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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1439-1444, (2002)
© 2002 American Thoracic Society

Original Article

Oligoclonal CD4+ T Cell Expansions in Lung Transplant Recipients with Obliterative Bronchiolitis

Steven R. Duncan, Colm Leonard, James Theodore, Mark Lega, Reda E. Girgis, Glenn D. Rosen and Argyrios N. Theofilopoulos

Department of Immunology, Scripps Research Institute, La Jolla; Department of Medicine, Stanford University Medical Center, Stanford, California; and Department of Medicine, Allegheny General Hospital, Pittsburgh, Pennsylvania

Correspondence and requests for reprints should be addressed to Steven R. Duncan, M.D., Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, 628 NW MUH, 3459 Fifth Ave., Pittsburgh, PA 15213. E-mail: duncsr{at}msx.upmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Obliterative bronchiolitis (OB) is a dreaded and frequent complication of lung transplantation with a poorly understood immunopathogenesis. To further evaluate disease mechanisms, we used T cell antigen receptor (TCR) ß-chain variable region RNase protection assays, after polymerase chain reaction amplification of TCR cDNA, to quantitate circulating CD4+ and CD8+ repertoires of transplant recipients with OB or no evidence of rejection (NER). All six recipients with OB had markedly abnormal CD4 expansions (2.5 ± 0.5 expansions/recipient) attributable to oligoclonal proliferations. Only two of six recipients with NER had a single, much lesser, CD4+ abnormality each (p < 0.01). Moreover, one of these patients developed OB shortly thereafter, and the other NER abnormality may have predated transplantation. In contrast, CD8+ expansions were common in both recipient populations. Findings of CD4+ expansions had 100% sensitivity and 80% specificity for the presence or imminent development of OB. These data suggest proliferations of CD4+ T cells are important in OB pathogenesis, and these are most likely part of a major histocompatibility complex Class II–dependent process of indirect alloantigen presentation. These CD4+ clones are likely to have facultative helper functions for the multiple and diverse immune processes that have been implicated in OB. Furthermore, the close association of CD4+ expansions with OB raises possibilities of development of novel diagnostic and therapeutic approaches.

Key Words: T lymphocytes • lung transplantation • graft rejection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The short-term successes of allogeneic pulmonary transplantation procedures continue to steadily improve due to ongoing technical advances. Nonetheless, eventual dysfunction of the allograft due to obliterative bronchiolitis (OB) remains a substantial problem, and graft survival rates have not improved during the last 20 years (1). OB is a syndrome of often severe fibroproliferative injuries in airways and small pulmonary blood vessels that eventually afflict one-half or more of long-term lung transplantation survivors (2). Although OB has a clearly immunologic pathogenesis and is generally considered to be a manifestation of chronic allograft rejection (3), details of the processes that lead to this disorder have not yet been defined.

Recently developed molecular methods for analyses of T cell antigen receptor (TCR) expression have yielded considerable insights into cellular immune mechanisms (4). An enormous potential diversity of T cells, each bearing unique TCR with finite specificity, results from random combinatorial rearrangements of TCR {alpha} and ß genes encoding variable (V), diversity (D) (only present in ß chains), and joining (J) segments during thymocyte maturation. The TCR repertoires of individuals are subsequently shaped by sequential intrathymic positive and negative selections and, following extrathymic export, effects of peripheral engagements with antigens (5). T cell responses to conventional antigenic peptide–major histocompatibility complex (MHC) complexes typically result in clonal expansions of the engaged lymphocyte(s). Hence, findings of clonal or oligoclonal T cell proliferations are presumptive evidence of cellular responses to stimulating antigens, and detailed analyses of these proliferations have provided important data relevant to disease mechanisms (5, 6).

We have previously shown that the TCR repertoires of peripheral blood lymphocytes are much more biased among lung transplant recipients with OB, in comparison with those of recipients with no evidence of rejection (NER) (7). Analogous findings have also been reported among other allograft recipients (6, 8–10). In general, these data implicate cellular immune responses in the development of chronic allograft rejection, but mechanistic details have not been completely elucidated.

