Nonchimeric HLA-Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers.

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)-identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34(+) stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection-free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow-up including 5-year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time-dependent increases of circulating CD4(+) CD25(+++) CD127(-) FOXP3(+) Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA-identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

Safety and Efficacy of Ultra-hypofractionation in Node-positive Prostate Cancer.

AIMS: The safety and efficacy of stereotactic body radiotherapy (SBRT) in localised prostate cancer are now established through phase III randomised trials. Its utility in node-positive prostate cancer is restricted due to a lack of controlled studies specifically addressing this subgroup. Herein we report the safety and efficacy of SBRT in this subgroup. MATERIALS AND METHODS: In total, 60 patients treated with SBRT to prostate and pelvis were analysed. All patients received neoadjuvant androgen deprivation therapy for at least 6 months and long-term adjuvant hormonal therapy (70% medical and 30% surgical). All patients were treated with daily image-guided rotational intensity-modulated radiotherapy. The dose delivered to the prostate and gross node was 35-37.5 Gy and 25 Gy in five fractions to the elective pelvic nodal region on alternate days. Acute and late toxicities were graded as per Radiation Therapy Oncology Group common toxicity criteria. RESULTS: Forty-one (68%) patients had a Gleason score ≥8. The median prostate-specific antigen level at diagnosis was 39 ng/ml. Twenty (33%) patients had common iliac nodal uptake on initial prostate-specific membrane antigen positron emission tomography-computed tomography. After the median follow-up of 30 months, no acute or late Radiation Therapy Oncology Group grade ≥3 gastrointestinal toxicity was noted. Acute grade 2 genitourinary and gastrointestinal toxicities were 8.3% and 11.7%, respectively. Late grade 2 genitourinary and gastrointestinal toxicities were 3.3% and 8.3%, respectively. Late grade 3 genitourinary toxicity was seen in two (3.3%) patients. Three-year overall survival and biochemical failure-free survival was 89% and 77%, respectively. CONCLUSION: SBRT to the prostate and pelvis is safe and efficacious in node-positive prostate cancer even with common iliac nodal involvement (stage M1a).

Analysis of T cell responses in liver allograft recipients. Evidence for deletion of donor-specific cytotoxic T cells in the peripheral circulation.

Analysis of cell-mediated lympholysis in long-term liver allograft recipients indicated that there was a donor-specific unresponsiveness that could not be reversed by the addition of rIL-2 and/or mixed lymphocyte culture supernatant or by nonspecific stimulation of the cultures with PHA. Stimulation of recipient cells with semisyngeneic cells having both donor and third-party HLA antigens failed to reveal the presence of cytotoxic T cells (CTL) specific to the donor, whereas the CTL response to third-party antigens remained normal. Removal of B lymphocytes from the responding cell population did not influence the responses. Furthermore, limiting dilution analysis showed that the liver transplant recipients did not have detectable levels of CTL precursors (CTLp) reactive to the donor antigens, whereas their CTLp to third-party antigens remained normal. Donor-specific CTLp were present before and during the early post-transplant period; these cells were eliminated from the peripheral circulation by 10 mo after transplantation. Taken together, these results indicate that there is a deletion of CTLp specific to donor MHC antigens in the peripheral circulation of long-term liver allograft recipients that may account in part for the success of liver transplantation across MHC barriers.

Abrogation of the alloreactive responses of cadaveric donor intestinal lymphocytes by intraoperative Campath-1H exposure.

