Peripheral CD4(+)CD25(+) TREG cell counts and the response to extracorporeal photopheresis in lung transplant recipients

Extracorporeal photopheresis (ECP) has been proposed as a possible alternative therapy for patients with bronchiolitis obliterans syndrome (BOS), with some evidence of efficacy. Although the mechanism by which ECP exerts its protective effects remains to be determined, two recent studies suggest that the modulation of transplant immune rejection may depend on the capacity to increase the number of peripheral T-regulatory (Treg) cells. We evaluated the effect of ECP treatment on the number of naturally occurring CD4(+)CD25(+) Treg cells in the peripheral blood of six lung transplant recipients: in five cases after failure of augmented or changed immunosuppression for BOS, and in one case owing to persistent acute rejection in a patient who contracted chronic hepatitis C viral infection after lung transplant. A functional stabilization was observed in three of our five patients with BOS, which was accompanied by a slight increase or stabilization of the number of peripheral blood CD4(+)CD25(high) cells with in vitro features of Treg cells. On the contrary, two patients with BOS who did not experience graft functional stabilization also showed a decline in the peripheral Treg subset. In the last patient Treg cell kinetics showed stabilization during the first 5 months of ECP treatment when lung function remained stable and graft histology normalized but showed a subsequent decrease, predating BOS diagnosis. In all, our results indicate that ECP may modulate peripheral Treg cell number but the time course of peripheral Treg cells varies according to graft function.     

Decrease of CD4+CD25+ T cells in peripheral blood after liver transplantation: association with immunosuppression

CD25 (IL-2 receptor alpha-chain) marks a population of CD4-positive T cells with a suppressor phenotype. These CD4+CD25+ regulatory T cells can suppress both effector T cells and antigen-presenting cells and have been identified as a principle regulator of tolerance in experimental transplantation models. In the setting of human liver transplantation, however, little is known about the dynamics of these cells in relation to rejection, tolerance, and immunosuppression. In the current study we determined CD4+CD25+ T cell in blood of liver transplant recipients using flow cytometry and investigated a possible link with immunosuppressive therapies. Peripheral blood mononuclear cells (PBMC) of 27 liver transplant patients (pretransplantation and 12 months posttransplantation) and 16 healthy controls were included. We found that the percentages of CD25+ cells within the CD4 T-cell population was significantly reduced in more than two-thirds of patients 1 year after transplantation. Also the total percentage of CD4-positive T cells declined significantly within this period, making the absolute reduction of regulatory T cells after transplantation even more profound. Comparing PBMC samples of patients and healthy controls revealed an increased percentage of CD4+ T cells in the patients before transplantation, probably related to the chronic liver illness. The reduction in CD4+CD25+ T cells after transplantation was similar for different immunosuppression regiments. All patients, however, received calcineurin inhibitors, suggesting a possible suppressive effect of this therapy on regulatory T-cell levels in peripheral blood. Currently, assays for regulatory T-cell activity are used to further support this hypothesis.     

The control of anti-donor immune responses by regulatory T cells in organ transplant patients

The role of regulatory T cells in the induction and maintenance of transplant tolerance has been widely investigated in experimental animal models. Their involvement in the regulation of allogeneic immune reactivity in immunosuppressed organ transplant patients, however, remains unclear. Measurements of regulatory T cells after clinical organ transplantation may contribute to understanding their role in anti-donor immune responses. Several studies have investigated the frequency and/or immune regulatory function of peripheral regulatory T-cell populations, such as, the CD4+CD25+ regulatory T cells or the CD8+CD28- suppressor T cells, in clinically stable organ transplant patients and patients with acute or chronic rejection. This review summarizes these studies and discusses the correlations found between the number and function of regulatory T cells and the immunological state of the patient. This knowledge is warranted for a better understanding of the control of allogeneic immune responses by regulatory T cells. Subsequently, monitoring regulatory T cells may help us to identify patients in whom the anti-donor reactivity is actively suppressed.     

