Further Assessment of Rosette Inhibition Test in Clinical Organ Transplantation

The rosette inhibition test was used in the clinical management of organ allografts to estimate the amount of immunosuppressive drugs necessary to prevent rejection. In patients surviving more than three months renal function appeared to be better than in a similar group of patients managed without the test. It is suggested that this was due to a reduction in the number of clinical or subclinical rejection episodes. On the other hand, the test indicates that in many cases the level of immunosuppression should be much higher, and if this advice is followed the patients become increasingly exposed to the risk of infection. In other words, those patients with good renal function remained well, whereas those who might otherwise have rejected their kidney and survived had in fact died of sepsis.     

Immunosuppressive drug-free operational immune tolerance in human kidney transplant recipients: Part I. Blood gene expression statistical analysis

Survival of solid organ grafts depends on life-long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous "operational tolerance" can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross-validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis.     

Spontaneous rosette and rosette-inhibition tests on fresh and cryopreserved lymphocytes.

Peripheral blood lymphocytes from 13 healthy adults were compared in the fresh state and after controlled freezing in liquid nitrogen. The mean percentage of cells forming spontaneous rosettes with sheep red blood cells was 52% in the fresh samples and 57% in frozen-thawed material. These figures are not significantly different. Fresh and frozen cells proved equally sensitive to the action of an antilymphocyte globulin preparation in inhibiting rosette formation. It is concluded that valid results may be obtained through the use of frozen preserved lymphocytes in rosette and rosette-inhibition tests.     

Differential inhibitory effect of iron on E, EA, and EAC rosette formation

The effect of ferric citrate on active E, EA, and EAC rosette formation by human peripheral blood mononuclear (PBM) cells was examined. Active E and EAC rosette formation was significantly inhibited at four of the five concentrations tested. Inhibition of EAC rosette formation was generally lower than that observed with active E rosette formation. Treatment with Fe³⁺ had no effect on EA rosette-forming cells.     

ES-cell derived hematopoietic cells induce transplantation tolerance

Background

Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells.

Methodology/Principal Findings

Here, we derived CD45⁺ HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts.

Conclusions

Our data show, for the first time, the efficacy of ES-derived CD45⁺ HPCs to engraft in allogenic recipients without the use of immunosuppressive agents, there by protecting cardiac allografts from rejection.     

Acid hydrolase activity of the blood in immunodepression

Immunosuppressants reduce the activity of acid RNase and acid phosphatase in the blood serum of mice and patients with kidney allografts. In reversible graft rejection crises, the activity of both enzymes ascends in the period preceding the crisis or at its height. However, following intensive immunosuppressive therapy it drops again. In irreversible crises, the activity of acid hydrolases goes on ascending in spite of immunosuppression intensification. The time course of the activity of these enzymes correlates with changes in "active" and "total" T-rosette-forming cells.     

Clinical outcome following nerve allograft transplantation

The clinical outcome of seven patients who underwent reconstruction of long upper- and lower-extremity peripheral nerve gaps with interposition peripheral nerve allografts is reported. Patients were selected for transplantation when the nerve gaps exceeded the length that could be reconstructed with available autograft tissue. Before transplantation, cadaveric allografts were harvested and preserved for 7 days in University of Wisconsin Cold Storage Solution at 5 degrees C. In the interim, patients were started on an immunosuppressive regimen consisting of either cyclosporin A or tacrolimus (FK506), azathioprine, and prednisone. Immunosuppression was discontinued 6 months after regeneration across the allograft(s) was evident. Six patients demonstrated return of motor function and sensation in the affected limb, and one patient experienced rejection of the allograft secondary to subtherapeutic immunosuppression. In addition to providing the ability to restore nerve continuity in severe extremity injuries, successful nerve allografting protocols have direct applicability to composite tissue transplantation.     

Spontaneous cytotoxicity of human peripheral blood mononuclear cells toward red blood cell targets. II. Time-dependent loss of suppressor cell activity.

In a previous paper we demonstrated that human peripheral blood mononuclear cells become strikingly cytotoxic toward a wide variety of red blood cell targets after 7 days of in vitro culture. The cell responsible for cytotoxicity does not rosette with SRBC and demonstrates both surface adherence and phagocytic properties. In this paper we wish to show that development of spontaneous cytotoxicity is due to a time-dependent loss of suppressor cell function. Fresh autologous lymphocytes, when added to cultured cells, abrogate the subsequent expression of spontaneous cytotoxicity toward RBC targets. The suppressor cell is radioresistant; requires 24 hr to suppress optimally; is inactivated by heating at 56 degrees C for 15 min, and is enriched in the non-T interface after SRBC rosette depletion over a discontinuous Ficoll-Hypaque gradient. Furthermore, the addition of a cell-free sonicate of fresh lymphocytes is capable of inhibiting spontaneous cytotoxicity toward RBC targets. However, if mononuclear cells are allowed to incubate in tissue culture medium for 7 days they are no longer suppressive after sonication. These data suggest that fresh mononuclear cells exert a potent negative regulatory influence on monocyte killing. Our culture conditions by removing this negative influence have produced a new model of spontaneous nonspecific killing by monocytes.     