A number of recent reports in clinical and experimental intestinal transplantation have suggested that the donor lymphocytes present in and around the gastrointestinal system are potent mediators of graft-versus-host (GvH) reactivity and that GvH responses may contribute to posttransplant morbidity. Therefore, we have tested the proliferative and cytotoxic capabilities of gut-associated lymphocytes from cadaveric donors obtained prerevascularization (pre-r) and about 6 hours postrevascularization (post-r) in recipients pretreated with Campath-1H antibody (alemtuzumab). Mixed lymphocyte reactions (MLR) were performed with lymphoid cells isolated from intestinal epithelial mucosa, lamina propria, and lymph nodes. The pre-r lymphocytes responded strongly to both the recipient and third-party alloantigenic stimulating cells. However, similar preparations from the post-r samples responded in MLR at significantly lower levels (P < .01). This post-r decrease in responsiveness was not observed in similar lymphocyte samples obtained from donors of recipients not treated with Campath-1H. Both the pre-r and post-r samples had similar flow cytometric profiles, suggesting that there was no receptor loss in these lymphoid tissues by the short-term 6-hour exposure to Campath-1H given to the recipient. Conversely, in preliminary experiments where the donor were treated with Campath-1H, it was observed that very few lymphocytes could be obtained from intestinal tissues (n = 3). These results suggested that Campath-1H treatment of the recipient could bring about a drastic reduction in an otherwise strong GvH reactivity by the donor intestinal immune cells.

Development of autoantibodies to T cell clonotypic structures in a liver-kidney allograft recipient.

To elucidate the mechanism of human liver allograft rejection and acceptance, T cell clones were established from liver biopsies of a liver-kidney transplant recipient during a rejection episode. Five of the clones were characterized and found to be CD4+, alpha/beta positive T cells that proliferated specifically to the mismatched donor HLAs. Using an immunofluorescence assay, it was observed that three of these clones were recognized by autologous antibodies developed during the post-transplant period between 18 months and the end of the testing period of 36 months. Furthermore, by capping experiments, it was determined that these antibodies were recognizing the CD3-TCR complex. This was confirmed by immunoprecipitation and sequential immunoprecipitation followed by SDS-PAGE and autoradiography of lysates of radiolabeled T cell clones. Thus, our data indicate that donor-reactive T cells infiltrate into the liver allograft during rejection episodes, and that autologous antibodies reactive to the CD3-TCR complex of these cells are developed in the post-transplant period. These results support the hypothesis that the development of auto-antibodies directed against or cross-reactive to the clonotypic structures of the donor-reactive lymphocytes may play an important role in the down-regulation of the immune response against the allograft.

A polymorphic human kidney-specific non-MHC alloantigen. Its possible role in tissue-specific allograft immunity.

Tissue specific non-MHC alloantigens play a crucial role in allograft immunity. However, their structural properties have remained elusive, largely due to their inability to induce a strong antibody response. We report the characterization of a monkey heteroantiserum, MHK-I, raised against human kidney cells, that serologically reacts specifically with kidney cells after extensive absorptions of anti-HLA class I and II reactivities. The non-MHC MHK-I-binding molecule(s) is expressed only in the renal cortex on the glomerulus, peritubular capillaries, venous endothelium, and tubular epithelium. Immunochemically, MHK-I recognizes a kidney-specific non-MHC alloantigen of Mr 90,000 to 100,000 (90 kD). These properties of MHK-I are similar to those of the previously characterized alloantibodies eluted from rejected kidneys. These alloantibodies bind to the kidney from which the antibody was eluted and to a few others but are unlike MHK-I, which binds to extracts prepared from all human kidneys. Biochemical analysis by two-dimensional electrophoresis (pI ranging between 4.5 and 5.5) and peptide fingerprinting provide further evidence that the alloantigen is polymorphic. These findings imply that the non-MHC kidney-specific molecule(s) may function as target(s) for immune destruction of renal allografts.

Modulatory effects of human donor bone marrow cells on allogeneic cellular immune responses.

