A broad and strong humoral immune response to donor HLA after implantation of cryopreserved human heart valve allografts

Cryopreserved human heart valves are used for valve replacement in patients with congenital or acquired heart disease. Although no blood group or human leukocyte antigens (HLA) matching is performed and no immunosuppression is administered, the clinical results are relatively good. After valve replacement, the majority of the patients develop HLA antibodies, whereas a smaller group of patients shows valve-related events at the long term after right ventricular outflow tract reconstruction. Therefore, we hypothesized that not the mere presence, but rather the titers of antidonor HLA antibodies may be related to valve allograft failure. The presence and specificity of HLA class I antibodies were determined by complement-dependent microlymphocytotoxicity (CDC) test in longitudinally taken peripheral blood samples of 35 valve allograft recipients. In eight patients with an antibody response specific against donor-HLA class I, the titers were measured by this CDC method after stepwise dilution of the plasma. Panel reactive antibodies of more than 10% were found in 31 of 35 (89%) valve allograft recipients. From these 31 patients, 24 (77%) developed donor-specific HLA class I antibodies. All eight selected patients had detectable donor-specific antibody titers, ranging from 1:2 to 1:8,000. Two donor valve recipients before retransplantation had (donor-specific) HLA antibodies and showed high titers of 1:256 and 1:8,000 shortly after the second allograft valve replacement, which was associated with an early graft failure in the latter patient. We conclude that transplantation of cryopreserved human heart valve allografts leads to a broad and strong humoral response, which is probably the result of a lack of immunosuppressive therapy after valve transplantation. Patients receiving a second or following valve allograft appeared to be sensitized and developed early and high allo-antibody titers after second valve allograft implantation. Valve failure was diagnosed in a patient with extremely high titers. These findings suggest that preoperative cross-matching may identify patients with high donor-specific HLA antibody titers and may reduce the risk for early recurrent graft failure.     

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.     

The blood transfusion effect: experimental aspects

In some animal models of organ transplantation, notably the rat, preoperative administration of donor strain blood may produce long-term allograft survival even in the absence of adjunctive immunosuppressive therapy. The ability of blood transfusion to prevent rejection is highly dependent on the strain combination and type of graft, as well as the nature, dose and timing of transfusion. Although the effect is donor-specific, partial sharing of MHC and/or minor antigens between the blood and organ donor may be sufficient to prolong graft survival. The mechanisms underlying the enhancing effect of blood transfusion and other protocols which lead to specific unresponsiveness in the adult animal are undoubtedly complex and still poorly understood. In contrast to neonatal tolerance, where there is complete clonal deletion or anergy, lymphocytes from enhanced animals often show normal alloreactivity in vitro. Blood transfusion provokes an accelerated immune response to an allograft with rapid leukocyte infiltration of the graft and associated induction of class I and class II MHC target antigens. Moreover, graft infiltrating cells obtained from non-rejecting grafts in transfused recipients show levels of in vitro specific cytotoxicity equivalent to or in excess of those found in rejecting grafts. Despite the heightened cellular response provoked by blood transfusion, host regulatory mechanisms override the rejection response and there is substantial evidence for the existence in transfused animals of T cells with antigen specific suppressor activity. However, the site in the rejection process at which suppression is mediated is unknown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulated donor-specific T-cell reactivity during successful tapering of immunosuppression after kidney transplantation

Stable cadaveric renal transplant patients were routinely converted from cyclosporin A (CsA) to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation to reduce the side effects of long-term immunosuppressive therapy. Thereafter, the AZA and MMF dose was gradually tapered to 50% at 2 years after transplantation. We questioned whether a reduction of immunosuppressive treatment results in a rise of donor-specific T-cell reactivity. Before transplantation (no immunosuppression), 1 year (high dose immunosuppression) and 2 years (low dose immunosuppression) after transplantation, the T-cell reactivity of peripheral blood mononuclear cells (PBMC) against donor and third-party spleen cells was tested in mixed lymphocyte cultures (MLC) and against tetanus toxoid (TET) to test the general immune response. We also measured the frequency of donor and third-party reactive helper (HTLpf) and cytotoxic (CTLpf) T-lymphocyte precursors in a limiting dilution assay. Donor-specific responses, calculated by relative responses (RR = donor/third-party reactivity), were determined. Comparing responses after transplantation during high dose immunosuppression with responses before transplantation (no immmunosuppression), the donor-specific MLC-RR (P = 0.04), HTLp-RR (P = 0.04) and CTLp-RR (P = 0.09) decreased, while the TET-reactivity did not change. Comparing the responses during low dose with high dose immunosuppression, no donor- specific differences were found in the MLC-RR, HTLp-RR and CTLp-RR, although TET-reactivity increased considerably (P = 0.0005). We observed a reduction in donor-specific T-cell reactivity in stable patients after renal transplantation during in vivo high dose immunosuppression. Tapering of the immunosuppressive load had no rebound effect on the donor-specific reactivity, while it allowed recovery of the response to nominal antigens.     