Increased frequency of regulatory T Cells and selection of highly potent CD62L+ cells during treatment of human lung transplant recipients with rapamycin

The currently available immunosuppressive agents applied in human transplantation medicine are highly potent in the protection from acute allograft rejection. However, long‐term allograft survival is still poor as these drugs fail to sufficiently prevent chronic allograft rejection. Naturally occurring regulatory T cells have been postulated as the key players to establish long‐lasting transplantation tolerance. Thus, the development of immunosuppressive regimens which shift the pathological balance of cytopathic versus regulatory T cells of human allograft recipients towards a protective T‐cell composition is a promising approach to overcome limitations of current transplantation medicine. Thirty‐three patients that received rapamycin (RPM) or calcineurin inhibitor treatment following lung transplantation were included and their T‐cell compartments analysed. Twelve healthy volunteers without history of lung disease served as controls. In this article, we show that treatment of human lung transplant recipients with RPM is associated with an increased frequency of regulatory T cells, as compared with treatment with calcineurin inhibitors or to healthy controls. Moreover, regulatory T cells during treatment with RPM were CD62Lhigh, a phenotype that displayed an enhanced immunosuppressive capacity ex vivo. Our data support the use of RPM in human lung transplant recipients and undertaking of further prospective studies evaluating its impact on allograft and patient survival.     

Noninvasive monitoring of human cardiac allograft rejection

The frequency of donor-reactive cytolytic T lymphocytes was measured in the peripheral blood mononuclear cell population of a group of 12 cardiac allograft recipients immediately before and at various time points after transplantation. At each of the time points after transplantation the donor heart was biopsied and the rejection status of the graft was determined by applying standard histological criteria. The results of this study showed that the preoperative frequency of donor-reactive cytolytic T lymphocytes in the blood was not predictive of a future tendency toward graft rejection. However, when all the data were examined it was apparent that the frequency of donor-reactive cytolytic T lymphocytes was significantly higher (P less than 0.05) in blood samples from patients whose simultaneous biopsy showed histological evidence of acute cardiac allograft rejection than in blood samples from transplant patients showing no evidence of rejection.     

Regulatory CD4+CD25+ T cells in the peripheral blood of lung transplant recipients: correlation with transplant outcome

BACKGROUND: The subset of CD4+CD25+ regulatory T cells, recently identified in humans, may play a central role in the regulation of immune tolerance to graft survival. METHODS: This study assesses the frequency and functional profile of CD4+CD25+CD69- cells in the peripheral blood of lung transplant recipients (>3 years from transplantation), 10 of whom were in a stable clinical condition and 11 of whom demonstrated chronic rejection (bronchiolitis obliterans syndrome). We also studied a group of seven healthy subjects. RESULTS: The frequency of CD4+ T cells expressing CD25 (CD4+CD25+) and the highest levels (CD25) were lower in patients with bronchiolitis obliterans syndrome compared with healthy subjects and subjects in a stable clinical condition (P < or = 0.01). Purified CD4+CD25+ cells exhibited a regulatory functional profile in vitro: they were hyporesponsive, suppressed the proliferation of CD4+CD25- cells, and produced interleukin-10. CONCLUSION: These results provide in vivo evidence that peripheral CD4+CD25+ T cells may represent an important regulatory subset in lung transplantation.     

