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Right arrow Transplantation - heart

Eur J Cardiothorac Surg 2005;27:783-789
© 2005 Elsevier Science NL

Impaired circulating dendritic cell reconstitution identifies rejecting recipients after clinical heart transplantation independent of rejection therapy

Petros Athanassopoulosa,*, Leonard M.B. Vaessenb, Aggie H.M.M. Balkc, Johanna J.M. Takkenberga, Alex P.W.M. Maata, Willem Weimarb, Ad J.J.C. Bogersa

a Department of Cardiothoracic Surgery, University Medical Center Rotterdam, Erasmus MC, Rotterdam, The Netherlands
b Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands
c Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands

Received 17 September 2004; received in revised form 20 December 2004; accepted 23 December 2004.

* Corresponding author. Present address: Room Ee563a, Erasmus MC, Dr Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands. Tel.: +31 10 4635421; fax: +31 10 4089443. (E-mail: p.athanassopoulos{at}erasmusmc.nl).


   Abstract
Top
 Abstract
1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Objective: Dendritic cell (DC) mediated allo-antigen presentation to host antigen specific T-lymphocytes initiates acute allograft rejection. We investigated peripheral blood DC (PBDC) incidence and DC subset reconstitution in relation to histological diagnosis of acute cellular rejection (AR) and administration of rejection therapy after clinical heart transplantation (post-HTx). Methods: Venous blood from 20 HTx recipients under standard immunosuppression was collected during serial endomyocardial biopsy (EMB) prior to administration of rejection therapy in a 9-month follow-up post-HTx. Echocardiographic assessment of allograft function during EMB was performed to distinguish clinical necessity for rejection therapy within histologically rejecting patients (R). Myeloid (mDC) and plasmacytoid (pDC) subsets identified by flow-cytometry were analysed for different ISHLT rejection grades. Circulating PBDC incidence and mDC/pDC ratio were compared sequentially between non-rejecting (NR) recipients and R patients treated (3A+) or not-treated (3A–) with rejection therapy during follow-up. Results: Eleven samples from biopsy-proven AR episodes (AR+: ISHLT≥3) were compared to 89 samples from non-rejection episodes (AR–: ISHLT grade 0, n=52; grade 1, n=29; grade 2, n=8). We observed an inverse correlation of mDCs (P<0.05) but not pDCs with increasing rejection grade. PBDC incidence and mDC/pDC ratio were low in blood samples obtained during AR (P<0.05 and P<0.01, respectively). Both PBDCs and mDC/pDC ratio decreased during each AR episode (P<0.05). Comparison of 3A+ and 3A– rejectors with NR patients after 12 weeks post-HTx revealed lower PBDC incidence (P<0.01) and mDC/pDC ratio (P<0.05) for R patients, independent of rejection therapy. Conclusions: Defective DC subset reconstitution by dendritic cell profiling identifies patients at risk for AR after 3 months post-HTx. This finding may contribute to further optimization of immunosuppressive treatment strategies after clinical heart transplantation.

Key Words: Dendritic cell reconstitution • Heart transplantation • Rejection


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Macrophages, neutrophils and natural killer (NK) cells are intrinsic components of early non-specific allograft damage induced by ischaemia-reperfusion injury after heart transplantation (HTx) [1,2]. Acute rejection (AR) through allo-specific T-cell graft infiltration remains the leading cause of cardiac allograft dysfunction in the first months post-HTx [3]. The latter is initiated by the presentation of foreign allo-antigen (allo-Ag) to T lymphocytes by antigen presenting cells (APCs) such as donor or recipient dendritic cells (DCs) [4].

Two major DC subsets have been identified in human circulation [5]. The CD11c+ subset belongs to the myeloid lineage, whereas the CD11c– subset, recognized as plasmacytoid cells, expresses the IL-3 receptor-{alpha} chain (CD123) and consists of the direct precursors of lymphoid DCs [6]. Peripheral blood DCs (PBDCs) emerge in an immature state from bone marrow progenitors and traffic via blood to non-lymphoid tissues, where they exhibit high phagocytic capacity for antigens (Ags). Upon Ag encounter, DC subsets undergo multi-step maturation and present Ags in the lymph nodes. In this process, DC chemokine receptor expression is altered from an inflammatory to a lymphoid homing mode [7]. Major histocompatibility class I and II molecules, several adhesion (CD11a, CD18, CD44 & CD54) and co-stimulatory molecules (CD40, CD80, CD86) are increased on their surface, while DCs start secreting pro-inflammatory cytokines like IL1ß, IL-6, IL-12, IL-10 and IL-23 [8].