In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.     

Longitudinal study of the frequency of cytotoxic T cell precursors in kidney allograft recipients

Clonal deletion or inactivation of donor-specific alloreactive cells are important mechanisms that are believed to account for acquired immune tolerance in allograft recipients. Serial assessment of precursor cytotoxic T lymphocyte frequencies (CTLpf) by limiting dilution analysis (LDA) provides information at the clonal level on changes in the alloimmune response of graft recipients. We performed a longitudinal study of 15 cadaveric kidney recipients before and every 3 months throughout the first year after transplantation (Tx). Pre-Tx values of donor CTLpf showed high interindividual variability without a predictive value for the clinical outcome. All patients with well functioning kidneys had decreased CTLpf at 3 months post-Tx in comparison with pre-Tx values. This decrease was donor-specific in four patients and was permanent in two cases throughout the study. Most patients presented decreased anti-donor CTLpf values from 6 to 9 months, whereas a partial recovery of donor CTLpf was observed in three patients. Reversible acute rejection was diagnosed in three patients, and it was associated with a marked increase in anti-donor CTLpf, returning to pre-Tx values by 9 months post-Tx. In addition, one patient with chronic rejection displayed a transient increase in CTLpf 6 months after Tx. The results of this sequential study indicate the establishment of a state of either hyporesponsiveness or functional clonal inactivation, transient or permanent, which could facilitate allograft acceptance.     

Immunology of renal allograft rejection

Allograft rejection remains the critical problem of renal transplantation. The immunologic mechanisms that underlie renal allograft rejection are heterogeneous and involve the humoral and cellular limbs of the immune response. Antibody-mediated hyperacute rejection is now rare owing to improved prospective cross-matching. Chronic rejection, characterized by intrarenal arterial fibrosis, is still poorly understood. Knowledge of the afferent and efferent processes involved in rejection has led to effective therapeutic and experimental strategies that employ monoclonal antibodies and other pharmacologic agents to reverse, or prevent, acute allograft rejection. In addition, allospecific tolerance has been achieved experimentally and clinically in a variety of manners. Preliminary studies on the mechanism of allograft tolerance induced by donor-specific blood transfusions before transplantation suggest a role for an immunoregulatory cell population that specifically down regulates cytotoxic lymphocyte responses to donor antigens in some recipients. A role for noninherited maternal antigens and anti-idiotypic antibodies in down regulating immune responses to allografts have also been reported by several studies. An improved understanding of allograft rejection and tolerance may identify approaches to prolong allograft survival without the morbidity and mortality associated with present-day immunosuppression.     

Desensitization for solid organ and hematopoietic stem cell transplantation

Desensitization protocols are being used worldwide to enable kidney transplantation across immunologic barriers, i.e. antibody to donor HLA or ABO antigens, which were once thought to be absolute contraindications to transplantation. Desensitization protocols are also being applied to permit transplantation of HLA mismatched hematopoietic stem cells to patients with antibody to donor HLA, to enhance the opportunity for transplantation of non-renal organs, and to treat antibody-mediated rejection. Although desensitization for organ transplantation carries an increased risk of antibody-mediated rejection, ultimately these transplants extend and enhance the quality of life for solid organ recipients, and desensitization that permits transplantation of hematopoietic stem cells is life saving for patients with limited donor options. Complex patient factors and variability in treatment protocols have made it difficult to identify, precisely, the mechanisms underlying the downregulation of donor-specific antibodies. The mechanisms underlying desensitization may differ among the various protocols in use, although there are likely to be some common features. However, it is likely that desensitization achieves a sort of immune detente by first reducing the immunologic barrier and then by creating an environment in which an autoregulatory process restricts the immune response to the allograft.     

