Witold Rybka

Bone marrow augmentation of donor-cell chimerism in kidney, liver, heart, and pancreas islet transplantation

We have previously postulated that donor cell chimerism in organ transplantation is needed to attain a tolerant state. Here we show that donor cell chimerism can be augmented in organ recipients if they are infused perioperatively with 3 x 10(8) per kg of unmodified donor bone marrow cells and are kept on a conventional immunosuppressive regimen of tacrolimus (FK506) and prednisolone. 36 patients took part, of whom the first 18 patients have good transplanted kidney (n = 10), liver (n = 7), and heart (n = 7) function when followed up between 4 and 16 months. All patients are well. We found persistent multilineage leucocyte chimerism in blood of 17 recipients by flow cytometry and PCR techniques to detect donor alleles or Y chromosomes in female recipients of male organs. The use of the 5-antigben HLA matched same sex donor precluded detection of chimerism in one patient.

Relationship of CD34+ cell dose to early and late hematopoiesis following autologous peripheral blood stem cell transplantation

We evaluated early and late hematopoietic reconstitution in 27 patients with advanced lymphoma, Hodgkin’s disease, and breast or ovarian cancer after treatment using high-dose/myeloablative conditioning regimens and autologous peripheral blood stem cell (PBSC) transplantation. Eighteen patients (67%) received G-CSF 5 μ g/kg/day following chemotherapy and nine (33%) were mobilized using G-CSF alone. Each patient had 7 × 108 mononuclear cells (MNC) per kg collected. G-CSF was administered post-PBSC infusion. While all patients showed prompt granulocyte recovery by day 14, platelet recovery failed to occur in four (15%) heavily pretreated patients with non-Hodgkin’s lymphoma. Retrospective analysis in 17 patients revealed that the infused number of CD34 surface antigen-positive cells correlated with time to granulocyte (r = 0.59, P = 0.012) and platelet (r = 0.58, P = 0.021) recovery. Patients receiving the higher numbers of CD34+ cells had consistently better hematologic parameters at all times examined. At 180 days post-transplant, the median Hb level was 124 g/l vs 88 g/l (P = 0.004); platelet count was 202 × 109/l vs 25 × 109/l (P = 0.004); and neutrophil count was 3100 × 106/l vs 1400 × 106/l (P = 0.15). Hemoglobin strongly correlated with the CD34+ cell dose at 360 days (r = 0.90, P = 0.01). We conclude that graft CD34+ cell content appears to be an indicator of the quality of late as well as early hematopoietic function.

Bone Marrow Contributes to Epithelial Cancers in Mice and Humans as Developmental Mimicry

Bone marrow cells have the capacity to contribute to distant organs. We show that marrow also contributes to epithelial neoplasias of the small bowel, colon, and lung, but not the skin. In particular, epithelial neoplasias found in patients after hematopoietic cell transplantations demonstrate that human marrow incorporates into neoplasias by adopting the phenotype of the surrounding neoplastic environment. To more rigorously evaluate marrow contribution to epithelial cancer, we employed mouse models of intestinal and lung neoplasias, which revealed specifically that the hematopoietic stem cell and its progeny incorporate within cancer. Furthermore, this marrow involvement in epithelial cancer does not appear to occur by induction of stable fusion. Whereas previous claims have been made that marrow can serve as a direct source of epithelial neoplasia, our results indicate a more cautionary note, that marrow contributes to cancer as a means of developmental mimicry.

Posttransplant adoptive immunotherapy with activated natural killer cells in patients with metastatic breast cancer.