Suppressor cell activity of peripheral mononuclear cells from patients undergoing chronic haemodialysis

The enhanced in vitro proliferative response of peripheral mononuclear cells (PMNC) when pre-cultured for 24 hours prior to the addition of Concanavalin-A has been used as an indirect parameter of suppressor cell activity in healthy subjects and in patients undergoing chronic haemodialysis. The proliferative response of PMNC from haemodialysis patients is lower than that of control subjects when Con-A is added at the initiation of culture but approximates to that of normal subjects when the addition of Con-A was deferred for 24 hours. The Suppressor Indices of PMNC from haemodialysis patients were at least as great and sometimes greater than those of control subjects but the absolute T-cell counts were lower in haemodialysis patients than in controls. These results suggest that the relative energy of haemodialysis patients is partly attributable to T-cell depletion but this is accompanied by retention, and in some cases augmentation, of suppressor cell activity.     

Dynamic expression of Qa-2 during acute graft rejection

Human leukocyte antigen (HLA)-G exhibits immunotolerogenicity and is related to allograft acceptance. Qa-2 is the murine homolog of HLA-G; it has structure and functions similar to those of HLA-G. We investigated the dynamic expression of Qa-2 in skin allografts by immunohistochemistry and on peripheral blood lymphocyte subsets by flow cytometry during the entire process of acute graft rejection (AGR) with a murine skin transplantation model to determine its relationship with the pathological changes of allografts and the influence of immunosuppressive therapy. In grafts without immunosuppressive treatment, Qa-2 did not exhibit obvious changes in syngeneic and allogeneic recipients. In contrast, with immunosuppressant-treated grafts, positive expression of Qa-2 was observed. It remained at high levels in the immunosuppressant-treated syngeneic group; however, it became weakly positive and even negative in infiltrating inflammatory cells as AGR advanced, but it remained strongly positive in other skin tissues throughout the AGR process. Qa-2 expression on CD4(+) and CD8(+) peripheral blood lymphocyte subsets remained stable at a normal level in the non-immunosuppressant-treated syngeneic group. Immunosuppressive treatment can also significantly upregulate Qa-2. In the allogeneic groups, decreased expression was observed when AGR was at histological grades 1 to 2 (well before gross rejection was observed). Qa-2 was upregulated again after the graft was rejected completely. The results suggest that the increase in Qa-2 may be attributed to the use of immunosuppressive treatments. Moreover, Qa-2 expression decreased significantly with AGR progression, suggesting that it may be a potential marker for predicting AGR, especially in the presence of immunosuppressive agents.     

JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants

The field of organ transplantation has had tremendous success because of the availability of immunosuppressive drugs that efficiently prevent acute organ rejection. Numerous and severe side effects are, however, associated with all current immunosuppressive therapies and justify a search for drugs with better efficacy and safety profiles. Janus kinase (JAK) 3, a tyrosine kinase that is crucial for mediating signals from the common gamma-chain of cytokine receptors, is peculiar in that its expression, contrarily to the targets of most current immunosuppressive drugs, is limited to cells that actively participate to the immune response to allografts. The recent demonstration in stringent preclinical models that JAK3 inhibition results in efficacy for the prevention of allograft rejection with a narrow side-effect profile might lead to a new era in the field of immunosuppression.     

Rosette formation of guinea pig lymphoid cells with rabbit erythrocytes—Differences between thymocytes and peripheral blood lymphocytes

Rosette formation of guinea pig thymocytes (Th) and thymus-derived peripheral blood lymphocytes (TBL) was tested under different experimental conditions. Up to 93% of Th and 38% of TBL showed an affinity to rabbit red blood cells (RRBC). Treatment with metabolic inhibitors like sodium azide and sodium cyanide or freezing and thawing nearly abolished rosette formation by TBL but was ineffective with respect to Th. Heating the cells destroyed rosette-forming capacity of both cell types. These results indicate that spontaneous rosette formation with RRBC by Th does not require the live cell.     