BACKGROUND: In order to evaluate whether immunoregulatory mechanisms are brought about by human donor bone marrow cell infusions accompanying organ transplantation, we established in vitro culture systems analogous to the transplant model. METHODS: Cell-mediated lympholysis (CML) and mixed lymphocyte culture (MLC) responses of peripheral blood lymphocytes or spleen cells stimulated with irradiated cadaver donor spleen cells in the presence of specific donor vertebral-body bone marrow cell (DBMC) modulators were tested. RESULTS: When compared with spleen cells as modulator controls, DBMC inhibited both the proliferative and cytotoxic responses in a dose-dependent manner. Use of unirradiated and T cell-depleted DBMC was required for detection of the inhibitory activity. Furthermore, DBMC had to be added within the first 2 days after the initiation of the cultures for the down-regulation of CML (MLC) to occur. The down-regulation of MLC responses could not be shown to be antigen (donor) specific. Physical separation of DBMC from the responder-stimulator cells using the transwell system abrogated modulation of the CML (and MLC) reactions, suggesting the requirement of cell-cell contact for modulatory effect. The inhibitory activity by DBMC could not be overcome by addition of up to 200 U/ml of exogenous recombinant interleukin 2 to the cultures. However, it could be abrogated by restimulation with donor spleen cells, indicating that donor reactive cells were not deleted by DBMC in short-term cultures. CONCLUSIONS: These results showed a regulatory role for DBMC in cellular immune responses against donor cell alloantigens, supporting the rationale for DBMC for facilitating clinical allograft acceptance.

Biochemical and immunological evaluation of donor-specific soluble HLA in the circulation of liver transplant recipients.

MHC antigens, normally expressed as integral membrane proteins, are also present in soluble form in the peripheral circulation. These soluble human leukocyte antigens (sHLA) are found at elevated levels in patients with a variety of infections as well as in organ transplant recipients. In liver transplant recipients, however, most of the circulating sHLA are of donor phenotype, especially during the early posttransplant period. Here we report the purification and characterization of sHLA of both recipient and donor origin from liver transplant recipients. It was observed that sHLA consisted of four major polypeptides having molecular mass of 44, 41, 35-37, and 12 kD complexed with IgM and IgG antibodies. Further analysis revealed that these immunoglobulins contained anti-HLA antibodies. Analysis of the affinity-purified materials by a number of approaches failed to detect any other fragment(s) of HLA class I heavy chain polypeptides smaller than 12 kD. No significant difference was observed in the biochemical nature of the sHLA of donor and recipient origin and they were similar to those found in normal individuals. Affinity-purified HLA-A3 inhibited the cytolytic activity of an HLA-A3-specific CD8+ T cell line, whereas, purified sHLA-A2 failed to inhibit anti-HLA-A3 CTL activity. Further, the proliferation of the T cell line was not inhibited by sHLA-A3. Thus, the inhibitory activity shown by sHLA was antigen-specific and directed against a functional subset of T lymphocytes. These results support the notion that sHLA may play an important regulatory role in the immune response to allograft in humans.

Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses.

BACKGROUND: The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. METHODS: Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. RESULTS: Compared with spleen cells as controls, un-irradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports). The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34+ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38+, CD2+, CD5+, and CD1+ lymphoid cells (all were depleted of CD3+ cells) as well as CD33+ (but CD15 negative) myeloid precursors. However, positively selected CD19+ B-cells and CD15+ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3+ TcR-alphabeta+ CD4+ and CD28+ cells. CONCLUSION: A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.

In vitro immunogenicity of cadaver donor bone marrow cells used for the induction of allograft acceptance in clinical transplantation.

