell reactivity to human hsp60 have been described in renal transplant patients (3840), but no studies on the modulation of the repertoire of autoreactive T cells have been reported, so far.
In the present work, we studied the repertoire of autoreactive T cells in renal transplant patients, analyzing the proliferative response to autologous peripheral blood mononuclear cells (PBMC) in the pre-Tx period and at different time-points post-Tx, by auto-MLR and autologous limiting dilution analysis (auto-LDA).
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Methods |
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HLA class I and class II typing
All patients and donors were typed for HLA class I (HLA-A and -B) by serology, and for HLA-DR class II antigens by PCR-SSP, as previously described.
Proliferation assays: auto-LDA and auto-MLR
The protocol used in this study was based on the method described by Lefkovits and Waldmann (41). Samples from different time-points of the same patient were all studied simultaneously, in the same assay. For auto-LDA, responder cells were cultivated with autologous irradiated (3000 rad) PBMC from the same time-point. Assays were performed in 21 wells for each cell concentration on average, using four different cell concentrations of responder cells (dilution factor equal to 2; initial concentration usually 5x104 cells/well) and a fixed concentration of irradiated stimulator cells (from 2.5x104 to 105 cells/well).
In auto-LDA, when the initial concentration of responder cells was <105 cells/well due to limitation of biological material (initial concentration from 2.5 to 5x104 cells/well), auto-MLR was also performed. Auto-MLR were performed simultaneously under the same culture conditions, in triplicates with 105 cells/well (patients 710: all time-points and patient 12: pre-Tx and 13 months post-Tx; exceptions: patient 4: pre-Tx and patient 11: all time-points). It should be pointed out that patients 5 and 6 were only studied by auto-MLR at all time-points.
Positive control experiments with phytohemagglutinin (PHA, 5 µg/ml; Sigma, St Louis, MO) were also performed for each cell concentration. For background controls, two backgrounds were calculated: responder cells alone for each cell concentration (background 1) and irradiated stimulator cells alone (background 2).
Briefly, both auto-LDA and auto-MLR were performed in 96-well U-bottom microplates (Costar, Cambridge, MA) in a total volume of 150 µl of complete DMEM medium with 2 mM L-glutamine, 10% vitamins, 10% non-essential amino acids (Gibco/BRL, Grand Island, NY) and 10% autologous plasma (collected at least at 6 months post-Tx). Cultures were supplemented with 510 U/ml of recombinant human IL-2 (Hoffman-La Roche, Nutley, NJ) at days 4 and 7. Assays were incubated at 37°C in a 5% CO2 humidified atmosphere, pulsed after 9 days with 0.25 µCi [3H]thymidine per well (Amersham, Arlington, Heights, IL) and 18 h later, plates were frozen until the moment of harvest (Cell Harvest; Packard, Canberra, Australia). Thymidine incorporation was measured by gas scintillation on a Matrix 96 direct ß-plate counter (Packard).
Analysis of data
The results were analyzed considering three parameters for each time-point: (i) stimulation index (SI; qualitative analysis), calculated as the means of c.p.m. of responder cells versus irradiated stimulator cells divided by the means of c.p.m. of responder cells alone, (ii) type of the proliferative response (single-hit, detection of a responder population but no regulatory cell population, or multi-hit, detection of a responder population and a regulatory cell population) and (iii) frequency of precursor cells (quantitative analysis). In the qualitative analysis, the response was considered positive when the SI 2 in at least one cell concentration and there was a significant difference from the background c.p.m. (Student's t-test or MannWhitney rank-sum test, P < 0.05).
