The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation.

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)-gamma producing cells determined by enzyme-linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN-gamma producing cells reactive to this pathway increased significantly (P = 0.04) during AR and the number decreased (P = 0.005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN-gamma producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.

The effects of renal transplantation on circulating dendritic cells.

The effects of immunosuppressive agents on T cell function have been well characterized but virtually nothing is known about the effects of renal transplantation on human dendritic cells (DCs). With the use of flow cytometry, we studied the kinetics of myeloid and plasmacytoid DCs in peripheral blood of 24 kidney allograft recipients before and after transplantation, and in 23 donors before and after kidney donation. All patients were treated with tacrolimus, mycophenolate mofetil and prednisone. Surgery resulted in a strong decline in the number of myeloid and plasmacytoid DCs, both in kidney donors and in their recipients. However, in donors this effect was transient, as the numbers of both DC subsets had normalized completely by the third postoperative month. In contrast, the recovery of myeloid DC counts in kidney transplant recipients was only incomplete at the end of the 3-month follow-up, despite tapering of immunosuppression. The seven patients who required additional immunosuppressive treatment because of acute rejection experienced an even more marked decrease in DC counts in the early postoperative period compared with patients who remained rejection-free. Surgical procedures markedly affect the numbers of circulating myeloid and plasmacytoid DCs. Immunosuppressive drugs have important additional in vivo effects on this cell type and impair the reconstitution of the myeloid DC subset in peripheral blood after renal transplantation.

Pretransplant donor-specific helper T cell reactivity as a tool for tailoring the individual need for immunosuppression.

BACKGROUND: A reliable immunological assay for quantification of donor-specific alloreactivity to identify patients at risk for future allograft rejection would be a helpful tool in organ transplantation. Therefore, we questioned whether the T cell reactivity in patients measured before transplantation was predictive for the occurrence of acute rejection during the first year after kidney transplantation. METHODS: The pretransplant T cell reactivity of peripheral blood mononuclear cells to donor and third-party antigens was tested in mixed lymphocyte cultures, and to tetanus toxoid. In addition, we measured the frequency of donor and third-party reactive helper T lymphocyte precursor and cytotoxic T lymphocyte precursors using limiting dilution analysis. RESULTS: Patients who experienced acute rejection had significantly higher donor-specific mixed lymphocyte cultures responses (n=38; median stimulation index): 113 vs. 15, P=0.005) and helper T lymphocyte precursor frequency (n=37; median 194/106 vs. 62/106, P=0.009) measured before transplantation compared to patients without acute rejection. All patients with a low mixed lymphocyte culture response (stimulation index

Increased numbers of circulating donor-specific T helper lymphocytes after human heart valve transplantation.

Implantation of cryopreserved human donor heart valves for either congenital or acquired cardiac disease has been performed since the last three decades. Although the clinical outcome is good, long-term valve degeneration resulting in dysfunction has been observed. A specific immunological response of the recipient against the allograft has been proposed as one of the factors involved in this process. Helper T lymphocytes play an important intermediate role in cellular and humoral immune response. Increasing numbers of circulating donor-specific helper T lymphocytes precursors (HTLp) correlate with graft rejection after organ transplantation. To investigate whether cryopreserved human donor heart valves are able to induce a donor-specific T helper response, we monitored the HTLp frequencies (HTLpf) in peripheral blood samples of 13 patients after valve allograft transplantation by use of a limiting dilution assay followed by an interleukin-2 bioassay. Prior to transplantation, HTLpf specific for donor and third-party antigens showed individual baseline levels. After allografting, the antidonor frequencies significantly increased in 11 of the 13 patients (P = 0.02). This was not found for stimulation with third-party spleen cells (P = 0.68), which indicates a donor-specific response. Maximal donor-specific HTLpf were already found at 1--2 months after operation. Valve allograft transplantation induces an increase in the numbers of donor-specific HTLp in peripheral blood of the patients. Analogous to organ transplantation, these HTLp may play a crucial role in events that lead to valve damage. Therefore, monitoring of HTLp in peripheral blood samples might be informative for donor valve degeneration (rejection) and subsequently valve allograft failure.

Immunological aspects of fresh and cryopreserved aortic valve transplantation in rats.