Follicular T helper cells and humoral reactivity in kidney transplant patients

Memory B cells play a pivotal role in alloreactivity in kidney transplantation. Follicular T helper (Tfh) cells play an important role in the differentiation of B cells into immunoglobulin-producing plasmablasts [through interleukin (IL)-21]. It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney transplant patients, therefore we investigated the absolute numbers and function of peripheral Tfh cells (CD4^POSCXCR5^POS T cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA), and the presence of Tfh cells in rejection biopsies. After transplantation peripheral Tfh cell numbers remained stable, while their IL-21-producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral Tfh cells still had the capacity to induce B cell differentiation and immunoglobulin production, which could be inhibited by an IL-21-receptor-antagonist. After transplantation the quantity of Tfh cells was the highest in patients with pre-existent DSA. In kidney biopsies taken during rejection, Tfh cells co-localized with B cells and immunoglobulins in follicular-like structures. Our data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu.     

Interactions between bronchoalveolar lymphocytes and macrophages in heart-lung transplant recipients

Bronchoalveolar lavages (BAL) were obtained from heart-lung transplant patients. Following transplantation, the number of lymphocytes and macrophages are considerably increased. BAL lymphocytes frequently exhibit donor specific secondary allogeneic proliferation measured in primed lymphocyte testing (PLT) assays. We report our findings regarding a persistence of donor derived macrophages and lymphocytes in BAL during the posttransplant period. The presence of donor specific macrophages causes a proliferative response of alloreactive BAL lymphocytes from the recipient. This "Bronchoalveolar Macrophage Lymphocyte Reaction," or "BMLR," may represent a unique aspect of in vivo interactions associated with lung allograft responses. Comparative studies showed considerably lower PLT responsiveness of peripheral blood lymphocytes than that of BAL lymphocytes. These studies suggest that functional assays on BAL cells may be useful in monitoring lung transplant rejection and other immunological phenomena that affect pulmonary function of heart-lung transplant patients.     

冷冻保存异体软骨移植免疫排斥反应的研究状况

背景：目前多采用冷冻保存方法来降低异体软骨移植免疫排斥反应,但有关异体软骨的取材、冷冻保存方法、冷冻保存条件仍然需要深入的研究探讨。 目的：对于冷冻保存异体软骨移植后免疫排斥反应机制的研究进行回顾分析,并对不同保存方法的特点进行比较分析。 方法：由第一作者检索1990/2008 PubMed数据及万方数据库有关异体软骨移植后免疫排斥反应及冷冻保存方法对软骨移植影响等方面的相关文献。 结果与结论：异体软骨组织移植治疗关节软骨缺损治疗效果明显优于其他治疗方法。冷冻保存异体关节软骨保持了软骨组织的性状和生物学活性,而且可择期完成关节重建,并且有充裕的时间完成多项指标检测,防止供体携带细菌病毒和传染性疾病的传播,并且降低了软骨组织的抗原性,具有较大的临床应用价值。但冷冻保存的各个环节,如:低温保护剂的应用、降温和复温速度等方面还存在诸多问题,软骨移植后仍然会出现软骨吸收、退变等现象。随着冷冻生物学的不断进步,冷冻损伤机制的不断揭示,这些问题终将会解决,软骨组织冷冻保存技术会得到进一步的完善。     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

Limiting dilution analysis of alloreactive T-cell status and distribution during allograft rejection

We have developed limiting dilution analysis techniques that discriminate between precursor T cells and T cells that have responded to prior contact with alloantigens. We used these techniques to monitor the behavior of functional T-lymphocyte subsets in sponge matrix allograft recipients. Thus, we were able to measure at the graft site and/or in the peripheral blood (1) all donor-reactive cytotoxic T lymphocytes, (2) the subset of donor-reactive cytotoxic T lymphocytes that has responded to graft alloantigens in vivo, (3) all donor-reactive helper T lymphocytes, and (4) the subset of donor-reactive helper T lymphocytes that has responded to graft alloantigens in vivo. In this communication, we describe some aspects of the in vivo T-cell response to allograft implantation that were revealed by this study.     

Relationship of donor-specific mixed lymphocyte culture reactivity to graft function in recipients of cadaveric renal allografts.

The response of twelve cadaveric renal allograft recipients was serially studied in one-way MLC using the specific donor spleen lymphocytes as stimulating cells. The stimulation index (SI) progressively decreased between the second and the eighteenth post-transplantation week in the presence of normal plasma. The appearance of MLC non-reactivity (SI of less than 2.3) correlated well with the achievement of excellent graft function. In nine recipients allograft rejections have not occurred once the non-reactive state in cultures with normal plasma was established. In two of the recipients reactivity reappeared after an interval of non-reactive phase. In both instances rejection followed such reactivity and non-reactivity followed successful management of rejection. In one patient severe irreversible allograft rejection occurred in spite of the appearance of an early MLC non-reactive phase. This patient developed donor-specific lymphocytotoxic antibodies and his rejection was perhaps of antibody mediated type. Donor-specific MLC reactivity may represent cellular immune response of host to an allograft and predict cellular allograft rejection.     