For estimation of precursor frequency, the number of negative wells was initially calculated for each cell concentration at each time-point. The number of negative wells was calculated as the number of wells for each cell concentration with the c.p.m. lower than the mean c.p.m. of the highest background + 2SD (background 1: responder cells alone; background 2: stimulator cells alone). The ratio between the number of negative wells and the total number of wells studied for each cell concentration was then used to estimate the precursor frequency, using the LDA computer program, Poisson (Rovensky et al., 1997). The 2 value for a given set of data was used as an objective measurement to evaluate the curve-fitting and the capacity of the curve to estimate the frequency of precursors. If all experimental points fit a linear regression curve (P > 0.05), it was concluded that it was a single-hit analysis and only one population was responsible for the response, LPC1 (Lymphocyte Cell Population 1). However, if experimental points did not fit the linear regression curve (P < 0.05), the culture was considered to display multi-hit kinetics. In such cases of LDA, it was assumed that at least two populations were involved in the response: LPC1, the autoreactive population, and LPC2 (Lymphocyte Cell Population 2), a population with regulatory activity over LPC1. These two populations are present in the well with the frequencies f1 (precursor frequency of LPC1) and f2 (precursor frequency of LPC2) 1,1 respectively. The second cells (LPC2) are able to proliferate independently (an m-hit process) and suppress the proliferation of LPC1 (an n-hit process). So, the fraction of positive (F+) cultures can be calculated as (42):
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where C is the number of cells per well, n is the number of regulatory cells (LPC2) necessary to inhibit the proliferation of precursors of the responder population (LPC1) and m is the number of cells of LPC2 or LPC1 necessary for the detection of its proliferation in vitro.
The single-hit case can be described by the use of this function when f2 = 0:
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To fit the data to the theoretical function 1,1 the nonlinear weighted least squares method was used. The computer program Regression V6.6 (Sidorov, 1997) was used as a tool for parameters estimation. The result of fitting is the set of estimated values (f1, f2, n and m).
The frequencies from different time-points were considered significantly different when the frequency intervals (value ± SD) did not overlap.
Internal validity of LDA
In order to evaluate eventual technical experimental variations, we performed both intra- and inter-experimental controls in LDA assays performed with PBMC from normal individuals during the establishment of this method in our laboratory. Our results showed reproducibility of data for experiments performed simultaneously by two different investigators (data not shown).
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Results |
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The serial analysis of precursor frequency showed that most patients (seven out of eight patients) displayed variations in the frequency of autoreactive T cells (f1) during the evolution (Table 3). Comparing the frequencies in the pre-Tx period with the latest period analyzed (> 6 months to
1 year post-Tx), five out of eight patients (62.5%) displayed an increase in the precursor frequency (patients 3, 7, 9, 10 and 11), three of whom, in fact, displayed no detectable precursors in the pre-Tx period.
Regulation of autoreactive repertoires
The kinetics of the proliferative response to auto-PBMC was predominantly multi-hit (total 57%; pre-Tx 50%; post-Tx 59%) and six out of 10 patients showed variation in the type of kinetics at the different time-points (patients 1, 2, 3, 8, 11 and 12), suggesting regulation over these autoreactive repertoires (Table 4).
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Association between transfusion and autoreactivity
No significant difference was found between patients with either positive or negative response to autologous PBMC in the period pre-Tx, in relation to the underlying disease or to renal function or prevalence of rejection episodes up to the last clinical evaluation (from 16 to 23 months, mean 21 months). Moreover, no association was found between reactivity to autologous PBMC and graft evolution, so far. To date, only patient 7 developed chronic rejection 5 months post-Tx. This patient displayed positive autoreactivity in the late period post-Tx (>1 year post-Tx) and an increase in the precursor frequency in this period.
All patients with detectable autoreactivity in the pre-Tx period (patients 1, 2, 3, 4, 8 and 11) (Table 4) had received several (from 3 to 28, mean of 9.8) allogeneic blood transfusions (not donor specific). In contrast, patients with no detectable autoreactive precursors (patients 7, 9 and 10) had received no blood transfusions before Tx. Therefore, we found a positive association between detection of autoreactive precursors in the pre-Tx period and a multiplicity of allogeneic blood transfusions before Tx (Fisher's exact test, P = 0.012).