BACKGROUND: The influence of immune activation on valve allograft degeneration remains unclear. We studied the combined effect of major histocompatibility complex (MHC)-incompatibility and cryopreservation on valve performance, histomorphology, and tissue antigenicity in rats. METHODS: Fresh or cryopreserved allogeneic aortic valves from WAG (RT1u) rats were transplanted to DA (RT1a) recipients and syngenic transplants served as controls. After 7 or 21 days, valves were examined for competence and morphology. Immune reactivity of the recipient was measured by concanavalin A (conA) stimulation and analysis of donor-reactive Helper T-lymphocyte frequencies (HTLf) in peripheral blood and spleen. RESULTS: Syngenic grafts demonstrated normal competence and structure. Allografts lost their competence over time caused by destruction of the leaflets combined with cellular infiltration in the vascular wall. Cryopreservation induces early loss of competence and retrovalvular thrombosis. Cryopreserved allografts were also heavily infiltrated. ConA stimulation indices and HTLf were higher in allogeneic recipients compared to syngenic recipients (p < 0.03). Cryopreserved allografts elicited a lower immune response compared with fresh allografts (p < 0.03). CONCLUSIONS: Aortic valve allografts are able to induce a donor-reactive immune response that is related to early graft destruction and incompetence. Cryopreservation appears to diminish but not eliminate the antigenicity of the allograft.

Reassuring effect of pravastatin on natural killer cell activity in stable renal transplant patients.

BACKGROUND: Administration of pravastatin soon after transplantation successfully lowers cholesterol levels, whereas a reduced number of acute rejection episodes is accompanied by a decrease in natural killer (NK) cell activity. As a consistent low NK cell activity caused by pravastatin might impair tumor surveillance leading to cancer, we studied the effect of pravastatin on NK cell activity in stable renal transplant patients. METHODS: From 14 cyclosporine (CsA)-treated and 11 azathioprine (AZA)-treated patients with hypercholesterolemia, more than 1 year after kidney transplantation, we determined NK cell number and cytotoxic activity before, and at 6 and 12 weeks after, initiating pravastatin treatment. Additionally, cholesterol levels and liver and kidney function parameters were assessed. RESULTS: During pravastatin treatment, total cholesterol and low-density lipoprotein-cholesterol levels decreased significantly in both patient groups. In the CsA group, the number and cytotoxic activity of the NK cells at 12 weeks after institution of pravastatin was in the same range as before pravastatin. Additionally, in the AZA group, pravastatin did not influence the number of NK cells. However, in the AZA group, both the number of NK cells and their cytotoxic activity were significantly (<0.002) lower compared to the values in the CsA group. CONCLUSIONS: In contrast to previous reports on decreased NK cell cytotoxicity caused by pravastatin treatment early after transplantation, we cannot confirm these results in stable kidney recipients. In our hands, NK cell cytotoxicity during pravastatin treatment was within the same range as in the absence of pravastatin. Thus, in view of the potential role of NK cells in tumor surveillance, these data are reassuring.

Heart valve dysfunction resulting from cellular rejection in a novel heterotopic transplantation rat model.

Structural failure of heart valve allografts may be related to technical factors or immunological reactions. To circumvent nonimmunological factors a new rat implantation model was developed to study whether alloreactivity results in histopathological changes and valve dysfunction. Syngeneic (WAG-WAG, DA-DA) and allogeneic (WAG-BN, WAG-DA) transplantation was carried out using this new technique, and the function of explanted valves was assessed 21 days later by retrograde competence testing. Additionally, grafts were examined using standard histological and immunohistochemical techniques. There was no leakage during retrograde injection in nine of tem syngeneic and two of ten allogeneic grafts. Microscopically, syngeneic valves appeared normal without fibrosis or intimal thickening, although CD8+ lymphocytes and macrophages were found in necrotic myocardial rim and adventitia. In contrast, allogeneic valves were deformed and noncellular, with extensive infiltration of CD4+, CD8+ and CD68+ cells in adventitia and media. Absence of fibrosis and intimal thickening in syngeneic transplanted valves indicated circumvention of nonimmunological factors. Allogeneic valve transplantation induces cellular infiltration in the graft with subsequent graft failure.

Induction of cytotoxic T lymphocytes with destructive potential after cardiac valve homograft implantation.