Accordingly, we undertook these investigations, using quantitative methodologies, to more completely define the T cell responses of pulmonary transplantation recipients. The present studies also show TCR ß-chain variable gene (TCRBV) repertoires of recipients with OB are biased compared with those of NER recipients and that these biases are due to oligoclonal expansions of T cells. A unique finding of the present study is the demonstration that circulating CD4+ clonal expansions are a distinguishing feature of OB, whereas both OB and NER subjects tended to have expansions of their CD8+ T cells. The current study also independently corroborates earlier indications that the abnormal expansions of T cells may precede clinical manifestations of OB by several weeks.

These findings are evidence that T cells of both recipients with NER and those with OB recognize alloantigenic determinants and undergo proliferative responses. Extreme oligoclonal CD4+ proliferations are much more frequent among recipients with OB, however, suggesting that the pathogenesis of OB is linked to the cognate recognition of donor alloantigens by recipient helper T cells. Moreover, these data are most consistent with an important role for indirect antigen presentation (i.e., donor alloantigens presented by recipient antigen-presenting cells [APC]) in the development of OB. In addition to insights into disease mechanisms, the observations also raise interest in possible novel approaches that could have utility for the early detection and/or prevention of OB in lung transplantation recipients.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Specimens
Lung transplant recipients were consecutively recruited during the course of their routine clinical evaluations. Normal subjects (n = 10) were recruited by advertisement. All subjects gave written informed consent under guidelines delineated by the appropriate Institutional Review Board. Clinical status of lung transplant recipients was established by lung biopsy, using standardized histologic criteria (11) and defined changes in pulmonary function tests (12). The diagnosis of bronchiolitis obliterans syndrome (BOS) (12) was used here interchangeably with OB. Transplant recipients had been maintained on stable doses of conventional immunosuppressants, as detailed elsewhere (7). Recipients were excluded if they had ongoing infections or acute rejection (AR) or had received treatments that could transiently alter TCR repertoires (e.g., augmented immunosuppression) within the preceding 8 weeks.

T cells subsets (CD4+ and CD8+) were positively selected from 20–50 ml of peripheral blood by use of antibody-coated magnetic beads (Dynal, Lake Success, NY) according to the manufacturer's protocol. This methodology has previously been shown to result in populations with greater than 95% purity (13). Cell pellets were frozen and stored at -70°C pending further processing.

TCRBV Reverse Transcription–Polymerase Chain Reaction
This methodology enables nonbiased amplifications of TCRBV chains from small numbers of cells. Details of this technique have been delineated elsewhere (7) and are schematized in Figure 1 . In brief, total cellular RNA was extracted from specimens using guanidinium thiocyanate, followed by sequential synthesis of cDNA, addition of 3' homodimeric (dG) tails, and seminested polymerase chain reactions (PCR). Synthetic RNA was subsequently generated and used in the multiprobe RNase protection assay (RPA).



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  		Figure 1. Nonbiased amplification of TCRBV mRNA. First-strand cDNA was synthesized from 3' poly-A mRNA using Moloney MuLV reverse transcriptase with oligo-dT primers. dG-tails were added by terminal deoxynucleotidyl transferase, and the purified product was subjected to seminested PCR amplifications using primers corresponding to TCRBC. BC1 depicts external (3') TCRBC primer, and BC2 is an internal (more 5') sequence. T7 Anchor denotes T7 promoter sequence. The product of the second PCR is purified, and synthetic cRNA is subsequently generated by T7 RNA polymerase and used in the multiprobe RPA (7).

 
RPA
Assays were performed as previously described (7, 13, 14). In brief, amplified synthetic RNA was transcribed in the presence of [35S]UTP, and an aliquot (1,000 cpm) was incubated overnight with one of three labeled TCRBV probe sets (1 x 106 cpm) and a labeled TCR ß-chain constant region (TCRBC) probe (1 x 105 cpm). After digestion of unhybridized RNA and ethanol precipitation, samples were electrophoresed on polyacrylamide sequencing gels for autoradiography and direct ß counting (Radioanalytical Imaging Apparatus; Ambis Systems, San Diego, CA). Results for each of the 24 gene probes were expressed as percentages of total measured TCRBV.