Controls: response to PHA and to donor cells
All 12 patients displayed a positive response to PHA. A donor-specific allogeneic response was positive at all cell concentrations in all patients studied who presented HLA-DR disparities with donors (data not shown).
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Discussion |
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A particularly interesting observation was the appearance of detectable autoreactivity after Tx in six out of eight patients (75%) with no reactivity before Tx. One interpretation to these data is that immune responses taking place post-Tx expand the repertoire of autoreactive T cells 1,1 interfere somehow in the peripheral regulation of this T cell repertoire, eventually resulting in breakdown of tolerance to self-antigens.
In concordance with this hypothesis, Benichou and co-workers (35) showed that some strains of mice immunized with allogeneic splenocytes developed a CD4+-mediated proliferative response to an immunodominant MHC selfpeptide, which was interpreted as breakdown of tolerance to autologous peptides following in vivo allostimulation. Molecular mimicry between allogeneic MHC and self-MHC antigens is discussed as a possible mechanism involved in this phenomenon. More recently, the same group also reported both T and B cell autoreactivity to cardiac myosin following allogeneic but not syngeneic heart Tx in mice (36).
This is an interesting hypothesis to be considered in the context of this study, where HLA or other allomolecules present on donor cells could stimulate T cells to cross-react with epitopes from autologous cells and induce autoreactivity after Tx.
Molecular mimicry between viral, parasitic, bacterial and self-antigens has been proposed to play a role in the pathogenesis of different autoimmune diseases, both by our group and others (3032). It should be remarked, however, that the detection of T cell autoreactivity per se does not mean immune dysfunction or imply the development of autoimmune disease, as autoreactive T cells have been described in normal individuals, both in human and murine systems (4344). Moreover, autoimmunity has also been implicated in immune regulatory mechanisms, as observed by our group in relation to Hsp autoimmunity in Tx (manuscript in preparation) and other investigators in other systems (24,45). In the present study, no correlation was found between detection of autoreactivity and the nature of underlying diseases nor with episodes of rejection or graft outcome, as only one patient who developed post-Tx autoreactivity presented with chronic rejection. However, it cannot be excluded that post-Tx autoimmunity is implicated in graft outcome over the long term. In the mouse study in which response to self cardiac myosin was induced post-allo-Tx, it was shown that anti-myosin autoimmunity contributed to graft damage (36).
Another interesting finding in our study was the significant positive correlation between detection of autoreactive precursors in the pre-Tx period and the number of blood transfusions before Tx. These observations are in accord with other reported data which indicated allogeneic blood transfusion to be a factor in the induction of autoreactivity in non-transplanted patients and suggest that an allogeneic stimulus could be related to the autologous response (46). Moreover, in our study, non-transfused patients also developed autoreactivity after Tx.
Although not shown in the present work, it should be mentioned that donor-allogeneic response was present in all patients who presented positive response to autologous PBMC, in assays performed simultaneously. Furthermore, donor-allogeneic response was also present in patients with no detectable autoreactivity. Despite the potential relation of post-Tx autoreactivity and donor-allogeneic reactivity, an important difference observed was that the donor-allogeneic response displayed single-hit kinetics in 77% of the assays (data not shown), while the response to auto-PBMC was multi-hit in 57% of LDA assays. This indicates stronger regulatory control over the autologous response, as reported in other systems (14,4750).
Regulatory control over the autoreactive population is also suggested in the qualitative analysis of LDA assays, when considering whether or not the SI was positive at the different cell concentrations (data not shown). In seven out of 10 patients, the SI was negative in the higher cell concentrations and positive in lower concentrations, suggesting that the proliferation of the autoreactive population was only detected when the concentration of the regulatory population was gradually diminished, as described in other models (46,48,50,51). Other groups have also proposed that regulatory cells or cells with suppressor activity over autoreactivity could be present in the peripheral blood and play an important role in keeping peripheral self-tolerance (21,22,5254).