BACKGROUND: The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions to induce allograft acceptance in clinical transplantation is not fully understood. Aside from acting as immune responding and regulatory cells, the infused DBMC also may sensitize the recipient to the donor antigens. METHODS: To analyze this stimulatory activity of DBMC, in vitro mixed lymphocyte cultures (MLC) and cell-mediated lymphocytotoxicity (CML) culture systems analogous to the transplant model with DBMC infusion were used. RESULTS: When responding peripheral blood lymphocytes (PBL) from normal volunteers were placed in culture with suspensions of Ficoll-purified, T cell-depleted, un-irradiated allogeneic DBMC (NT-DBMC), a reaction was seen in both MLC and CML. However, when compared to allogeneic spleen cells as stimulating cells, the responses to NT-DBMC were of markedly lower magnitude and were not seen when the NT-DBMC was irradiated (3000 R). When responding PBL were stimulated with either NT-DBMC that had been previously cultured with irradiated cells from the responders for 1 week (activated NT-DBMC), NT-DBMC further depleted of CD15+ and glycophorin A-positive cells (NT-LP/DBMC), or purified CD34+ and CD2+ DBMC subsets, stronger lymphoproliferative and cytotoxic responses were observed. Moreover, these responses were not abrogated by irradiation of the stimulating DBMC subpopulations. Depletion of antigen-presenting cells by positive selection of CD3+ cells from the responding PBL abrogated MLC and CML reactivity, even when purified NT-LP/DBMC, the most stimulatory cells, were used. This latter observation was in contrast to the responses seen with cultures containing allogeneic stimulating spleen cell populations. This indicated the requirement for indirect alloantigen presentation, i.e., the failure of these DBMC to stimulate by direct alloantigen presentation. NT-DBMC was able to stimulate responding PBL in secondary MLC and CML responses with an equivalent magnitude, irrespective of whether the stimulators were spleen cells or NT-DBMC. Finally, the MLC and CML responses were inhibited by tacrolimus (FK506), mycophenolic acid (MPA), and cyclosporine (CsA) in a dose dependent manner, in contrast to previously observed refractoriness of DBMC preparations to these agents if DBMC was tested as responder cells or in modulatory assays. CONCLUSIONS: These results indicate that DBMC are able to function as effective in vitro stimulators, but only by indirect antigen presentation, and that the immune responses mediated by them can be down-regulated by their own inherent suppressive nature, an effect that can be enhanced by the presence of immunosuppressive drugs.

Cellular immune responses of human cadaver donor bone marrow cells and their susceptibility to commonly used immunosuppressive drugs in transplantation.

BACKGROUND: The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions in human organ transplantation, especially in the context of continuous pharmacologic immunosuppression, is not fully understood. Yet, in inbred rodents and even primates, administration of specific bone marrow cells has caused a state of acquired immunologic tolerance. METHODS: In vitro mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) culture systems were used to compare the responding and regulatory properties of DBMC and individual bone marrow cell subsets versus spleen cells in the presence or absence of pharmacologic immunosuppression. RESULTS: In the absence of immunosuppressive drugs, the DBMC proliferated in MLC and in response to phytohemagglutinin, but to a lower magnitude than donor spleen cells. In CML assays, DBMC failed to function as cytotoxic cells. Removal of both CD3+ and CD34+ cells together (not just singly) had to occur for complete abrogation of the proliferative response of DBMC evoked in the presence of allogeneic stimulating cells. Testing several experimental variables using flow cytometric analysis led to the conclusion that when purified DBMC CD34+ cells were placed in coculture with irradiated allogeneic peripheral blood mononuclear cells, such CD34+ cells give rise both to CD3- TCRalphabeta+ as well as to dimly staining CD3+ TCRalphabeta+ cells. Low pharmacologic concentrations of tacrolimus/cyclosporine (CsA) and mycophenolic acid (MPA) singly or in combination had no effect on the spontaneous proliferation of DBMC and had significantly less inhibitory activity on MLC responses of DBMC and its purified CD3+ or CD34+ subpopulations, compared with the responses of spleen cells. Moreover, the previously described regulatory effects of DBMC on the MLC responses of peripheral blood or splenic responding cells were not inhibited by these immunosuppressive drugs. CONCLUSIONS: Taken together, these results support the notion that in vitro DBMC subpopulations, which proliferate as responding cells in co-culture with x-irradiated allogeneic cells and which cause regulatory effects when added as a third component to MLC reactions, seem to be culture-generated lymphoid cell lineage(s) progeny of CD34+ cells. This possibly includes unique CD3+ "primitive" (dimly staining) T cells, which are not as inhibited in their function by tacrolimus/CsA and MPA, as are postthymic (splenic) T cells.