Impact of psoralen/UVA-treatment on survival, activation, and immunostimulatory capacity of monocyte-derived dendritic cells

BACKGROUND: Extracorporeal Photopheresis (ECP) has been shown to be an effective treatment of graft-versus-host disease, solid organ graft rejection, and other T-cell-mediated diseases. The mechanisms of action of ECP include lymphocyte apoptosis, cytokine modulation, and the induction of regulatory T cells. It has been suggested that dendritic cells (DCs) are more resistant to ECP-induced apoptosis and might be directly modulated by ECP. We tested this hypothesis using in vitro Psoralen/UVA (PUVA) treatment as an in vitro model of ECP. METHODS: Monocyte-derived DCs (mo-DCs) were treated with 8-methoxypsoralen /UVA and analyzed for surface molecule expression, apoptosis markers, endocytosis, and migratory and immunostimulatory capacity. Mo-DC phenotype and cytokine secretion was tested after CD40L stimulation. Naive T cells stimulated with PUVA-treated mo-DCs were tested for Th1/Th2 cytokine secretion and associated chemokine receptor patterns. RESULTS: DCs underwent apoptosis after in vitro PUVA and in vivo ECP. In vitro, the induction of apoptosis was preceded by partial maturation of immature mo-DCs. PUVA-treated immature mo-DCs also exhibited enhanced migratory and immunostimulatory capacity. However, mo-DCs stimulation through CD40 ligation was abrogated and interleukin (IL)-12 secretion was abolished 24 hr after PUVA treatment. PUVA-treated mo-DCs skewed naive T cells toward a Th2 response as defined by increased IL-4, IL-10, and IL-13 and decreased interferon-gamma levels, and the expression of the Th2-associated chemokine receptors CCR4 and CCR10. The observed Th2 shift was partially reversed by exogenous IL-12. CONCLUSION: These data suggest that direct modulation of DC function as well as apoptosis contribute to the immunoregulatory effects of ECP.     

Regulatory T cells and T cell depletion: role of immunosuppressive drugs

Allogeneic immune responses are modulated by a subset of host T cells with regulatory function (Treg) contained within the CD4(+)CD25(high) subset. Evidence exists that Treg expand after peritransplantation lymphopenia, inhibit graft rejection, and induce and maintain tolerance. Little, however, is known about the role of Treg in the clinical setting. IL-2 and activation by T cell receptor engagement are instrumental to generate and maintain Treg, but the influence of immunosuppressants on Treg homeostasis in humans in vivo has not been investigated. This study monitored Treg phenotype and function during immune reconstitution in renal transplant recipients who underwent profound T cell depletion with Campath-1H and received sirolimus or cyclosporine (CsA) as part of their maintenance immunosuppressive therapy. CD4(+)CD25(high) cells that expressed FOXP3 underwent homeostatic peripheral expansion during immune reconstitution, more intense in patients who received sirolimus than in those who were given CsA. T cells that were isolated from peripheral blood long term after transplantation were hyporesponsive to alloantigens in both groups. In sirolimus- but not CsA-treated patients, hyporesponsiveness was reversed by Treg depletion. T cells from CsA-treated patients were anergic. Thus, lymphopenia and calcineurin-dependent signaling seem to be primary mediators of CD4(+)CD25(high) Treg expansion in renal transplant patients. These findings will be instrumental in developing "tolerance permissive" immunosuppressive regimens in the clinical setting.     

Identification of Epstein-Barr virus-specific CD8+ T lymphocytes in the circulation of pediatric transplant recipients

BACKGROUND: Pediatric transplant recipients are at increased risk for Epstein Barr virus (EBV)-related B cell lymphomas. In healthy individuals, the expansion of EBV-infected B cells is controlled by CD8+ cytotoxic T cells. However, immunosuppressive therapy may compromise antiviral immunity. We identified and determined the frequency of EBV-specific T cells in the peripheral blood of pediatric transplant recipients. METHODS: HLA-B*0801 and HLA-A*0201 tetramers folded with immunodominant EBV peptides were used to detect EBV-specific CD8+ T cells by flow cytometry in peripheral blood mononuclear cells from 24 pediatric liver and kidney transplant recipients. The expression of CD38 and CD45RO on EBV-specific, tetramer-binding cells was also examined in a subset of patients by immunofluorescent staining and flow cytometry. RESULTS: Tetramer-binding CD8+ T cells were identified in 21 of 24 transplant recipients. EBV-specific CD8+ T cells were detected as early as 4 weeks after transplant in EBV seronegative patients receiving an organ from an EBV seropositive donor. The frequencies (expressed as a percentage of the CD8+ T cells) of the tetramer-binding cells were HLA-B8-RAKFKQLL (BZLF1 lytic antigen peptide) tetramer, range=0.96 to 3.94%; HLA-B8-FLRGRAYGL (EBNA3A latent antigen peptide) tetramer, range=0.03 to 0.59%; and HLA-A2-GLCTLVAML (BMLF1 lytic antigen peptide) tetramer, range=0.06 to 0.76%. The majority of tetramer reactive cells displayed an activated/memory phenotype. CONCLUSIONS: Pediatric transplant recipients receiving immunosuppression can generate EBV-specific CD8+ T cells. Phenotypic and functional analysis of tetramer cells may prove useful in defining and monitoring EBV infection in the posttransplant patient.     