Relapse after high-dose chemotherapy is the main cause of therapeutic failure in patients with metastatic breast cancer. Adoptive immunotherapy with interleukin-2 (IL-2) plus activated natural killer cells may eliminate residual disease without excessive toxicity. The authors sought to determine if immunotherapy immediately after transplantation would affect engraftment and the toxicity associated with transplantation. Fifteen consecutive patients with metastatic breast cancer were allocated to three cohorts. Cohort 1 (five patients) received high-dose cyclophosphamide, thiotepa, and carboplatin (CTCb) followed by peripheral blood stem cell infusion and granulocyte colony-stimulating factor at 10 micrograms/kg. Cohort 2 (five patients) received in addition rhIL-2 (2 x 10(6) IU/m2/day) for 4 days intravenously via continuous infusion after peripheral blood stem cell infusion. In cohort 3 (five patients), peripheral blood stem cell transplant was followed by infusion of autologous activated NK cells and rhIL-2 (2 x 10(6) IU/m2/day) for 4 days (via continuous intravenous infusion). Generation of activated NK cells was possible in all patients in cohort 3. All patients has successful engraftment. Median time to absolute neutrophil count more than 0.5 x 10(9)/L was 8 days (range, 8 to 11 days) in cohort 1, 9 days (range, 7 to 11 days) in cohort 2, and 9 days (range, 8 to 9 days) in cohort 3. Median time until the platelet count was more than 20 x 10(9)/L was 14 days (range, 9 to 22 days) in cohort 1, 11 days (range, 6 to 14 days) in cohort 2, and 12 days (range, 11 to 21 days) in cohort 3. All patients developed neutropenic fevers, but the overall toxicity associated with the infusion of IL-2 (cohort 2) or IL-2 plus activated NK cells (cohort 3) did not differ from that observed in cohort 1. Complete responses were achieved in one patient in cohort 1, in two patients in cohort 2, and in one patient in cohort 3. In conclusion, post-transplant adoptive immunotherapy with activated NK cells plus IL-2 is feasible, well tolerated, and does not adversely affect engraftment.

Combined simultaneous kidney/bone marrow transplantation.

On the basis of observations in patients with long-term (28-30 years) renal allograft survival, all of whom had evidence of systemic microchimerism, we began a program of combined simultaneous kidney/bone marrow transplantation. Between 12/14/92, and 10/31/94, 36 kidney transplant recipients received 3-5 x 10(8) unmodified bone marrow cells/kg; 6 patients also received pancreatic islets, and 7 patients also received a pancreas. The mean recipient age was 39.0 +/- 10.8 years, and the mean donor age was 31.8 +/- 16.1 years; the mean cold ischemia time was 23.0 +/- 9.1 hr. Twenty control patients received kidneys alone, mainly because of refusal by the donor family to consent to vertebral body recovery; 3 of these patients also received a pancreas. The mean recipient age was 47.9 +/- 11.7 years, and the mean donor age was 41.5 +/- 17.9 years; the mean cold ischemia time was 28.6 +/- 6.2 hr. All patients received tacrolimus-based therapy, without radiation, cytoreduction, or induction antilymphocyte preparations. Blood was drawn prior to and at regular intervals after transplantation for detection of chimerism and for immunologic studies. With a mean follow-up of 11.1 +/- 5.8 months, all 36 study patients are alive, and 33 (92%) have functioning allografts with a mean serum creatinine of 1.9 +/- 1.2 mg/dl and a BUN of 26 +/- 9 mg/dl. Graft vs. host disease was not seen in any patient. The incidence of rejection was 72%; 11% of the patients required OKT3 or ATG for steroid-resistant rejection. The incidence of CMV was 14%, and that of delayed graft function was 17%. A total of 18 (90%) control patients are alive, and 17 (85%) have functioning allografts, with a mean serum creatinine of 2.1 +/- 1.3 mg/dl, and a BUN of 30 +/- 13 mg/dl. The incidence of rejection was 60%, and 10% required OKT3 or ATG. CMV was seen in 15%, and delayed graft function in 20% (P = NS). In the study patients, chimerism was detected in the peripheral blood of 30 of 31 (97%) evaluable patients by either PCR or flow cytometry. In the control patients, chimerism was seen in 9 of 14 (64%) evaluable patients (P < .02). Decreasing donor-specific responsiveness was seen in 6/29 (21%) evaluable study, and 4/14 (29%) evaluable control patients (P = NS). We conclude that combined kidney/bone marrow transplantation is associated with acceptable patient and graft survival, augmentation of chimerism, and no change in the early events after transplantation.