After transplantation, DCs induce anti-donor responses through the direct or indirect presentation of donor Ags [9]. Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) enter the T-cell areas of secondary lymphoid organs via the blood and drive the proliferation and differentiation of donor specific T cells [10]. As mDC primed T lymphocytes are able to differentiate into effector CD4+ or cytotoxic CD8+ cells [11] it is postulated that mDCs are the primary instigators of allograft rejection post-HTx. On the other hand, as pDC primed T lymphocytes also differentiate into regulatory T cells, it is suggested that pDCs may promote allograft tolerance [12]. Accordingly, peripheral blood DC subset balance is believed to be critical for the development of allograft acceptance. However, clinical doses of induction and maintenance immunosuppressive regimens are known to negatively affect peripheral blood DC subset Ag presenting capacity as well as maturation, migration and differentiation from their haemopoietic progenitors after transplantation [13]. Interestingly, patients at risk for AR are known to differ substantially in the mDC/pDC ratio from patients successfully withdrawn from immunosuppression [14].

The aim of this study was to look into circulating DC subset kinetics in relation to histological grading of AR and administration of rejection therapy after heart transplantation. Given that 1 week post-HTx circulating mDC and pDC numbers decrease significantly [15], we investigated peripheral blood DC numbers and the mDC/pDC ratio as a measure of DC subset reconstitution in a prospective 9-month follow-up post-HTx. Non-rejecting recipients were compared to patients with biopsy proven AR episodes in order to explore whether DC content might reflect their status with respect to risk for AR. Rejecting recipients were analysed according to rejection treatment in order to investigate dependence of DC profile on rejection therapy post-HTx.


   2. Patients and methods
Top
 Abstract
 1. Introduction
 2. Patients and methods
3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
2.1. Patients
We studied 20 adult HTx recipients (11 men and 9 women, mean age 51.6 years, range 37–65 years) operated between June 2002 and December 2003. All patients were classified with chronic symptomatic but clinically stable NYHA III–IV heart failure and had experienced a standardised medical regimen before transplantation [15]. One patient had already received maintenance immunosuppression pre-HTx, as he was re-transplanted due to graft vascular disease of an 11-year-old cardiac allograft. None of the patients presented with any major co-morbidity pre-HTx. The study protocol was approved by the local medical ethical committee on human research, (MEC 215.732/2002/157). All patients were recruited from the Thoraxcenter, Erasmus Medical Center (Rotterdam, The Netherlands) and gave written informed consent before entering the study.

2.2. Endomyocardial biopsies
Scheduled right venticular endomyocardial biopsies (EMB) were performed weekly until week 6, once every 2 weeks until week 10, monthly up to 6 months and once every 2 months up to the end of the first year post-HTx. Clinical course and prior rejection profile of each individual patient triggered EMB rescheduling in 2- to 4-weekly intervals late during follow-up. Using light microscopy, rejection grade was evaluated by two cardiac pathologists on 6 tissue samples extracted during each EMB procedure, according to the standard working formulation of the International Society for Heart and Lung Transplantation (ISHLT) [16]. Hematoxylin and eosin stained sections were also analysed to exclude Quilty effect as the etiology of lymphocytic infiltration. Patients with ISHLT grade ≥3 episodes were considered to experience acute cellular rejection.

2.3. Echocardiography
All patients underwent trans-thoracic echocardiography in order to assess left ventricular (LV) function within 4h of each EMB procedure, using a Hewlett Packard Sonos 5500 ultrasonograph with a 3.75MHz transducer (Hewlett Packard, Andover, MA, USA). LV wall dimensions were obtained by parasternal M-mode recordings combined with an electrocardiogram. Mitral flow velocities were measured within the valve orifice near the leaflet tips by two-dimensional Doppler echocardiography through an apical four-chamber view. M-mode echocardiograms were analysed for end-diastolic total wall thickness (TWT), calculated by adding posterior left ventricular wall thickness and end-diastolic interventricular septum thickness. Overall changes in systolic function were assessed by ‘eyeballing’ technique. Diastolic function was evaluated by peak early (E)/peak atrial (A) mitral flow velocity (E/A ratio) and deceleration time of E (DT). Recordings were analysed by a single investigator without knowledge of EMB outcome.