P-glycoprotein activity is decreased in CD4+ but not CD8+ lung allograft-infiltrating T cells during acute cellular rejection

BACKGROUND: Modulation of P-glycoprotein (P-gp) activity in graft-infiltrating T cells may alter their susceptibility to immunosuppression. METHODS: P-gp activity was measured by rhodamine efflux in T-cell subsets from bronchoalveolar lavage (BAL) of five healthy volunteers and 27 lung allograft recipients. The effect of T-cell activation on P-gp activity was modeled by stimulation of peripheral blood mononuclear cells with staphylococcal enterotoxin B. RESULTS: Most BAL T cells expressed memory-effector markers. Patients had a lower proportion of CD4 T cells (P = 0.005), whereas control subjects had CD4-to-CD8 ratios similar to peripheral blood. In controls, basal P-gp activity was greatly increased in both CD4 (35% P-gp active) and CD8 (63%) lung T cells compared with peripheral T cells. Basal P-gp activity was elevated in patient BAL T cells but was lower than control BAL activity (CD4, P = 0.07; CD8, P = 0.03). Lung T cells from transplant patients had modest (CD4) or marked (CD8) increases in substrate-induced P-gp activity compared with normal lung, indicating that P-gp was not irreversibly inhibited. Patients with acute cellular rejection (ACR) had reduced P-gp activity in CD4, but not CD8, BAL T cells compared with patients without ACR (P = 0.004). To determine the relationship between T-cell activation on P-gp modulation, P-gp activity was measured in staphylococcal enterotoxin B-stimulated peripheral blood mononuclear cells. P-gp activity was abrogated in CD71 cycling cells but remained high in a persistent but minor population of resting naive T cells. CONCLUSIONS: Lung T cells have increased in vivo P-gp activity and therefore may eliminate substrate drugs, resulting in local resistance to immunosuppressive therapy. However, P-gp function is reduced during T-cell activation, providing a window of susceptibility to treatment during ACR.     

Expression of HLA molecules on peripheral blood lymphocytes: a useful monitoring parameter in cardiac transplantation

During the rejection process of cardiac allografts, the expression of HLA antigens increases on various graft tissues, ie, the myocardium and the interstitial structures. However, in this type of transplant there is a paucity of knowledge about HLA expression on recipient cells, such as peripheral blood mononuclear cells. In the present study expression of HLA class I and class II antigens was monitored on peripheral blood lymphocytes prior to and during a 12-month follow-up, using flow cytometry. In our series, the frequency of acute rejection episodes was greater from the fourth to the ninth month after transplantation, coinciding with a reduction in cyclosporine blood levels. At the same time, expression of HLA class I and class II antigens significantly increased among recipients suffering from more severe acute rejection episodes compared with those showing acceptance of their grafts (P < .01). In conclusion, acute rejection episodes in cardiac transplantation were associated with up-regulation of HLA molecules on recipient peripheral blood cells. Monitoring the expression of HLA molecules on peripheral blood lymphocytes may represent an easy, noninvasive practice to individualize immunosuppressive therapy.     