T-Cell Immunoglobulin and ITIM Domain (TIGIT) Associates with CD8+ T-Cell Exhaustion and Poor Clinical Outcome in AML Patients

Purpose: T-cell immunoglobulin and immunoreceptor tyrosine–based inhibitory motif (ITIM) domain (TIGIT) is a recently identified T-cell coinhibitory receptor. In this study, we aimed to determine the clinical impact of TIGIT in patients with acute myelogenous leukemia (AML) and dissect the role of TIGIT in the pathogenesis of leukemia progression. Experimental Design: TIGIT expression on T cells from peripheral blood collected from patients with AML was examined by flow cytometry. The correlation of TIGIT expression to clinical outcomes, including rate of complete remission and relapse post-allogeneic stem cell transplantation (alloSCT) in AML patients, was analyzed. Phenotypic and functional study (cytokine release, proliferation, killing, and apoptosis) of TIGIT-expressing T cells were performed. Using siRNA to silence TIGIT, we further elucidated the regulatory role of TIGIT in the T-cell immune response by dissecting the effect of TIGIT knockdown on cytokine release and apoptosis of T cells from AML patients. Results: TIGIT expression on CD8+ T cells is elevated in AML patients and high-TIGIT correlates with primary refractory disease and leukemia relapse post-alloSCT. TIGIT+ CD8+ T cells display phenotypic features of exhaustion and exhibit functional impairment manifested by low production of cytokines and high susceptibility to apoptosis. Importantly, their functional defects are reversed by TIGIT knockdown. Conclusions: TIGIT contributes to functional T-cell impairment and associates with poor clinical outcome in AML. Our study suggests that blockade of TIGIT to restore T-cell function and antitumor immunity may represent a novel effective leukemia therapeutic. Clin Cancer Res; 22(12); 3057–66. ©2016 AACR.

Pure red cell aplasia following ABO-incompatible bone marrow transplantation: response to erythropoietin.

Allogeneic bone marrow transplants with major ABO incompatibility may be associated with delayed erythroid engraftment. A case of a male patient with erythroleukemia (blood group O) who received a bone marrow transplant from an HLA‐identical sibling (blood group AB) is reported. The bone marrow transplantation was followed by normal myeloid and megakaryocytic engraftment, but pure red cell aplasia was present for more than 230 days after bone marrow transplant. Despite documentation of an elevated endogenous erythropoietin level (360 mU/mL; normal value, < 19 mU/mL) during the period of absent erythropoiesis, erythroid engraftment was observed soon after the initiation of human recombinant erythropoietin at a dose of 50 U per kg daily. This experience suggests that high‐dose erythropoietin may stimulate sufficient production of erythroid precursors to overcome circulating inhibitors resulting in the correction of pure red cell aplasia.

PD-1(hi)TIM-3(+) T cells associate with and predict leukemia relapse in AML patients post allogeneic stem cell transplantation.

Prognosis of leukemia relapse post allogeneic stem cell transplantation (alloSCT) is poor and effective new treatments are urgently needed. T cells are pivotal in eradicating leukemia through a graft versus leukemia (GVL) effect and leukemia relapse is considered a failure of GVL. T-cell exhaustion is a state of T-cell dysfunction mediated by inhibitory molecules including programmed cell death protein 1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3). To evaluate whether T-cell exhaustion and inhibitory pathways are involved in leukemia relapse post alloSCT, we performed phenotypic and functional studies on T cells from peripheral blood of acute myeloid leukemia patients receiving alloSCT. Here we report that PD-1hiTIM-3+ cells are strongly associated with leukemia relapse post transplantation. Consistent with exhaustion, PD-1hiTIM-3+ T cells are functionally deficient manifested by reduced production of interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). In addition, these cells demonstrate a phenotype consistent with exhausted antigen-experienced T cells by losing TN and TEMRA subsets. Importantly, increase of PD-1hiTIM-3+ cells occurs before clinical diagnosis of leukemia relapse, suggesting their predictive value. Results of our study provide an early diagnostic approach and a therapeutic target for leukemia relapse post transplantation.

Combined kidney/bone marrow transplantation : evidence of augmentation of chimerism