2.4. Immunosuppressive regimen
After transplantation, all HTx patients received anti-thymocyte globulin (ATG) and triple therapy consisting of steroids, Cyclosporine A (CsA, Novartis, Basel, Switzerland) or Tacrolimus (FK506, Fujisawa GmbH, Munich, Germany) and Mycophenolate Mofetil (MMF, Roche, Basel, Switzerland) as maintenance immunosuppression, for the whole post-HTx period. Horse-ATG (Imtix Sangstat BV., Lyon, France) at 3–8 i.v. dosages daily (1 dosage=212.5 lymphocytotoxic units/kg/24h) was administered until adequate CsA or FK506 trough levels were achieved. All patients received 75mg prednisolone i.v. in the first post-transplant day, 50mg/day for 5, 40mg/day for 3 and 30mg/day for 2 consecutive days post-HTx. Thereafter, orally administered prednisone was tapered with 5mg decrements up to 20mg/day for 3 days and then with 2.5mg decrements every 7 days up to 10mg/day. CsA and FK506 were started at 8mg/kg/24h and 0.3mg/kg/24h oral dosages respectively, divided in 2 doses daily which were titrated further according to the corresponding trough levels. MMF was started at the end of the induction therapy in doses between 2000 and 3000mg, daily. CMV syndrome, as manifested by symptoms and a positive PCR, was treated with ganciclovir (5mg/kg, 2 times daily) for 10–14 days. In case of resistance to this therapy, MMF was withdrawn from the immunosuppressive regimen. Patients presenting with ISHLT grade ≥3A biopsies were considered for treatment of rejection with additional immunosuppression in the form of methyl-prednisolone i.v. for 3 days at 1000mg/day (Pfizer, Pharmacia & Upjohn, Sandwich, UK). In case of minor infiltrates and when myocardial damage was considered too mild to warrant rejection therapy, echocardiographic assessment was used to decide upon rejection treatment: in such cases rejection therapy was omitted when systolic and diastolic LV function were normal or unchanged compared to earlier measurements.

2.5. Monoclonal antibodies
Allophycocyanin (APC)-conjugated CD11c (clone S-HCL-3), phycoerythrin (PE)-conjugated anti-IL-3 receptor {alpha} chain (CD123; clone 9F5), peridinin chlorophyll protein (PerCP)-conjugated anti-HLA-DR (clone L243) and fluorescein isothiocyanate (FITC)-conjugated lineage cocktail 1 (Lin 1) were commercially obtained (Becton Dickinson Biosciences, San Jose, CA, USA). The Lin 1 contained monoclonal antibodies (mAbs): CD3 (T cells; clone SK7), CD14 (monocytes/macrophages; clone M{Phi}P9), CD16 (natural killer cells; clone 3G8), CD19 (B cells; clone SJ25C1), and CD56 (natural killer cells, clone NCAM16.2). Mouse anti-human CD45 FITC (clone F10-89-4)/CD14 PE (clone UHCM-1) reagent (Serotec, Oxford, UK) was used to monitor lymphocyte, monocyte and granulocyte presence in each sample.

2.6. Dendritic cell characterisation
Patient peripheral blood cells were collected at 1, 4, 12, 24 and 38 weeks post-HTx, during the EMB procedure and before histological diagnosis of AR. Whole blood cells were incubated with mAbs followed by erythrocyte lysis with FACS lysing solution (BD Biosciences) at room temperature. After washing with FACSflow (BD Immunocytometry Systems), 200,000–300,000 events were analysed in a FACSCalibur fluorescence-activated cell sorter with the CellQuest Pro software program (BD Biosciences). White blood cell (WBC) numbers were determined by a Sysmex Microcellcounter F-300 automated cell counter, (Goffin Meyvis, Etten Leur, The Netherlands). DC characterization was blinded for occurrence of AR at the time of blood sampling. CD11c and CD123 expression was determined within Lin– HLA-DR+ cells in order to define mDC (CD11chigh CD123low) and pDC (CD11clow CD123high) subsets, as described before [15]. Absolute mDC and pDC numbers were calculated from the WBC count multiplied by the proportion of each subset within WBCs. DC incidence was calculated by adding absolute mDC and pDC numbers. The mDC/pDC ratio was used as a means to express circulating DC reconstitution post-HTx. Assay reproducibility has been confirmed previously, as DC sample analysis in healthy controls revealed stable % DC, absolute numbers of mDCs and pDCs as well as a constant mDC/pDC ratio, within the period of 1 month [17].