Donor bone marrow-derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses.

BACKGROUND: Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. METHODS: Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 living-related-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. RESULTS: The yield of RdD cells ranged from 0.1 to O.9% of the starting material with the majority being TcRalphabeta, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 23 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated alogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. CONCLUSIONS: These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.

Analysis of post-transplant immune status in recipients of liver/bone marrow allografts.

The aims of this study were to assess the effect of donor bone marrow infusion on the reactivity of recipient peripheral blood lymphocytes (PBL) to mitogen and to donor and third-party cells after primary liver allotransplantation and to identify any correlation between altered immunoreactivity and HLA mismatches, occurrence of rejection, and immunosuppression. The immunoreactivity of recipient PBL toward frozen donor splenocytes was evaluated in mixed lymphocyte culture (MLC) (n = 29) and cell-mediated lympholysis (CML) (n = 27) assays in time intervals ranging from 0.7 to 27 months after transplant. Overall, the mean anti-donor MLC stimulation index (SI) fell from 25.6 +/- 5.2 preoperatively to 4.8 +/- 1.7 after transplantation (p < 0.002), with 14 out of 29 (48.3%) patients developing donor-specific MLC hyporeactivity. HLA class II mismatches were significantly associated with recipient post-transplant immune profile (p < 0.05): MLC donor specific hyporesponsiveness was observed in 70%, versus 37% of patients who shared a class II antigen, versus those that did not. Of the control group, 61.1% developed donor-specific nonreactivity versus 27.2% in the donor bone marrow cells (DBMC) group (p = 0.02). Donor-specific CML hyporeactivity was observed after transplantation, independent of DBMC infusion, with mean percentage values of pre- and post-transplant donor-specific lysis of 22.4% +/- 4.1% versus 3.1% +/- 1.6%, p = 0.0004, respectively. Our results suggest that DBMC infusion favors development of nonspecific MLC hyporesponsiveness to donor and third-party alloantigen, with maintenance of reactivity to mitogen and no additional effect on T-cell cytotoxicity.

Functional analysis of complement receptor 1 using a new monoclonal antibody, KuN241.

A monoclonal antibody (MAb), KuN241, recognized an antigen present on all monocytes, polymorphonuclear leukocytes (PMNs), B cells, and a subpopulation of T cells. Cell surface expression pattern and immunoprecipitation studies indicated the molecule recognized by KuN241 to be complement receptor 1 (CR1). This was confirmed by sequential immunoprecipitation using E11, a CR1-specific monoclonal, and by immunoprecipitation analysis of a truncated transfection product of CR1. In vivo activation of PBLs for 7 days with pokeweed mitogen (PWM) abrogated surface expression of CR1 on B cells. Overnight culture with phorbol 12-myristate 13-acetate (PMA) downregulated the expression of CR1 detected by KuN241 both on PMNs and monocytes. Addition of MAb KuN241 to purified PMN and monocytes resulted in a transient increase in intracellular calcium ([Ca2+]i). This was blocked by the Fab fragment of MAb KuFc79 directed against the Fc gamma receptor. Further, Fab fragment of KuN241 cross-linked with F(ab')2 goat anti-mouse Ig failed to increase [Ca2+]i levels. Taken together, these results suggested a novel mechanism for transmembrane signaling events by dual binding of KuN241, the Fab region to CR1 and the Fc region of Fc gamma RII.

Primary melanoma of the pineal gland.

Primary leptomeningeal melanoma presenting as a lesion in the region of the pineal gland is rare. The MRI characteristic of this interesting condition is described and its value in studying spread or other foci is emphasized.