Recent studies on patients who have maintained stable hepatic and renal allograft function for 10–28 years have revealed systemic microchimerism in all (1, 2). With the belief that this is the essential mechanism of allograft acceptance, we sought to augment the spontaneous chimerism in newly transplanted recipients, and began a program of combined simultaneous kidney/bone marrow transplantation without pretransplant radiation or other cytoreduction therapy (3). Postoperative immunosuppression was with FK506 and prednisone, using these drugs in the same way as in standard practice (4). Seventeen cadaver kidney recipients were entered into the bone marrow augmentation series up to June 1994. All are well. We report here the first 10, whose simultaneous kidney-bone marrow transplantations were between December 14, 1992 and December 14, 1993. Two of them also received pancreatic islets. The mean recipient age was 41.3 ± 13.7 years (range 24.6–63.4). All patients were undergoing their first transplantation and had a panel-reactive antibody level of less than 15%. The mean number of HLA matches and mismatches was 2.4 ± 1.3 (range 1–5) and 3.2 ± 1.8 (range 1–6), respectively. The mean donor age was 25.0 ± 7.5 years, and the mean cold ischemia time was 26.5 ± 9.4 hr. Eight control patients undergoing their first cadaveric kidney transplantation but not receiving bone marrow were also studied. The most common reason for not performing the combined procedure in the control cases was refusal of the donor family to consent to vertebral body recovery. Their mean age was 44.0 ± 11.0 years (range 29.7–59.8). The mean number of HLA matches and mismatches was 3.5 ± 1.9 (range 1–6) and 2.4 ± 2.0 (range 0–5). The mean donor age was 50.3 ± 12.4 years, and the mean cold ischemia time was 31.3 ± 3.6 hr. Neither irradiation nor any other kind of recipient preconditioning was used in the bone marrow recipients. All blood transfusions, if needed, were with irradiated packed red blood cells. After completion of the renal transplant procedure, 3 × 108 unmodified bone marrow cells/kg, isolated from the donor vertebral bodies, were infused via central line (3). Bone marrow infusion was performed at the time of closure of the transplant incision, or shortly thereafter in the recovery room. In the 2 patients who received islets, isolation was according to previously described techniques (5). The islets were infused into the portal vein, after completion of the kidney transplant. All of the protocols were approved by the Institutional Review Board of the University of Pittsburgh. Just prior to transplantation, 40 ml of blood was drawn and placed in heparinized tubes. Postoperatively, blood was drawn weekly for the first month, and monthly thereafter, for chimerism studies, which included fluorescent activated cell sorter analysis (FACS), polymerase chain reaction (PCR), and Y-chromosome analysis by both competitive PCR (cPCR) and fluorescent in-situ hybridization (FISH) of cytospin samples (in the case of female recipients of kidneys from the male donors). The mean follow-up for the 10 kidney/bone marrow recipients is 9.9 ± 4.7 months: all are alive and well, and all have functioning allografts. The mean serum creatinine and BUN are 1.6 ± 0.4 mg/dl, and 23 ± 8 mg/dl. Five (50%) patients have been weaned off steroids. The two islet recipients remain on insulin; they both have evidence of C-peptide production (0.44 and 0.11 pmol/ml), but at levels insufficient to allow for insulin independence. Two (20%) patients experienced early kidney nonfunction and required dialysis during the first postoperative week. The incidence of acute rejection was 50% (5 patients). All rejection episodes were responsive to steroids and/or an increase in the FK 506 dosage. No patient required antilymphocyte therapy. Cytomegalovirus (CMV) was seen in 2 (20%) patients and was treated successfully with intravenous gancyclovir. Graft vs. host disease (GVHD) was not seen any patient. The control recipients of a kidney only have been followed for 4.9 ± 0.5 months; all are alive and have functioning allografts (Table 1). The mean serum creatinine and BUN are 2.4 ± 1.0 mg/dl and 50 ± 32 mg/dl. None of these patients has been weaned off prednisone as of yet—however, this is most likely related to the relatively short follow-up. Two (25%) patients had acute tubular necrosis (ATN), although only 1 (13%) required dialysis during the first week after transplantation. The incidence of acute rejection was 63% (5 patients). All rejections were responsive to steroids and an increase in FK506. CMV was seen in 2 (25%) patients; both patients were successfully treated with gancyclovir. GVHD was not seen. TABLE 1 Summary of clinical course In the kidney/bone marrow patients, evidence of chimerism was present by at least one of the three modalities in 9 of 9 (100%) evaluable patients (Table 2). The tenth patient, who received a 1-B, 2-DR matched same-sex allograft, was not evaluable for microchimerism by any of these technologies. FACS was positive in 5 of 7 patients in whom evaluation was feasible, with a range of 0.9–3.0%. PCR for HLA disparities was positive in 8 of 8 patients in whom