Although molecular mimicry leading to breakdown of tolerance to self-antigens is an attractive hypothesis, other mechanisms may account for our observations, such as weakening of regulatory control, immunosuppressive drugs, cytokine-driven bystander activation either due to rejection or infection and indirect alloreactivity. In this study, some patients displayed a change from multi-hit to single-hit response at some time-points post-Tx, suggesting either loss or fluctuations of regulatory control over the autologous response. The use of immunosuppressive drugs such as cyclosporin A (CsA) could alter the T cell repertoire and contribute to the development of autoreactivity post-Tx. Indeed, CsA has been reported to be associated with breakdown of tolerance to self-antigens in lethally irradiated mice, reconstituted with either allogeneic or syngeneic bone marrow and subsequently treated with CsA (5556). Although indirect alloreactivity could be contributing to the proliferative response detected, the marked difference with respect to the kinetics of the response, predominantly single-hit to donor cells (data not shown) and multi-hit to autologous cells, also indicates a relevant distinction between allogeneic and autologous proliferative responses, reported in this study.
Finally, another interesting point to be discussed is the variability of response. Eight out of 10 patients displayed fluctuations in the autologous response, presenting autoreactivity at some time-points and not in others. We favor that this variability can be attributed to dynamic regulatory mechanisms involved in maintenance of peripheral tolerance. In fact, following the development of a response with time has allowed the observation of phenomena such as T cell epitope spreading or shifting, in the context of autoimmune diseases and in indirect alloreactivity, by other groups (5759) and by our own (submitted manuscript). The sequential analysis of a particular biological phenomenon may, indeed, give a more dynamic and complex picture of the biological process, perhaps closer to what may be happening in vivo, and may be an approach to explore the biological significance of variability in the context of immune regulation.
Considering the biological complexity of human Tx, a combination of multiple connected mechanisms is likely to occur. Whichever stimuli are relevant in triggering or increasing post-Tx autoreactivity, it should be mentioned that not all patients developed autoreactivity. This diversity of response was also reported in the animal model mentioned earlier, in which it was shown that breakdown of tolerance to self-antigens occurred in some allogeneic strain combinations and not in others (35). In humans, it is also possible that some, but not other stimuli, such as certain HLA disparities, infections or immunosuppressive drugs, may constitute an immunological context in which the threshold of regulatory mechanisms is overcome, culminating in a significant alteration in the autoreactive T cell repertoire and, eventually, breakdown of tolerance to self-antigens.
In conclusion, in the present work we show that heightened autoreactivity is detected in human Tx, suggesting that post-Tx immune mechanisms may alter the repertoire of autoreactive T cells. The detection of proliferative responses exclusively in the period post-Tx suggests a significant alteration of the self-reactive T cell repertoire. In the context of Tx, it is important to clarify if autoreactive populations may down-regulate alloreactivity and inflammation, as described for Hsp-reactive T cells in rheumatoid arthritis and experimental arthritis (45,60), or if they contribute to aggression to allografts, as reported in the post-Tx anti-myosin autoreactivity in the murine system.
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Acknowledgments |
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Abbreviations |
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auto-LDA autologous limiting dilution analysis |
auto-MLR autologous mixed lymphocyte reaction |
CsA cyclosporin A |
f1 precursor frequency of LPC1 (autoreactive population) |
f2 precursor frequency of LPC2 (regulatory population) |
LPC1 Lymphocyte Cell Population 1 (responder population) exhibiting single-hit kinetics |
LPC2 Lymphocyte Cell Population 2 (regulatory/suppressor population) exhibiting multi-hit kinetics |
m number of LPC2 or LPC1 cells necessary for the detection of its proliferation in vitro |
PBMC peripheral blood mononuclear cell |
n number of regulatory cells (LPC2) necessary to inhibit the proliferation of precursors of the responder population (LPC1) |
PHA phytohemagglutinin |
SI stimulation index |
Tx transplantation |
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Notes |
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Received 18 January 2000, accepted 19 February 2001.
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References |
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