Expression of HLA-DR antigens on peripheral blood T lymphocytes and renal graft tubular epithelial cells in association with rejection

Since the expression of HLA-DR antigens on peripheral blood T lymphocytes and renal graft tubular epithelial cells may be associated with immunological stimulation, we investigated the expression of these antigens on blood T lymphocytes and graft tubular epithelium in 84 renal transplant patients. Peripheral blood T lymphocytes were monitored by flow cytometry during the first 3 months after transplantation. Since the DR+ lymphocytes were selected by a double-labeling technique used for the Leu2a phenotype, the majority of the DR+ lymphocytes were also Leu2a+ with a small percentage of unidentified Leu2a- lymphocytes. Forty-one patients were treated with cyclosporine (CsA) and 43 with azathioprine (Aza), while both groups received low-dose steroids. Frozen sections of 57 renal biopsies from 43 patients (21 on CsA and 22 on Aza) were stained for HLA-DR antigens. In the Aza group, clinical rejection episodes correlated with an increased percentage of DR+ peripheral lymphocytes (P = 0.0005), and the expression of DR antigens on graft epithelial cells (P less than 0.001). In the CsA group, no relation between the expression of DR antigens on blood lymphocytes and clinical rejection episodes was evident, and the correlation between tubular DR staining and clinical rejection episodes was weaker than in the Aza group (P = 0.03). In both the Aza and CsA group, an increase in DR+ peripheral lymphocytes correlated with positive staining of the renal tubular cells for HLA-DR antigens (P less than 0.001).     

Human leukocyte antigen-G, a new parameter in the follow-up of liver transplantation

Human leukocyte antigen-G (HLA-G) displays immunotolerogenic properties toward effector cells in graft rejection through inhibition of natural killer (NK) and cytotoxic T lymphocyte (CTL)-mediated cytolysis and CD4+ T-cell alloproliferation. CD4(+)CD25(+)high regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance of pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. The aim of this study was to investigate whether there was an association between soluble and membrane-bound HLA-G levels on Treg cells and liver graft prognosis. For this purpose, we studied 37 liver transplant patients and 13 healthy blood donors. To investigate the expression of HLA-G on the surface of peripheral mononuclear (PMNL) cells, we have used monoclonal antibodies in flow cytometry to estimate CD4, CD25, CD45, and HLA-G content. HLA-G serum levels were determined by ELISA. We observed a correlation between sHLA-G serum levels and liver function tests. After a month of HLA-G decrease in serum levels, liver function tests such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), direct bilirubin (DB), total bilirubin (TB), and alkaline phosphatase (ALP) were above normal levels, suggesting liver dysfunction or rejection. Considering these results, we concluded that the increased sHLA-G in serum and on cell surfaces may afford preliminary data on the prognosis and response to treatment in liver transplant patients.     

Reduced numbers of blood natural regulatory T cells in stable liver transplant recipients with high levels of calcineurin inhibitors

INTRODUCTION: In solid organ transplantation, most efforts are directed to achieve a state of tolerance and reduce the immunosuppressive load. Since the liver is thought to be more tolerogenic than other grafts, we examined the impact of various immunosuppressant regimens on induction of CD4+CD25(high)FOXP3+ regulatory T cells (Tregs). MATERIALS AND METHODS: We divided 35 liver transplant recipients with stable function and free of rejection episodes for at least 8 years into two main groups according to the blood levels of calcineurin inhibitors (CNIs) at the time of the study: 15 patients showing high concentrations of either cyclosporine or FK506 (high CNI: cyclosporine >80 ng/mL or FK506 >6 ng/mL) and another 20 patients with low levels (low CNI). Thirty-eight age-matched healthy subjects were used as normal controls. RESULTS: Circulating T cells of a regulatory phenotype (CD4+CD25(high)FoxP3+) were decreased in peripheral blood of liver transplant recipients compared with healthy donors. Importantly, those patients with high CNI demonstrated significantly lower levels of Tregs compared with those with low CNI. Furthermore, low CNI recipients showed no significant difference from healthy donors. CONCLUSIONS: A high load of immunosuppression may hamper the induction of tolerance in liver transplantation through interference with induction of Tregs in stable liver transplant recipients. Thus, quantification of blood Tregs may help to decide whether to lower immunosuppression.     