BACKGROUND AND AIM OF THE STUDY: Clinical and experimental studies have shown that a specific immunological response may play a role in the degeneration of human cardiac valve homografts. In heart and corneal transplantation, cytotoxic T lymphocytes (CTL) with high avidity for donor antigens are presumed to be the major effector cells causing graft destruction. We studied the kinetics of these donor-specific CTL precursors (CTLp) and their high-avidity fraction in peripheral blood of patients receiving a cryopreserved valve homograft. METHODS: Limiting dilution analysis (LDA) was used to enumerate donor-specific CTLp in peripheral blood samples of 15 patients, obtained up to 12 months after valve implantation. Donor-specificity was proven by using donor-HLA mismatched third-party stimulation cells as controls. CD8 monoclonal antibodies were used to distinguish high- and low-avidity CTLp. RESULTS: A significant increase in total donor-specific CTLp among the peripheral blood mononuclear cell population occurred in 14/15 patients (93%) at 3-6 months (p = 0.045) after implantation and remained so for up to 12 months (p = 0.015). In addition, a significant increase was seen in the fraction of circulating CTLp with high avidity for donor antigens (p<0.026) within the first 3 months after implantation. CONCLUSION: Implantation of cryopreserved valve homografts increases the number of donor-specific CTLp and their high-avidity fraction, in the peripheral blood. These cells have the capacity to destroy organ and tissue grafts.

Immunologic monitoring of the beneficial effect of one HLA-DR-shared blood transfusion in heart transplant patients.

BACKGROUND: Pre-transplant blood transfusion (BT) results in better graft survival in organ transplant recipients, especially when BT donor and allograft recipient share an HLA-DR antigen. Although the immunologic mechanisms involved are still poorly understood, we wanted to know whether down regulation of donor-reactive T cells play a role. METHODS: In a retrospective study, we analyzed the clinical effects of BT for 45 heart transplant (HTx) patients who had each received 1 BT that shared an HLA-DR-antigen with the recipient, and 55 who had a DR-mismatched BT before heart transplantation. From 30 patients, 15 with DR-shared BT and 15 with DR-mismatched BT, peripheral blood lymphocytes (PBL) were available. From each patient, we analyzed PBL samples taken at the day of transplantation (pre-transplant), and 1 to 2 months, 5 to 7 months, 9 to 14 months, 2 years, and 6 to 7 years after transplantation. Cytotoxic T-lymphocyte precursors (CTLp) and helper T-lymphocyte precursors (HTLp) were measured in a combined limiting dilution assay. RESULTS: Analysis of survival during the first 10 years revealed a significantly (p = 0.016) better survival rate in the group of patients who had received HLA-DR-shared BT compared with the group who had received HLA-DR-mismatched BT. Patients of the DR-shared group experienced significantly (p = 0.042) less acute rejections compared with the patients who received DR-mismatched BT. We found no differences in the development of graft vascular disease. Frequencies of CTLp specific for the organ donor did not change with time after transplantation in the individual patients, nor did we detect any differences between the two BT groups. We found the same for organ donor-specific HTLp frequencies. CONCLUSIONS: These data suggest again that transfusion effect depends on HLA-DR compatibility between the heart transplant recipient and the pre-transplant BT donor. The mechanism that caused better survival rate was not down regulation of the donor-reactive T-cell frequency.

Circulating donor-specific cytotoxic T lymphocytes with high avidity for donor human leukocyte antigens in pediatric and adult cardiac allograft valved conduit recipients.