Alloreactivity and the predictive value of anti-recipient specific interleukin 2 producing helper T lymphocyte precursor frequencies for alloreactivity after bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) is a treatment modality with the potential of curing otherwise lethal diseases. The predominant indications for BMT are haematological malignancies. In BMT alloreactivity plays a pivotal role for the outcome. Graft-versus-host disease (GvHD) and graft-versus-leukaemia (GvL) are correlated manifestations of alloreactivity. Severe GvHD is one of the main causes of morbidity and mortality post-BMT. In the absence of GvL the risk of relapse is high. The main effector cells are T lymphocytes. Donor leukocyte infusion (DLI) for treatment of leukaemic relapse after BMT can induce durable remissions. DLI causes GvHD in the majority of the responding patients. However, a GvL effect may be present without evidence of GvHD and vise versa. The importance of alloreactivity for the treatment outcome prompted the interest for a predictive test of alloreactivity. Interleukin 2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis (LDA) in the graft-versus-host direction were explored. The HTLp assay was optimized and the sources of error minimized to ensure sensitive and reproducible results. The IL-2 dependent cell line, CTLL-2 was optimized to detect 0.6 pg IL-2. UV-B irradiation of the cells was demonstrated to effectively terminate proliferation of the responder cells and thus allow IL-2 to be detected in the whole culture volume. The design of the assay was explored by Monte Carlo simulations resulting in a design yielding frequencies with a coefficient of variation of 20% in the range of 1:20,000-1:1,000,000. The influence of autoreactivity of the donor and recipient cells was minimized as well as the risk of the stimulator cells producing IL-2. The HTLp frequencies correlated with the degree of human leukocyte antigen (HLA) disparity and the assays were able to detect minor histocompatibility antigen mismatches. The HTLp frequencies of 28 HLA-identical sibling BMT pairs and 20 HLA-matched unrelated and partially HLA-matched related BMT pairs were determined. HTLp frequencies from the HLA-identical sibling BMT pairs had a median of 1:557,362 (range 1:9.511 to < 1:2,500,000). The HTLp frequencies from the HLA-matched unrelated and partially HLA-matched related BMT pairs had a median of 1:88,110 (range 1:4.139-1:736,123). Analysis of the HLA-identical sibling BMT pairs in a high and a low HTLp frequency group above and below 1:500,000 showed a trend towards a higher risk of acute GvHD > or = grade II and a significantly higher risk of chronic GvHD in the high HTLp frequency group. This group had a significantly lower risk of relapse as well as a significantly better overall survival and leukaemia free survival. The HLA-matched unrelated and partially HLA-matched related BMT pairs were split evenly in a high and a low HTLp frequency group above and below 1:90,000. There was a significantly higher risk of acute GvHD > or = grade II and a trend towards a higher treatment related mortality (TRM) in the high HTLp frequency group. There were no differences in chronic GvHD, risk of relapse, overall survival and leukaemia free survival. Analyzing all 48 patients the risk of acute GvHD > or = grade II and TRM was significantly higher with HTLp frequencies > 1:100,000 and there was a trend towards a higher risk of relapse with low HTLp frequencies < 1:400,000. Patients in the intermediate HTLp frequency group 1:100,000-1:400,000 had a trend towards improved survival. The HTLp frequency seems to detect clinically significant differences in alloreactivity, that can be useful in donor selection, graft-engineering, T cell add-back and the pharmacological immunosuppression used after BMT.     

Transplantation tolerance in heart transplant recipients as demonstrated by unresponsiveness in cell-mediated lympholysis.

Transplantation tolerance or adaptation to an allograft is associated with unresponsiveness to donor-specific transplantation antigens measured in in vitro cell-mediated lympholysis (CML). We here demonstrate in a longitudinal follow-up that CML nonreactivity develops in seven of ten patients following heart transplantation. The first manifestation of this nonreactivity manifested between 3 and 27 months after transplantation. CML nonreactivity correlated with time after transplantation and the percentage of activated lymphocytes in peripheral blood. CML nonreactivity was also associated with good graft function, i.e., in condition of nonresponsiveness patients did not manifest acute rejection. The only exception was seen in one patient in whom the immunosuppressive therapy was strongly reduced. A more detailed evaluation of this patient indicated that the underlying mechanism for CML nonreactivity is clonal anergy or active suppression of the alloreactive cells.     

Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristics as a means of monitoring the development of rat small bowel allograft rejection.