Alloreactivity of natural killer cells in allogeneic liver transplantation

BACKGROUND: The cytolytic attack of natural killer (NK) cells is blocked by recognition of the idiotypic phenotype of certain polymorphisms in HLA class I molecules, specifically by HLA-C alleles (Asn77, Lys80 or Ser77, Asn80) or HLA-Bw4 allotypes. Because liver allograft rejection is associated closer with mismatch in HLA class I than class II, we investigated the role of NK cells in acute hepatic allograft rejection in vivo/in vitro. METHODS: The HLA pattern was typed with serological and polymerase chain reaction (PCR) techniques. In 31 liver transplantations, mononuclear cells from donor spleen and peripheral blood of recipients (before/after transplantation) were cultured in mixed lymphocyte cultures (MLC). MLC-derived effector cells were analyzed by flow cytometry and tested in 51Cr-release assays. RESULTS: Patients with NK allospecific constellations tended to have higher numbers of NK cells in peripheral blood during the first 4 weeks after transplantation, and patients' lymphocytes stimulated with donor cells had a significantly higher cytotoxic activity on days 14 and 21 compared with patients without NK allospecificity. However, acute rejection occurred with similar frequency in both groups (31% with allospecific constellations vs. 40% without). Moreover, acute rejection episodes were not associated with an increase in NK cells in vivo or enhanced cytotoxicity of NK cells to donor target cells. CONCLUSIONS: Under standard immunosuppressive therapy, NK allospecific constellations did not seem play a major role in acute hepatic allograft rejection. Strategies to prevent or treat NK allospecific constellations after liver transplantation are not likely to reduce the incidence or severity of acute allograft rejection.     

Lung transplantation: pulmonary cell lysis mediated by alveolar mononuclear cells

Methods were developed to monitor graft rejection in a porcine model of unilateral lung transplantation. The ability of peripheral blood mononuclear cells and lavage-derived mononuclear cells to lyse donor pulmonary tissue was determined by standard chromium release assays at various times after transplantation. Effective antigraft activity was observed in the local environment of a rejecting graft, but not in the periphery. Since transplant rejection is a reversible process, with the administration of suitable immunosuppressive regimes frequently restoring graft function, it was reasoned that immunological assays based on the lysis of individual cells may not be relevant to the in vivo situation. We therefore describe an assay of the lung barrier function; perturbations of the tight intraepithelial junctions which compose the air-blood barrier can be determined in vitro by the measurement of transmonolayer resistance values.     

Induction of regulatory T cells after prophylactic treatment with photopheresis in renal transplant recipients

Extracorporeal photopheresis (ECP), originally used to treat cutaneous T-cell lymphoma, also has been applied to the therapy of transplant rejection. Our aim was to investigate the biologic response in two children who underwent kidney transplantation with ECP as prophylactic treatment. They received conventional immunosuppressive therapy and ECP immediately after transplantation: six applications over the course of 3 weeks. During a 12-month follow-up, the clinical course was favorable in both patients; renal histology was normal 6 months after transplantation. When compared with four transplanted controls, the ECP-treated patients showed lower tumor necrosis factor-alpha serum levels in the short-term and a marked increase of Foxp3-positive T-regulatory cells. T-regulatory cells were still higher than in the controls 1 year after transplantation. These preliminary results suggest that the addition of ECP to standard immunosuppressive therapy induces a tolerogenic shift in the immune system of kidney transplanted patients and may pave the way to preventing chronic rejection.     