Cloning and Sequencing
Standard methodologies, as delineated elsewhere (7, 13, 14), were employed. In brief, the gene sequence of interest was amplified by PCR using TCRBV-specific and TCRBC primers. Products of this reaction were cloned (TA Cloning Kit; Invitrogen, San Diego, CA), and plasmid DNA sequences were determined by an automated fluorescent sequencer (Prism 377; Applied Biosystems, Foster City, CA).

Statistics
Two-group comparisons of continuous or ordered data were analyzed by the Mann–Whitney test. Nominal values were compared by the chi-square test. Correlation coefficients were established by linear regression and Pearson product–moment correlation (r). The normal range for individual TCRBV gene expressions was defined as the mean ± 3 SD of values obtained from normal volunteers. Data are denoted as means ± SEM. Statistical significance was defined as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lung Transplantation Recipients
A total of 24 specimens were collected from 10 lung transplant recipients. Details of the study populations are shown in Table 1. Serial specimens were obtained from one NER recipient 44 months after transplantation and again 8 months later after a diagnosis of BOS had been established (with interval decrements of forced expiratory volume in 1 second and mean forced expiratory flow during the middle half of the forced vital capacity from 81 and 91% of predicted values, respectively, to 35 and 28% of predicted). The corresponding specimens from this patient are included in both NER and OB analyses. CD4+ TCR profiles were available from all 10 recipients, whereas complete CD8+ profiles were available from seven recipients (three with OB and four with NER). There were no differences between groups with respect to the number of previous episodes of serious infections or acute allograft rejection.


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  		TABLE 1. Patient demographics

 
TCRBV Analyses
CD4+ T cells. The CD4+ repertoires of recipients with OB were considerably more biased than those of recipients with NER (Figures 2 and 3) . Among the recipients with OB, 15 of 144 individual BV gene measurements (10.4%) were expanded beyond normal ranges, whereas only two of the determinations in recipients with NER (1.4%) were similarly expanded (p < 0.01). Abnormal CD4+ values were present in all six recipients with OB (2.5 ± 0.5 expansions/recipient), compared with two recipients with NER with one abnormality each. There were no correlations between number of BV abnormalities and elapsed time since transplantation (r = 0.08), and there was no apparent trend for preferential expansions in particular gene families (Figure 3). The magnitude of the CD4+ repertoire biases in the OB is considerable in that expansions accounted for 35.6 ± 4.9% of all measured BV among those afflicted. In contrast, the two expansions constituted 8.5 ± 4.2% of the total BV among the recipients with NER with TCRBV abnormalities (p < 0.05). The greater magnitude of biases in the recipients with OB is also reflected in intergroup comparisons of the coefficients of variation for individual BV gene values (90.3 ± 8.1 versus 60.4 ± 7.8, OB and NER, respectively, p < 0.01).



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  		Figure 2. Composite autoradiograph depicting typical results using all three RNase multiprobe sets (A–C). Lanes 1, 3, and 5 are autoradiographs of CD4+ specimens with normal gene expressions. Lanes 2, 4, and 6 depict CD4+ specimens from lung transplant recipients with abnormal TCRBV expansions (denoted with asterisks).

 


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  		Figure 3. TCRBV repertoires of peripheral blood CD4+ T cells from lung transplant recipients. Data are denoted as gene expression levels, referenced to total TCRBV expression in the specimen. Determinations from recipients with OB are depicted as circles, and specimens from recipients with NER are denoted as squares. Open symbols correspond to gene expression levels within normal limits (WNL), whereas closed symbols represent abnormal expressions. Upper limits of normal values for individual gene expressions are depicted by horizontal lines.

 
Three patients with OB had duplicate determinations, after intervals of 4–9 months, that were moderately concordant (r = 0.75 ± 0.13). The concordance between specimens that were obtained before and after the recognition of OB (Subject 5) was also relatively high (r = 0.73). A single abnormal gene expansion (BV3) was found at initial analysis and, although expression of this BV remained fairly high on subsequent testing, this particular finding was overshadowed by the presence of four new abnormal BV expansions (Figure 4) .