OBJECTIVE: Specific immunological responses may be involved in the process of cryopreserved allograft valved conduit (AVC) degeneration, which is more frequently seen in young recipients. Rejection of heart and corneal allografts is preceded by an increase in the fraction of cytotoxic T lymphocytes (CTL) with high avidity for donor human leukocyte antigens (HLA) circulating in both peripheral blood and the affected graft. These donor-specific high-avidity CTLs are regarded as the destructive cells capable of causing graft damage. To monitor the precursors of these cells (CTLp) in young and adult AVC recipients, in vitro quantitative tests were performed on sequentially taken blood samples to quantitate CTLp frequencies and their avidity for donor antigens. METHOD: Six children and nine adults who received a cryopreserved AVC in the period between 1994 and 1997 were included in the study. From these patients, two to six blood samples were obtained up to 3 years after valve implantation. The number of circulating CTLp present within the peripheral blood mononuclear cell (PBMC) population was determined by limiting dilution analysis (LDA). The fraction of CTLp with high avidity for donor HLA class I was determined by addition of CD8 monoclonal antibodies (mAb) during the cytotoxic phase of the assay. Third-party stimulator cells were used to verify the donor-specificity of the response. RESULTS: The number of donor-specific CTLp increased significantly in the period 6-12 months after AVC implantation, while third-party-specific CTLp frequencies were not affected. Additionally, we found a significant increase of the high-avidity fraction of CTLp directed against donor antigens as early as during the first 6 months after AVC implantation. The fraction of high-avidity CTLp remained significantly higher post- compared with pre-implantation, even after 12 months. We observed no significant difference in the kinetics of CTLp frequencies between pediatric and adult AVC recipients. CONCLUSION: Implantation of cryopreserved human AVC induces an increase in the total number of circulating CTLp directed against donor HLA class I in both adults and children. The shift towards more destructive high-avidity CTLp in the peripheral blood indicates their potential damaging effect towards the heart valve allograft.

Modulation of the T cell receptor beta chain repertoire after heart transplantation.

BACKGROUND: In a previous study it was shown that pre-transplant blood transfusion was associated with a better clinical outcome after heart transplantation (HTx). In this study the effect of heart transplantation (HTx) on the T cell receptor V beta chain (TCRVbeta) repertoire was investigated. Therefore, we analyzed the TCRVbeta repertoire of patients after HTx to see whether a correlation with clinical outcome could be observed. METHODS: Patients were analyzed at four different time points: pre-HTx, less than 1 month post-Htx, between 1 month and 2.5 month post-Htx and more than 2.5 months post-HTx. CD4+ and CD8+ T cells were purified from patient peripheral blood mononuclear cells (PBMC). TCR beta chain usage was analyzed semiquantitatively by Southern blot analysis. RESULTS: HTx affected the TCRVbeta repertoire in both the CD4+ and CD8+ T cell compartments in all patients. Changes in the TCRVbeta repertoire were most pronounced within the CD8+ T cell subset. Interestingly, one patient showed modulation in TCRVbeta chain usage predominantly in the CD4+ T cell compartment. CONCLUSIONS: Modulation of TCRVbeta chain usage was detected in all patients analyzed. No clear-cut relation was observed between TCRVbeta modulation after transplantation and clinical outcome. In some cases modulations appeared to concur with observed immunological events (clinically and/or in-vitro).

Donor-specific T-cell reactivity identifies kidney transplant patients in whom immunosuppressive therapy can be safely reduced.

BACKGROUND: To reduce the side effects of long-term immunosuppressive therapy, stable renal transplant patients were routinely converted from cyclosporine to either azathioprine or mycophenolate mofetil. Thereafter, the azathioprine and mycophenolate mofetil dose was reduced to 75% at 4 months and to 50% at 8 months after conversion. We questioned whether the T-cell reactivity before conversion was able to predict which patients could be safely converted and tapered in their immunosuppressive load, while remaining free from acute rejection. METHODS: Before conversion, the T-cell reactivity of peripheral blood mononuclear cells against donor and third-party spleen cells were tested in mixed lymphocyte cultures. We measured the frequency of donor and third-party reactive helper T-lymphocyte (HTLpf) and cytotoxic T-lymphocyte (CTLpf) precursors and their avidity for HLA class I antigens using limiting dilution analysis. Peripheral blood mononuclear cells were also stimulated with tetanus toxoid to test the general immune response. RESULTS: The tetanus toxoid response, reactivity to donor and third-party cells as measured in mixed lymphocyte cultures and HTLpf, and the avidity of cytotoxic T-lymphocyte precursors were not predictive for the development of acute rejection. However, significant differences were found in donor-specific CTLpf before conversion, between patients with and without acute rejection after conversion in immunosuppression. The donor-specific CTLpf was significantly lower in patients without compared to those with acute rejection (P=0.01). Additionally, when no CTLpf was detectable before conversion, acute rejection did not occur after conversion. Acute rejection was only diagnosed in patients with detectable CTLpf before conversion. CONCLUSION: The number of donor-specific cytotoxic T-lymphocytes identifies patients in whom the immunosuppressive load can be safely reduced.