This investigation used flow cytometry to monitor peripheral blood lymphocyte morphology after rat small bowel transplantation. Preliminary studies demonstrated that in vitro activated peripheral blood lymphocytes exhibited increased cell size and granularity as measured by flow cytometric analysis of forward (FSc) and side (SSc) light scatter characteristics. The formation of distinct 'activated' light scatter regions by such lymphoblastoid transformation occurred concomitantly with up-regulated p55IL-2R expression. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats without immunosuppression. Animals receiving isografts served as controls. Peripheral blood lymphocyte subsets were identified using appropriate MoAbs, and the light scatter characteristics of each cell subset were determined by backgating strategies. Increased proportions of activated alpha/beta T cell receptor (TCR)-positive cells could be detected in allografted animals as early as day 2 post-transplantation. B cells showed peak activation by day 4, at which time the proportion of activated cells was over two-fold greater than that seen in untransplanted animals--few activated B cells were detected in isografted animals. Resting natural killer (NK) cell light scatter regions only partially overlap with those of resting T and B lymphocytes, but in allografted animals almost the entire NK population fell outside the resting lymphocyte gate by day 2 post-transplantation, an activation state which was maintained until day 4. These findings associate peripheral blood cell subset lymphoblastoid transformation with developing small bowel allograft rejection. Importantly, changes were detected early and prior to the onset of overt rejection. These data suggest that analysis of peripheral blood lymphocyte light scatter properties may provide an insight into in vivo immune status after small bowel transplantation.     

In Situ Effector Mechanisms in Rat Kidney Allograft Rejection: III. Kinetics of the Inflammatory Response and Generation of Donor-directed Killer Cells

We have isolated and identified the infiltrating inflammatory cells from rejecting rat kidney allografts. The first host cells lo appear in the graft, already a few hours after the transplantation are monocytes and lymphocytes. Both T and B lymphocytes contribute to the infiltrate: at early stages of rejection most of the infiltrating lymphocytes have the high electrophoretic mobility of (resting) T cells, whereas later during the rejection most of the infiltrating lymphocytes display the slow mobility of (resting) B cells. The blast response follows 2 days after the influx of lymphocytes. The (B) plasmablast response lakes place somewhat earlier and is higher in magnitude than the (T) lymphoblast response. Macrophages appear 1.5 days after the influx of monocytes. The inflammatory cells proliferate rapidly: after 1 h of pulse-labelling with ³H-TdR in vivo up to 24% of the infiltrating leucocytes are labelled. Most labelled cells are blast cells or lymphocytes. although a small but distinct population of labelled monocytes is also detected in situ. The in situ blast and proliferative responses precede the corresponding responses in the host central lymphatic system, i.e. spleen, blood and lymph nodes. The inflammatory leucocytes arc isolated from the allograft parenchymal cells via I e velocity sedimentation. They are strongly and specifically cytotoxic in the 6 h ⁵¹Cr release assay to donor-derived lymphoid target cells in vitro. The peak in situ cytotoxic activity in the graft lakes plate already on day 5 after the transplantation, whereas in the central lymphatic system the cytotoxic cells are detected later and peak values are obtained only after the activity In situ has declined. The findings emphasize the role of the graft as the site of sensitization of kidney transplantation (peripheral sensitization) and the complex nature of the inflammatory response responsible for allograft rejection.     

Transplantation of hematopoietic stem cells for induction of unresponsiveness to organ allografts

Although it has been recognized since the early days of Owen and Medawar that engraftment of donor stem cells, induced in utero spontaneously or intentionally neonatally, results in life-long unresponsiveness to donor alloantigens. However, successful induction of transplantation tolerance in adult life still represents an unsolved problem. Engraftment of donor stem cells using conventional modalities involves intensive myeloablative or lymphoablative immunosuppression, which is associated with toxicity and mortality and such methods are not suitable for organ allograft recipients. In this chapter, we present an innovative approach for induction of donor-specific unresponsiveness to bone marrow and organ allografts without myeloablative conditioning. Our methods is based on cyclophosphamide-induced, alloantigen-primed lymphocyte depletion. Cyclophosphamide is administered 1 day following infusion of donor hematopoietic cells, thus eliminating predominantly host T lymphocytes reacting against donor cell challenge, and resulting in relative unresponsiveness to donor alloantigens. Subsequently, life-long tolerance to fully mismatched donor skin allografts can be accomplished by a second infusion of stem cells from the same donor, with donor T cells displacing residual alloreactive host cells that may have escaped deletion. Taken together, we believe that induction of true permanent and specific tolerance to organ allografts using donor hematopoietic cells could become a clinical reality in the foreseeable future.