OKT4/8 ratio in the blood and in the graft during episodes of human renal allograft rejection

We have analyzed the frequency of T helper (Th) and T suppressor/killer (Ts/k) lymphocytes in the blood and in the renal allograft during episodes of rejection and during quiescence. Monoclonal OKT4 and OKT8 antibodies were used to mark the Th and Ts/k cells, respectively. Density centrifugation-separated mononuclear leukocytes and FACS IV cell sorter or the Staphylococcus aureus rosette assay were used to determine the ratio in the blood, with concordant results. Fine needle aspiration biopsy (FNAB) and the Staph. assay were used to demonstrate the lymphocyte subtypes in the graft. The mean OKT4/8 ratio in the blood was significantly lower in the transplant recipients than in healthy controls (1.1 +/- 0.7 vs 1.8 +/- 0.2, respectively, P = 0.000). The individual variation was, however, high and no correlation between the OKT4/8 ratio in the blood and the inflammatory episodes in situ was observed. During 19 of the 25 episodes of inflammation, the dominant lymphocyte subtype in the graft was the Ts/k cell. In the remaining six cases it was the Th cell. All rejection episodes of the former type were reversible, in the latter type, four out of six were irreversible.     

Measles virus induced immunosuppression: targets and effector mechanisms

A profound, transient suppression of immune functions during and after the acute infection is the major cause of more than one million cases of infant deaths associated with measles worldwide. Concommittant with the generation of an efficient measles virus (MV) specific immunity, immune responses towards other pathogens are strongly impaired and provide the basis for the establishment and severe course of opportunistic infections. The molecular basis for MV-induced immunosuppression has not been resolved as yet. Similar to other immunosuppressive viruses, MV is lymphotropic and viral nucleic acid and proteins are detectable in peripheral blood mononuclear cells (PBMC). It is considered central to MV-induced immunosuppression that PBMC isolated from patients largely fail to proliferate in response to antigen specific and polyclonal stimulation. The low abundancy of MV-infected PBMC suggests that MV-induced immunosuppression is not directly caused by infection-mediated cell loss or fusion, but rather by indirect mechanisms such as deregulation of cytokines or surface contact-mediated signaling which may lead to apoptosis or impair the proliferative response of uninfected PBMC. Evidence for a role of any of these mechanisms was obtained in vitro, however, much has still to be learned about the tropism of MV and its interactions with particular host cells such as dendritic cells in vivo.     

Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α-produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA-positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA-positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.     

Differentiation between chronic lymphocytic leukaemia (B-CLL) and non-Hodgkin lymphomas in the leukaemic phase by the mouse red blood cell rosette assay

A distinction between B-CLL and other malignant B-cell lymphomas in the leukaemic phase may be difficult. Mouse red blood cell rosette formation of lymphocytes from 97 patients with B-CLL and 19 patients suffering from other B-cell lymphoproliferative disorders was examined together with lymphocyte rosette formation of healthy controls. The majority of circulating lymphocytes of B-CLL patients formed rosettes with mouse red cells, whereas there was no relationship between the number of peripheral neoplastic B-cells and that of rosette forming cells in other lymphoproliferative diseases. The relatively simple mouse red blood cell rosette assay proved to be of value in the differentiation of otherwise nearly related conditions.     

Relationship between chimerism and tolerance in a kidney transplantation model

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.     

Recovery of renal function after prolonged dialysis and transplantation.

Out of 250 patients with renal failure, seven (2.8%) treated by regular haemodialysis alone (four) or given cadaveric allografts (three) later showed recovery of function of their own kidneys lasting from one to four years. In the patients receiving haemodialysis alone recovery was easily recognised from their serum creatinine concentrations, but in those with transplants recovery was discovered unexpectedly during radionuclide scanning. These findings suggest that recovery of renal function may be more common than generally recognised, which should be borne in mind when beginning renal replacement treatment and particularly when contemplating bilateral nephrectomy.     

Evaluation of Rosette Inhibition Test in Renal Transplantation

Serial rosette inhibition tests were performed on 11 renal transplant patients in an attempt to predict graft rejection. The rosette inhibition titre was higher in immunosuppressed patients than in normal subjects. The test was of predictive value in only two out of 12 rejection episodes, where a fall in titre to normal levels occurred 48 hours and 24 hours, respectively, before biochemical evidence of rejection. In two further rejection episodes the titre fell at the time of rejection. The titre changes in all tests were small and there were frequent inconsistencies in the results of individual tests. A study of the variables was undertaken, with standardization of the technique, and improvements were made in reading the test. Despite these changes the test was still not sufficiently accurate or reliable to be used as the basis of treatment of rejection episodes.