2.7. Statistical analysis
Results are expressed as mean ± SEM of absolute white blood cell, lymphocyte, monocyte, granulocyte and total DC numbers. All continuous data sets were tested before comparisons in order to assess whether the assumption of normality was met. The Mann Whitney U-test and the paired Wilcoxon samples t-test were employed to compare differences between means as appropriate. Correlation of mDC/pDC ratio with ISHLT grade was performed by linear regression analysis after log10 transformation of the data using the Pearson's (r) correlation coefficient. One-way analysis of variance (ANOVA) was used for comparisons of clinical parameters between groups of patients and DC counts or mDC/pDC ratio between different rejection grades. Post-hoc analysis was performed to compare DC counts and mDC/pDC ratio for each rejection grade separately, using the Tukey's test for multiple comparisons. Continuous DC number and mDC/pDC ratio data of different groups of patients were compared by repeated measurements ANOVA. A P-value<0.05 was considered significant. SPSS 11.0.1 software (Chicago, IL, USA) and the GraphPad statistical program (San Diego, CA, USA) were applied for analyses and graphics, respectively.


   3. Results
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
4. Discussion
 Appendix A. Conference...
 References
 
3.1. Clinical characteristics
After transplantation, 14 of the 20 patients developed rejection in the whole period of 9-months follow-up. Within this group, 11 patients presented with impaired diastolic function during AR as assessed by echocardiography and therefore received rejection therapy (3A+) on one or more occasions during this period. However, 3 of the 14 rejecting patients, revealed once histological signs of AR but did not receive any rejection therapy after normal diastolic heart function assessment at the time of EMB (3A–). Infection was the most prominent complication for both non-rejecting (NR) and rejecting (R) groups of patients post-HTx (Table 1). In total 11 patients presented with CMV syndrome post-HTx, and for 8 of those MMF therapy was discontinued. Mean time of CMV syndrome onset between non-rejectors (83.4±12.8 days) and the 3A+ or 3A– rejectors (96.7±17.7 and 84.0±13.0 days, respectively) was not significantly different (P=0.79). Similarly, mean time for MMF withdrawal was comparable (P=0.66) between the NR (147.6±12.7 days) and the 3A– or 3A+ rejecting subjects (124.7±21.7 and 158.4±16.8 days, respectively). Seven patients were converted to FK506 after experiencing calcineurin inhibitor related renal insufficiency post-HTx (2 in the 1st, 1 in the 2nd, 2 in the 3rd, 1 in the 13th and 1 in the 18th week follow-up). Maintenance steroid dosages and acquired trough levels of immunosuppressive drugs were comparable between R and NR patients. However, 3A+ rejecting patients tended towards higher CsA trough levels (310.0±19.8ng/ml) than 3A– rejectors (272.3±25.8ng/ml) and NR patients (233.6±19.5ng/ml) for the whole period of follow-up (P=0.06). No differences were observed in immunologic or operation-related parameters between the 3 patient groups (Table 2).


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  		Table 1. Complications for rejecting and non-rejecting patients after heart transplantation
 

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  		Table 2. Clinical outcomes for non-rejecting and rejecting recipients post-HTx
 
3.2. DC numbers after heart transplantation
Table 3 shows the transplant recipient haematologic characteristics pre- and at 1, 4, 12, 24 and 38 weeks post-HTx. Leukocytes were elevated significantly at 1-week post-HTx compared to the pre-HTx condition (P=0.02), as granulocytes increased (P=0.04) early post-HTx. Blood monocyte counts remained unaffected while lymphocyte counts, initially decreased by 41–48% but returned to pre-HTx levels at 12 weeks post-HTx, long after termination of h-ATG therapy. White blood cell counts normalised again at 2 weeks post-HTx and remained within a normal range for the rest of the follow-up while total DC numbers decreased significantly at 1-week post-HTx and remained lower than the pre-HTx condition for the whole post-HTx period (P<0.001).