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  		Figure 4. Duplicate TCRBV determinations in a lung transplantation recipient before (NER) and after clinically evident OB. High expression levels of BV3 at initial testing were overshadowed by four other extreme expansions (denoted with asterisks) after development of OB.

 
Four of the recipients with OB (67%) have died during the follow-up interval (all but one within 7 months after BV testing). One recipient with NER died 5 months after BV testing from post-transplant lymphoproliferative disease and pneumonia, and another died 60 months later from complications of renal failure; both remained free of OB at the time of their deaths. Similarly, none of the living recipients with NER with normal CD4+ TCRBV profiles have developed OB during observation periods that range from 15 to 24 months.

Based on concurrent diagnoses at the time specimens were obtained, the finding of an abnormal CD4+ BV expansion in lung transplant recipients had perfect sensitivity (100%) and specificity of 67%. The positive and negative predictive values for presence of OB with abnormal BV expansions were 75 and 100%, respectively. Calculations based on categorizations of BV expansions as markers for current or subsequent OB result in a sensitivity of 100%, specificity of 80%, and positive predictive value of 88%.

CD8+ T cells. Unlike the results of CD4+ analyses, there were no striking differences between recipients with OB and those with NER with respect to biases among their CD8+ TCR repertoires (Figure 5) . The percentage of abnormally expanded BV was slightly, albeit insignificantly, greater among recipients with NER (10 of 96 BV determinations, i.e., 10.4%) than in those with OB (6 of 72 values, or 8.3%). All subjects studied here had one or more BV expansions (2.5 ± 0.9 per recipient with NER versus 2.0 ± 0.6 per recipient with OB, NS). There was no apparent association between numbers of BV abnormalities and time since transplantation (r = 0.31, p = 0.5), and the expansions also appeared to be randomly distributed among BV families.



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  		Figure 5. CD8+ TCRBV repertories of lung transplant recipients. Data depicted as in Figure 3. Although CD8+ expansions tended to be more extreme in recipients with OB, there were no significant intergroup differences.

 
Whereas the frequency of BV expansions was slightly greater among recipients with NER, the magnitude of repertoire skewing tended to be greater in those with OB, although again these differences were not statistically significant. Values of the abnormal BV among recipients with OB were 14.5 ± 8.3 SD greater than mean normal values, compared with 6.8 ± 1.1 in those with NER. The abnormal values accounted for 24.5 ± 9.0% of total measured BV in recipients with OB, compared with 11.9 ± 1.7 in the recipients with NER. The coefficients of variation for individual BV gene expressions were 99.8 ± 8.5 and 78.7 ± 7.4, for recipients with OB and NER, respectively.

The concordances between CD4 and CD8 repertoires were fairly high among individual recipients with OB (r = 0.77 ± 0.13). There was a much weaker correlation between these repertoires in recipients with NER (r = 0.48 ± 0.07), a likely reflection of their normal CD4+ profiles.

Clonality of TCR Repertoires
Analogous analyses of TCR repertoires in lung transplant patients and other populations (7, 13, 14) have consistently shown that marked BV expansions are almost invariably attributable to clonal or oligoclonal T cell expansions. To confirm that the BV expansions in these particular recipients are also due to oligoclonal proliferations, we performed VDJ sequence determinations in selected specimens. Given that the most striking intergroup differences were among CD4+, we focused our sequence determinations on this lymphocyte population.

BV3 sequences were individually amplified, cloned, and sequenced from the recipients with the most striking expansions of this particular BV (Figure 2) as well as another (NER) recipient with normal values of this BV. Specimens from a third subject with a lesser, but still abnormal, expansion of CD4+ BV17S1 were also cloned and sequenced.

As delineated in Table 2, there was a high degree of concordant sequences (oligoclonality) among the BV that were abnormally expanded. In contrast, there were no common VDJ sequences among 11 BV3 sequences from the subject with NER with normal expression levels of this BV. The extent of clonality (number of clones/number sequenced) was positively associated with both the absolute BV expression level (r = 0.79) and the number of SD by which these levels exceeded mean normal values (r = 0.95).