Donor-derived soluble HLA plasma levels can not be used to monitor graft rejection in heart transplant recipients.

OBJECTIVE: Increased levels of both donor- and recipient-derived HLA class I molecules (sHLA-I) can be found in serum or plasma of transplanted patients during rejection. Earlier data indicate that levels of donor-derived sHLA-I (dsHLA-I) correlate better with graft rejection than total sHLA Class I (Zavazava N, Kraatz E, Gassel AM, Muller-Ruchholtz W. Plasma MHC class I expression in cardiac graft patients: donor-specific soluble antigen in a pre-sensitized graft patient. Transplant Proc 1991;23:2258-2260; Claas FHJ, Jankowska-Gan E, DeVito LD, et al. Monitoring of heart transplant rejection using a donor-specific soluble HLA class I ELISA. Hum Immunol 1993;37:121). Therefore, quantifying donor-derived soluble counterparts of HLA Class I (sHLA-I) in the plasma of the recipient may offer a new possibility for non-invasive monitoring of rejection after organ transplantation. METHODS: In an extended study with 34 heart transplant recipients, we used sHLA-I specific ELISAs to monitor donor-derived soluble sHLA-A2, -A3, -A9, -B7, -B12 and B51. RESULTS: The assays were sensitive enough to detect dsHLA Class I in plasma of the recipients. However, the levels of sHLA were not found to be a useful tool for monitoring rejection. Rejection was often associated with low levels of donor sHLA. The recent finding that antibodies can inhibit the detection of sHLA molecules might explain this discrepancy. In order to test this hypothesis, patient sera were screened for the presence of anti-HLA antibodies and the results were related to the donor-derived sHLA levels. Only in four out of 34 patients HLA Class I specific antibodies could explain the low sHLA levels during rejection. CONCLUSIONS: In heart transplantation increased donor-derived sHLA levels are not a suitable marker for rejection and that antibody formation can not explain these results. Therefore, monitoring rejection episodes on the basis of donor-derived soluble HLA molecules is not a realistic approach to decrease the number of biopsies after heart transplantation.

A longitudinal study of TGF-beta1 protein levels in renal allograft recipients converted from CsA to MMF or AZA.

Cyclosporine (CsA) is thought to enhance transforming growth factor (TGF)-beta1 production in vitro and in vivo and this may have a negative effect on long-term graft survival. Therefore, we studied TGF-beta1, plasma levels in 30 patients before kidney transplantation, after transplantation during CsA treatment and after conversion from CsA to azathioprine (AZA) or mycophenolate mofetil (MMF). We questioned whether TGF-beta1 plasma levels would decrease after the discontinuation of CsA and whether the TGF-beta1 plasma levels did correlate with CsA trough levels and kidney function, measured by serum creatinine levels. TGF-beta1 plasma levels measured 1 yr after transplantation were lower compared to levels measured before transplantation, however not significantly (p = 0.08). After conversion from CsA to MMF or AZA, a slight increase was observed in some patients, but in the total group TGF-beta1 levels remained unaffected. No correlation was found between the TGF-beta1 levels and CsA trough levels nor with creatinine levels. In conclusion, we did not observe higher TGF-beta1 plasma levels in plasma levels of patients receiving CsA treatment compared to blood from the same patients while on AZA or MMF.

T helper frequencies in peripheral blood reflect donor-directed reactivity in the graft after clinical heart transplantation.

We describe the usefulness of a fast (48-h) limiting dilution assay (LDA) for the enumeration of human alloreactive helper T lymphocytes (HTL) in the peripheral blood, in relation to histologically defined rejection grades after heart transplantation. HTL frequencies (HTLf) in pretransplant samples varied from patient to patient, ranging from 106 to 625 HTL/106 peripheral blood mononuclear cells (PBMC). In the first week after heart transplantation (HTx), when immunosuppression was instituted, HTLf were significant lower (range 30-190 HTL/106). The level of HTL in the first week after HTx when rejection grade was 0 or 1A (ISHLT score) was considered to be the baseline frequency. This frequency did not correlate with the number of subsequent rejection episodes. During rejection (grade 3), donor-specific HTLf were increased above their baseline frequencies (P = 0.01). Expressed as percentage of baseline frequencies, HTLf increased significantly during acute rejection (AR) compared with 1-2 weeks before rejection (P = 0.003). The increase was specific, since viral infections did not result in a rise of donor-specific HTL, while also HTLf specific for third party HLA antigens were not elevated during rejection. Monitoring HTLf in peripheral blood with a shortened (48-h) assay may serve as a non-invasive method for detecting intragraft immunological reactivity. Demonstrating absence of donor-specific reactivity may limit the number of invasive endomyocardial biopsy (EMB) procedures and allow tapering of immunosuppressive treatment.