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  		Table 3. Recipient hematological characteristics after heart transplantation
 
3.3. Circulating DC reconstitution and EMB rejection grade
In total 100 right ventricular biopsies became available for study. Fourteen patients experienced a total of 20 biopsy-proven AR episodes post-HTx. No severe (grade 4) AR episodes were documented during follow-up. Blood samples corresponded to 52 grade 0, 27 grade 1A, 2 grade 1B, 8 grade 2, 10 grade 3A and 1 grade 3B biopsies. Table 4 shows a cross-tabulation of DC counts and rejection grade. We observed a negative association between absolute total DCs and the diagnosed ISHLT rejection grade for the whole period of follow-up (P=0.001). This effect was seen only in the mDC fraction (P<0.001), but not in the pDC fraction (P=0.55). Consequently the mDC/pDC ratio decreased significantly by increasing rejection grade (P=0.002). At the peak of the AR period (12 weeks post-HTx), mDC/pDC ratio was inversely correlated with ISHLT grade (r=–0.59, P=0.006) (Fig. 1). From the 11 AR episodes (grade 3A/B) caught in our follow-up, 2 occurred in the 1st, 2 in the 4th, 4 in the 12th, 2 in the 24th and 1 in the 38th week post-HTx. Complete data before, during and after rejection were available for 8 AR episodes. Paired samples analysis in the rejecting HTx-recipients for all episodes before, during and after AR (n=11) revealed that DC numbers and mDC/pDC ratio decreased during each rejection episode (Fig. 2A and 2B).


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  		Table 4. Peripheral blood dendritic cells in relation to EMB rejection grade after clinical heart transplantation
 


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  		Fig. 1. Correlation of mDC/pDC ratio with endomyocardial biopsy ISHLT infiltration grade for 20 heart transplant (HTx) recipients at 12 weeks post-HTx.

 


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  		Fig. 2. Total peripheral blood DC numbers (A) and circulating mDC/pDC ratio (B) of 14 rejecting patients post-HTx before, during and after 11 acute rejection (AR) episodes caught during 9 months follow-up. *P<0.05 represents the outcome of paired samples analysis for DC numbers or mDC/pDC ratio before vs during and during vs after AR.

 
3.4. Circulating DC reconstitution and rejection therapy post-HTx
Fig. 3 shows absolute DC counts and the mDC/pDC ratio at 1, 4, 12, 24 and 38 weeks post-HTx. Rejecting patients exhibited impaired DC recovery post-HTx when compared to NR patients. This phenomenon was independent of rejection therapy as in both 3A+ and 3A– rejectors circulating DC levels were equally lower than the PBDC counts of NR patients at 24 weeks (P=0.01) and 38 weeks post-HTx (P=0.0006) (Fig. 3A). Both mDC and pDC numbers for all patients remained significantly lower than the pre-HTx condition (mDCs: range 3.63–7.88cells/µl; pDCs: range 1.00–2.91cells/µl) for the whole period of follow-up. Interestingly, the mDC/pDC ratio of NR patients in the 1st week post-HTx appeared to be lower, compared to the ratio of 3A+ or 3A– rejecting recipients, but this difference did not reach statistical significance (P=0.09). For all patients mDC/pDC ratio decreased equally at 4 weeks post-HTx. For the whole period of follow-up, PBDC counts in patients who experienced clinical CMV syndrome were not different from patients who did not have symptomatic CMV infection (P=0.98). Later during follow-up it became evident that NR patients attained a higher mDC/pDC ratio over time than 3A+ or 3A– rejectors patients. Repeated measures ANOVA analysis showed that the difference in mDC/pDC ratio between NR vs. 3A– and 3A+ rejector groups became significant at 38 weeks post-HTx (P=0.04) (Fig. 3B).



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  		Fig. 3. Temporal analysis of absolute DC numbers (A) and mDC/pDC ratio (B) in peripheral blood of 6 non-rejecting (NR) recipients and 14 rejecting (R) patients treated (3A+, n=11) or not-treated (3A–, n=3) with rejection therapy up to 9 months post-HTx. *P<0.05, **P<0.01, ***P<0.001 represent the outcome of between groups one-way ANOVA comparisons.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
Appendix A. Conference...
 References
 
In the present study, we investigated PBDC counts and DC subset ratio as a means to explore circulating DC reconstitution after HTx. We observed that PBDC numbers do not return to pre-HTx levels for up to 38 weeks post-HTx. In addition, during AR, mDC levels decrease significantly, suggesting selective usage of this subset for induction of AR. The latter finding may be useful for early detection of AR after heart transplantation.