the study could be performed (Fig. 1). In the three female recipients of kidneys from male donors, Y-chromosome detection by PCR was positive in all cases and in one, competitive PCR (6) demonstrated a concentration of 0.5% donor DNA in PBMCs at 1 year. Using FISH for Y-chromosome detection, a level of 0.2–1.4% was seen (Fig. 2). FIGURE 1 Polymerase chain reaction (PCR) in a kidney/bone marrow recipient with a disparity for HLA-A29, demonstrating persistent chimerism. FIGURE 2 Fluorescent in-situ hybridization (FISH) of a cytospin preparation of PBMCs of a female recipient of a kidney/bone marrow from a male donor: orange represents X-chromosome; green represents Y-chromosome. TABLE 2 Testing for microchimerism In the kidney alone patients, 3 of the 8 patients could not be evaluated for microchimerism by any modality, because of complete DR matching and lack of donor sex disparity. In the 5 evaluable patients, 3 (60%) were positive for microchimerism by PCR, 1 by HLA disparity, and 2 by Y-chromosome analysis. However, FACS failed to detect microchimerism in any of 3 evaluable patients by 3 months after transplantation. Donor-specific reactivity could not be accurately assessed in 3 of the ten kidney/bone marrow recipients because there was complete DR-matching and a decreased in vitro immunologic response both pre- and post-transplantation. In the other 7 patients studied over time, there was one example of donor-specific nonresponsiveness and 2 of decreased donor-specific responsiveness that waxed and waned (Fig. 3). The other 4 patients retained donor-specific responsiveness. Only 5 of the 8 kidney-alone recipients were evaluable. One of the 5 has some evidence of decreasing responsiveness. FIGURE 3 Serial MLR responses of a kidney/bone marrow recipient. These results and those with 7 subsequently treated kidney-bone marrow recipients have shown that kidney/bone marrow transplantation is straightforward to perform and is safe. There were no examples of GVHD. Stable chimerism estimated to be 1000-fold more dense than that occurring spontaneously (3) was regularly achieved. It should be emphasized that (as was expected) bone marrow infusion did not influence the early events after transplantation. The incidence of acute rejection, the rates of early nonfunction, and the number cytomegalovirus infections were similar to those seen in patients receiving kidneys alone. However, treatment of these early problems was straightforward. After passing through this phase of convalescence, which was equally volatile with or without bone marrow augmentation, the more densely chimeric cohort of kidney-marrow recipients appeared to be an advantaged group. There was 100% patient and graft survival, and early completion of steroid weaning in half the cases. The full benefit of the chimeric state is expected to take one or 2 years, or longer. This trial was begun with patients undergoing primary transplantation who had low PRAs. We are now broadening our indications for kidney/bone marrow transplantation to include candidates undergoing retransplantation and/or those with high PRAs in order to assess the utility of adjuvant bone marrow in a more complex transplant setting. Historically important clinical attempts to facilitate kidney graft acceptance have been reported using adjunctive preoperative donor blood transfusions (7) or delayed (by 3 weeks) cryopreserved cadaveric donor bone marrow (8, 9). Unlike the premises of these earlier trials, our hypothesis was that long-term engraftment of the infused donor cells could occur without “making space” by host preconditioning, without an undue risk of GVHD, and using the same immunosuppressive strategy that fostered the previously unrecognized spontaneous development of microchimerism (1–3). The observations in the kidney recipients herein reported, as well as in recipients of livers and hearts (3) are confirmatory of these predictions.

Busulfan and cyclophosphamide (BU/CY2) as preparative regimen for patients with lymphoma.

The combination of busulfan and cyclophosphamide has seldom been employed as a conditioning regimen for patients with lymphoma. Twenty patients with relapsed or refractory lymphoma were treated with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (BU/CY) followed by peripheral blood stem cell rescue in 19 patients or autologous bone marrow in one patient. There were 12 females and eight males, with a median age of 48 years (range 30–65). Four patients had Hodgkin’s disease, and 16 patients had non-Hodgkin’s lymphoma. Disease status at the time of BU/CY was: first relapse in 10 patients (four patients with chemosensitive disease and six patients with chemoresistant disease), primary refractory disease in six patients, and more advanced disease in four patients. Excessive treatment-related toxicity was not noted. There were no cases of interstitial pneumonitis, but three cases of veno-occlusive disease occurred. At 2 years, the estimated overall survival and event-free survival are 50% and 33%. We concluded that BU/CY seems to have sufficient anti- lymphoma activity, is devoid of excessive toxicity and warrants further investigation in this patient population.