A size-matching heterotopic aortic valve implantation model in the rat.

BACKGROUND: Structural failure of cardiac valve allografts may be related to technical factors such as size mismatch, resulting in early intimal proliferation and fibrosis or immunological reactions against the transplanted valves, featuring lymphocytic infiltration. OBJECTIVE: To develop a heterotopic aortic valve implantation model in the rat to study the immunological factors leading to graft failure in the setting of a technical adaptation for size mismatch. METHODS: Syngeneic (WAG-WAG or DA-DA) and allogeneic (WAG-BN or WAG-DA) rat strain combinations were used to study the effect of the allogeneic response on valve properties. An end-to-side anastomosis was made between the U-shaped aortic root graft and the recipient's abdominal aorta to resolve the problems of size matching. RESULTS: No animals suffered from ischemic or neurological complications during the study period. One hundred percent survival and patency of the aortic grafts were achieved at the end of a 21-day observation period. In the syngeneic group 9 of 10 valves were still competent when assessed during retrograde injection. In contrast, 2 of 10 allogeneic valve grafts were competent on postoperative Day 21. Microscopic evaluation revealed no fibrosis or intimal thickening in the syngeneic valve grafts while the allogeneic valve grafts demonstrated rejection-like morphology. CONCLUSION: The absence of fibrosis and intimal thickening in the syngeneic transplanted valve grafts indicates that this implantation model is not influenced by nonimmunological-based structural changes. Therefore, this new model enables us to study the association between donor-directed immune responses and allograft degeneration in a technically unbiased manner.

Kinetics of circulating cytotoxic T lymphocyte precursors that have a high avidity for donor antigens: correlation with the rejection status of the human cardiac allograft.

Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7-11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating donor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the immune status of the cardiac allograft.

C1.7 monoclonal antibody designates high-avidity CD4+ cytotoxic T lymphocytes involved in clinical heart rejection.

BACKGROUND: It is assumed that not all donor-specific cytotoxic T lymphocytes (CTLs), but only those with a high avidity for donor antigens, can function as terminal effector cells in transplant rejection. METHODS: In the present study, we searched for markers that would exclusively designate these high-avidity CTL. RESULTS: FACS analysis of donor-specific CTL clones obtained from heart transplant patients revealed that high- and low-avidity CTL varied in their expression of p38, a surface molecule involved in signal transduction, which is stained by the antibody C1.7. High- and low-avidity CD8+ CTL and high-avidity CD4+ CTL expressed p38, whereas low-avidity CD4+ CTL did not. Noncytotoxic and naive CD4+ lymphocytes also lacked p38 surface expression. CONCLUSION: Therefore, we conclude that p38 is a marker for CD4+ lymphocytes with the potency to damage the transplanted heart. Accordingly, p38 might be used to analyze the contribution of CD4+ CTL in immune responses, such as transplant rejection.

Donor-specific CTL frequencies in peripheral blood in relation to graft vascular disease after clinical heart transplantation.

Cellular mechanisms may play a role in the development of graft vascular disease (GVD). We previously demonstrated that GVD correlated with an increase of donor-specific T-helper 1 cytokine production by graft-infiltrating lymphocytes but not by peripheral blood mononuclear cells (PBMC). These T-helper 1 cytokines aid the generation of cytotoxic T-lymphocytes (CTL). In the present report, we investigated whether there is a relationship between the frequency of donor-specific CTL precursors (pCTL) in PBMC and the development of GVD. We tested PBMC samples of five patients with GVD and five patients without GVD in the periods 3-6 months, 1 year, and 3 years after heart transplantation. At all time points, GVD was not related to the number of pCTL. In conclusion, donor-specific cellular tests in peripheral blood could not be related to GVD. Apparently, donor-specific reactions associated with the induction of GVD can only be monitored in the graft.