As surgery and stress impose a minimal and transient PBDC increase respectively [18,19], low circulating mDC, pDC and thus PBDC numbers post-HTx, must be ascribed to the administered immunosuppressive regimen. After heart transplantation, circulating mDC kinetics are seemingly affected by the ISHLT rejection grade at the time of the EMB procedure. As pDCs remained constantly low post-HTx, we observed that mDCs, PBDCs and consequently the circulating mDC/pDC ratio were negatively associated with a higher number of infiltrating lymphocytes and presence of myocyte damage diagnosed by ISHLT histological grades ≥3. Furthermore, both PBDC counts and mDC/pDC ratio decreased markedly during the majority of AR episodes. We hypothesize that altered PBDC kinetics reflect an intrinsic mechanism of circulating mDC depletion during AR. We can speculate that selective homing of this subset to secondary lymphoid tissues precedes in order to induce T cell differentiation into CD4+ helper or cytotoxic CD8+ cells and therefore initiate rejection.

Rejecting patients did not exhibit the PBDC recovery that the NR recipients experienced after 12 weeks post-HTx. Interestingly, within R recipients PBDC numbers remained low in both patients treated (3A+) or not-treated (3A–) with rejection therapy during follow-up. The fact that rejecting HTx recipients undergo circulating mDC depletion over time is supported by the observation that the mDC/pDC ratio decreases after 24 weeks post-HTx, independent of rejection therapy. This suggests that defective circulating DC reconstitution post-HTx, is inherently related to the immunological process of AR and not to additional immunosuppression administered as rejection treatment post-HTx.

On the other hand, it is unlikely that the temporal differences seen between NR and R recipients depend on infection onset or tapering of maintenance immunosuppression post-HTx. Opportunistic infections experienced by both groups of patients, were evenly distributed during follow-up and all recipients were treated according to the same local antibiotic or anti-viral therapeutic protocol. No significant differences were seen in the dosages or the acquired trough levels of maintenance immunosuppressants administered post-HTx, although 3A+ rejectors tended towards higher trough levels of CsA than 3A– rejectors and NR recipients for the whole period post-HTx. Nor was the manifestation and onset time of a major post-transplant infectious complication, such as CMV syndrome, different between the patient groups. Furthermore, time of MMF withdrawal from the immunosuppressive regimen for patients with CMV was similar between 3A+ or 3A– rejectors and NR recipients.

Emerging evidence suggests that abberant DC reconstitution is indeed related to adverse clinical outcomes after transplantation. For example, PBDCs decrease during acute graft-versus-host disease [20] and low DC counts predict relapse and even death after allogeneic hematopoietic stem cell transplantation [21]. In our hands, cardiac allograft rejectors, exhibited mean total DC numbers of 2.5cells/µL during AR, for the whole period of follow-up. At 6 and 9 months post-HTx, low mean total DC numbers of 6.5 and 7.7cells/µL identified patients that had undergone AR independent of rejection therapy, whereas higher mean total DC numbers (15.7 and 15.1cells/µL, respectively) characterised cardiac allograft non-rejectors. Similarly, shortly after kidney transplantation non-rejecting recipients exhibit higher percentage PBDCs than their rejecting counterparts [22]. Indeed, in our patient cohort we observed lower DC numbers for rejecting patients at 1 week post-HTx. At the same time DC subset distribution revealed that patients who rejected later during follow-up, had a higher circulating mDC/pDC ratio than NR patients. The differences seen during the early post-transplant period were not significant. However, these observations maybe biased by post-operative infections or influenced by clinical confounders such as induction immunosuppression in the form of h-ATG, high-doses of steroids or normalization of trough levels for CsA, FK506 and MMF, which may interfere with peripheral blood DC data acquired during the first month post-HTx.