Rapid identification of preformed alloreactive T cells for use in a clinical setting

BACKGROUND: In clinical practice, HLA matching is generally applied to minimize the incidence of graft rejection after transplantation. Recently, graft rejection has been directly associated with the presence of preformed alloreactive T cells before transplantation. Despite this knowledge, assays to rapidly quantify preformed alloreactivity are not available for use in a clinical setting. In this study, such an assay was developed and evaluated in a large cohort to correlate alloreactive T-cell reactivity with HLA matching. METHODS: Stimulator peripheral blood mononuclear cells were prestained with CD45-fluorescein isothiocyanate antibody and mixed with responder peripheral blood mononuclear cells. Activation-induced cytokine secretion was blocked using brefeldin A. After 6 hr, functionally active alloreactive responder CD4 and CD8 T cells were quantified among fluorescein isothiocyanate-negative cells by their expression of interferon-gamma on flow cytometry. RESULTS: Directly alloreactive CD4 and CD8 T cells among both stimulators and responders were easily distinguished after 6 hr of stimulation without being affected by bystander activation. Among 128 paired combinations, 23.4% of individuals had alloreactive CD8 T cells, 15.7% had alloreactive CD4 T cells, and 12.5% had alloreactivity in both T-cell subpopulations. Alloreactive T cells decreased from circulation within a few days after transplantation. In line with well-known clinical observations that associate HLA matching with graft outcome, the number of HLA-A and -B mismatches correlated with alloreactive CD8 T-cell frequencies in the whole study population, whereas it did not predict alloreactivity on an individual basis. CONCLUSION: Alloreactive T cells may rapidly be quantified after 6 hr of stimulation. Thus, the flow cytometric approach may be applied in a clinical setting to facilitate the individualization of immunosuppressive therapy and studies on the identification of patients who are at increased risk to develop graft rejection.     

CD4-veCD8-ve CD30+ve T cells are detectable in human lung transplant patients and their proportion of the lymphocyte population after in vitro stimulation with donor spleen cells correlates with preservation of lung physiology

INTRODUCTION: Survival following lung transplantation is less than 50% at 5 years, mainly due to immune-mediated chronic rejection. Recently a novel subset of T cells, CD4-veCD8-ve CD30+ve, so-called double negative (DN) CD30+ve T cells, has been described and shown to be responsible for tolerance in an animal model of skin transplantation. METHODS: We investigated 18 lung transplant recipients for the presence of DN CD30+ve T cells in resting peripheral blood and also following in vitro stimulation of recipient peripheral blood mononuclear cells (PBMCs) with donor spleen cells. RESULTS: Small percentages (0.2% to 6%) of DN T cells are detectable in resting PBMCs of human transplant patients (n = 18), but these did not correlate with allograft function, acute rejection episodes, HLA mismatch, or CMV status. On repeated stimulation of recipient PBMCs (two exposures) in vitro by donor spleen cells (2:1 ratio stimulators to responders) the percentage of DN CD30+ve T cells within the lymphocyte pool correlated with preservation of allograft lung function (both for FEV(1), P = .009, and FEF(25-75), P = .036) and was inversely correlated with grade of chronic rejection. On repeated exposure of recipient PBMCs to donor spleen cells with a 1:1 ratio the percentage of DN CD30+ve T cells correlated with the number of acute rejection episodes of grade 2 or greater. The total number of HLA mismatches correlated with the percentage DN CD30+ve T cells present after primary stimulation of recipient PBMCs with donor spleen cells (1:1 ratio). The number of mismatches at the B locus inversely correlated with the percentage of DN CD30+ve T cells after primary stimulation of recipient PBMCs with donor spleen cells (1:1 ratio; P = .031, n = 18). CONCLUSION: Percentages of DN CD30+ve T cells present following repeated stimulation of recipient PBMCs by donor spleen cells correlated with preservation of graft function following lung transplantation.