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  		TABLE 2. Sequences of cloned tcrbv

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present data are a novel demonstration that there are very distinct differences of CD4+ TCRBV profiles between lung transplant recipients with OB and those with NER. Earlier reports indicating biased TCR repertoires in lung and other transplant recipients employed methodologies that did not allow segregation or analyses of individual T cell subsets (7–10). Given that CD4 clonal expansions are extremely rare in the absence of pathogenic immune responses (15), the proliferations seen here among the recipients with OB are undoubtedly representative of alloantigen-driven processes in the allograft per se or regional lymph nodes. It has previously been shown that identical T cell clones can be identified in both allograft and circulation (7), and other cases wherein lymphocytes from intense inflammatory foci also traffic among peripheral blood lymphocytes have been reported (14, 16). Our findings that CD4 clonal expansions are much more frequent and extreme in recipients with OB suggest that the etiologic mechanism of chronic rejection is likely linked to alloantigen-driven proliferations of this lymphocyte subpopulation. Thus, these findings enable inferences to be drawn regarding likely immune mechanisms that are relevant to the development of OB.

The association here between CD4+ proliferations and OB is consistent with an increasingly compelling body of data that suggest indirect alloantigen presentation plays an important role in the immunopathogenesis of chronic allograft rejection (7, 8, 17). According to prevailing hypotheses, allogeneic peptides (notably including those derived from donor MHC molecules) are taken up by recipient APC that infiltrate the graft and are presented in the context of MHC Class II molecules to recipient CD4+ T cells (17). In experimental models, exogenous antigens can also be cross-presented by MHC Class I molecules of certain APC (some dendritic cells and possibly macrophages), in which case recognition by CD8+ T cells is predominant (18). The observation here that CD8+ responses are similar in both recipients with NER and those with OB, however, suggests that cross-presentation to these lymphocytes does not necessarily play a unique role in development of chronic rejection in vivo. Similarly, the presence of CD8+ expansions in both patients with and without chronic rejection would also dispel the singular significance of direct MHC Class I–dependent antigen presentation (recognition and responses to donor cells per se) in the etiology of OB, although this particular immune mechanism has been implicated elsewhere in the pathogenesis of acute allograft rejection (19). The findings that CD8+ expansions are ubiquitous among lung transplant recipients could thus be explained by the occurrences of AR or viral infections that had occurred earlier in the course of nearly every study patient. These data did not ascertain the absolute numbers or functional capabilities of the various cell populations. Findings elsewhere indicate increased numbers and/or augmented cytolytic activity among CD8+ of recipients with OB (20). These changes among the CD8+ subpopulation could, in turn, be due to the facultative help provided by CD4+ clones that have been activated and are rapidly proliferating (21).

The expanded CD4+ T cells found in recipients with OB could also mediate allograft injury by a variety of other mechanisms. Direct cytolytic activities have been observed among comparatively rare CD4+ subsets (22). The special importance of these effects in the pathogenesis of OB seems less likely, given the already considerable cytolytic potential of the expanded CD8+ clones that are present in all recipients. Perhaps more importantly, the expanded CD4+ may initiate and focus intragraft injuries by elaboration of activating factors and/or chemoattractants for diverse other cellular immune effectors that more directly cause airway injuries (23). The fibroproliferation that typifies OB could also result from or be augmented by CD4+-associated mediators (24). Allograft injuries may also be promoted by facultative CD4+ help that enhances production and specificity of allo-specific antibodies that are elaborated by B-cells (25).

In addition to providing insights into disease pathogenesis, these findings raise possibilities of the development of clinically applicable diagnostic tests. The early diagnosis of OB is rendered difficult due to the inhomogeneity of lesions and inherent limitations of transbronchial biopsies (26). The development of expiratory airflow obstruction (in the absence of other causes) is believed to be relatively specific (12), but spirometric abnormalities are not evident until there has already been considerable, and often irreversible, airway remodeling. If, in fact, OB represents the culmination of serial immune-mediated injuries, the appearance of physiologic abnormalities is likely to represent a late stage in the cascade that may no longer be dependent on the initial trigger(s). This hypothesis could explain the relative paucity of infiltrating T cells in well-developed lesions (11) and account for the usual refractoriness of mature OB to antilymphocyte therapeutics (3, 12).