The inclusion of small groups of patients and the restricted numbers of rejection episodes examined in this investigation, may limit the extrapolation of our results to clinical outcomes after heart transplantation. However, our long-term findings seem not to be affected by the clinical presentation of the patients after transplantation, with regard to post-operative infections and CMV syndrome manifestation. The analysis of fresh human material, the prospective nature of follow-up and blinding for AR grade during blood sampling, were all advantageous attributes to this study.

In summary, our results show that immunosuppression renders peripheral blood DC numbers decreased after heart transplantation. We have demonstrated that incidence of mDCs as well as the circulating mDC/pDC ratio are negatively associated with ISHLT infiltration grade. Both PBDC numbers and mDC/pDC ratio decreased markedly during AR. Rejecting patients exhibited impaired circulating DC reconstitution after 12 weeks post-HTx when compared to NR patients, independent of rejection therapy. In light of these findings, peripheral blood mDCs may be attributed an important role in eliciting and maintaining allograft rejection post-HTx. Although this technique is prone to bias from immunological complications such as opportunistic infections and CMV syndrome after transplantation, PBDC monitoring may identify patients with high risk for rejection after 3 months post-HTx. This might prove clinically relevant as appropriate adjustments in immunosuppressive regimens may avoid over-immunosuppresion of patients at a lower risk for rejection long after clinical HTx.


   Appendix A. Conference discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
References
 
Dr G. Laufer (Innsbruck, Austria): What type of induction therapy did you use, what specification of ATG?

Dr Athanassopoulos: We used horse ATG, which was administered for minimally 5 days, to all patients included in this study.

Dr Laufer: And do you know or do you have any idea why the count of this myeloid-type dendritic cells remained low in the patients who rejected their grafts? Do you think that the trafficking of these cells is routed to the lymphatic organs and they stimulate—this would be a very mechanistic theory, a surgical theory, for this low count—or do you have any other ideas how it is working or why the count is so low?

Dr Athanassopoulos: Well, our current postulation is that indeed these cells are being depleted from blood in order to induce acute rejection. We have used chemokine receptors, in order to characterize their expression on both dendritic cell subsets. We have data on chemokine receptor CCR7, which is a marker for mature dendritic cells migrating towards the lymphoid organs, and we saw a decrease in this subset of myeloid dendritic cells, as well. So we think that, during acute rejection, we always have selective usage of a mature fraction of circulating myeloid dendritic cells, towards the lymphoid organs. But, of course, under the current immunosuppressive regimen of these patients, we also think that there is a major problem in the differentiation of both dendritic cell subsets from the bone marrow. We believe, this explains why we also see a total decrease in the dendritic cells, as well.

Dr Laufer: To your knowledge, or do you know, if polyclonal ATG is also depleting both types of dendritic cells?

Dr Athanassopoulos: Yes.

Dr Laufer: There's a dramatic dropping in lymphocyte count with induction therapy, this polyclonal induction therapy?

Dr Athanassopoulos: Yes.

Dr Laufer: And they also targeted a magnitude of antigens on the lymphocytic surface and I think also as well on the dendritic cell surface.

Dr Athanassopoulos: It is true. There is a group, which has a publication in The Annals of Oncology. They have demonstrated that such antibody depleting protocols have a prolonged effect on both dendritic cell subsets, which can be induced for up to 6 months. However, we think that our results are important because nonetheless, all of the patients received ATG therapy with comparable intensity and at the end of the follow up one could still see the differences between the rejecting and the non-rejecting recipients.

Dr Laufer: Okay. I think somebody would also think that the donor-type dendritic cells and not the recipient-type dendritic cells are more important if you can achieve microchimerism, according to the results from Starzl. But anyhow, I think these cells are very important.


   Acknowledgments
 
This study was financially supported by the Biomedical Engineering Foundation, Erasmus MC, Rotterdam, The Netherlands.


   Footnotes
 
{star} Presented at the joint 18th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 12th Annual Meeting of the European Society of Thoracic Surgeons, Leipzig, Germany, September 12–15, 2004.


   References
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 

  1. Fairchild PJ, Waldmann H. Dendritic cells and prospects for transplantation tolerance. Curr Opin Immunol 2000;12(5):528-535.[CrossRef][Medline]
  2. Hosenpud JD, Bennett LE, Keck BM, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: eighteenth official report-2001. J Heart Lung Transplant 2001;20(8):805-815.[CrossRef][Medline]
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