The association of CD4 TCRBV expansions with the presence of OB here has seemingly greater sensitivity than transbronchial biopsy (12, 26). Of particular interest, the present data corroborate a previous anecdotal observation that T cell expansions may predate other clinical evidence of OB (7). If, in fact, the appearance of CD4 TCRBV proliferations was a sufficiently reliable predictor of imminent OB, the clinical management of lung transplant recipients may be considerably improved by serial TCR profile determinations. These minimally invasive analyses could facilitate predictions of OB and enable earlier and possibly more effective intervention while the process was still T cell dependent, before the development of widespread and fixed airway injury. Cytomegalovirus, other infections, and AR are frequent in this population, and the diverse immunologic effects of these events have often confounded assays based on detection of nonspecific immune activation or late-cascade immune mediators (12, 27). The CD4 TCRBV assays in the present series did not appear to be adversely affected by prior cytomegalovirus infections, several of which had also occurred in recipients with NER before sampling for the TCR analyses.

It is further possible that the study design here, based on cross-sectional rather than longitudinal sampling, could have negatively biased the specificity of these findings if the sole recipient with NER with a true false-positive assay had an antecedent (pretransplant) and persistent BV expansion. This particular recipient underwent lung transplantation for complications of cystic fibrosis and was the only subject in the study population with this disease. The TCR repertoire of patients with cystic fibrosis has not been evaluated systematically, but it is widely appreciated that the airways of these patients are chronically colonized/infected with pathogens (Staphylococcus aureus and Pseudomonas spp.) that have considerable immunogenecity, including elaboration of superantigens that could profoundly affect TCR profiles (28, 29).

Nonetheless, these data resulted from cross-sectional analyses of a comparatively small group of lung transplant recipients. Although these results are promising, they need additional corroboration in a larger cohort of recipients within the context of serial, longitudinal studies, before considering the use of TCR analyses as a clinical tool.

At least two other immunologic considerations with potential clinical significance emerge from these data. First, the marked biases evident in TCR repertoires of lung transplant recipients, in which a relatively few clonotypes account for disproportionately large proportions of circulating lymphocytes, imply that frequencies of T cells with other, potentially useful, antigen specificities are reduced (or absent). In effect, florid expansions of a limited number of alloantigen-reactive cells lead to diminutions of other T cells by poorly understood homeostatic mechanisms (30), and some of these reduced/absent lymphocytes undoubtedly have activities against disease pathogens. The "gaps" in TCR repertoire specificities generated by clonal expansions may account for some of the predilection for infections that typifies lung transplant recipients with OB (3, 12). Second, the finite number of highly expanded CD4+ clones seen among recipients with OB could generate interest in exploring the efficacy of more specific immunotherapies directed against highly select, disease-associated T cell subpopulations, including anti-clonotypic monoclonal antibodies or inhibiting peptides (30–34).

In summary, these investigations have shown that a distinguishing characteristic of TCR profiles among lung transplant recipients is the ubiquitous and near exclusive presence of marked CD4 clonal expansions among those patients with OB. Although causality has not been proven by these findings, the observations are additional, suggestive evidence that T cell–mediated responses are important in the development of this morbid complication and further implicate indirect antigen presentation as a critical mechanism in the immunopathogenesis of OB. The findings here, as well as anecdotal observations elsewhere, also raise the possibilities that the presence of CD4 clonal proliferations may have diagnostic and/or predictive value in assessments for OB, open avenues for new therapeutic considerations, and are an impetus for conducting larger, serial surveillances for TCRBV perturbations in a lung transplantation recipient cohort.


   Acknowledgments
 
Supported in part by U.S. National Institutes of Health grants 1KO8HL03016 and 1RO1HL64192.

Received in original form July 9, 2001; accepted in final form January 9, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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