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We report a prospective phase II clinical trial in 35 adult patients (median age 40.5 years) with hematologic malignancies who received T cell–depleted, hematopoietic stem cell transplants from HLA-compatible, unrelated donors. The cytoreductive regimen consisted of hyperfractionated total-body irradiation, thiotepa, and fludarabine. The preferred graft source was granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC). PBSC were CD34+ selected, followed by sheep erythrocyte rosetting to deplete residual T cells. Anti-thymocyte globulin provided graft rejection prophylaxis. No additional graft-versus-host disease (GVHD) prophylaxis was planned. Estimated disease-free survival at 4 years is 56.8% for the entire group and 75% in patients with standard-risk disease. The cumulative incidence of relapse is 6%. Acute GVHD grade II-III developed in 9% and chronic GVHD in 29% of patients. Fatal infections occurred in 5 of 35 (14%) patients. There was 1 late graft failure. This study demonstrates durable engraftment with a low overall incidence of GVHD. Its curative potential is reflected in the remarkably low relapse rate at 4 years.

T-cell depleted allogeneic hematopoietic stem cell transplants (TCD-HSCT) have demonstrated durable disease-free survival with a low risk of graft vs. host disease (GVHD) in patients with AML. We investigated this approach in 61 patients with primary refractory or relapsed non-Hodgkin lymphoma (NHL), who underwent TCD-HSCT from January 1992 through September 2004. Patients received myeloablative cytoreduction consisting of hyperfractionated total body irradiation, followed by either thiotepa and cyclophosphamide (45 patients) or thiotepa and fludarabine (16 patients). We determined the second-line age-adjusted International Prognostic Index score (sAAIPI) prior to transplant. Median follow-up of surviving patients is 6 years. The 10-year overall (OS) and event-free-survival (EFS) were 50% and 43%, respectively. The relapse rate at 10 years was 21% in patients with chemosensitive disease and 52% in those with resistant disease at time of HSCT. Nine of the 18 patients who relapsed entered a subsequent CR. Overall survival (p=0.01) correlated with the sAAIPI. The incidence of grade II-IV acute GVHD was 18%. We conclude that allogeneic TCD-HSCT can induce high rates of OS and EFS in advanced NHL with a low incidence of GVHD. Furthermore, the sAAIPI can predict outcomes and may be used to select the most appropriate patients for this type of transplant.

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182 780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner—responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

Graft failure is associated with a high mortality rate. To date, regimens invoked for second transplants have resulted in inconsistent engraftment with high transplant-related mortality (TRM). We here report 16 consecutive patients, aged 4-59 years, who received second HSCT (HSCT-2) at a median of 45 days following primary or secondary failure of an initial unmodified (N = 3) or T cell-depleted (TCD) (N = 13) HSCT (HSCT-1). HSCT-1 was administered after myeloablative total body irradiation (TBI)- or alkylator-based conditioning for acute leukemias (N = 7), MDS (N = 6), CML (N = 2), and Fanconi anemia (N = 1). All patients experienced 1 or more infectious complications between HSCT-1 and HSCT-2, and 10 patients had active infections at the time of HSCT-2. Cytoreduction regimens used for HSCT-2 included fludarabine (Flu) in combination with cyclophosphamide (CTX) (N = 9), or thiotepa (Thio) (N = 5). In addition, 1 patient received Flu alone and 1 patient Thio combined with CTX. Antithymocyte globulin (ATG) (N = 11) or Alemtuzumab (N = 3) was added pretransplant to prevent rejection. For HSCT-2, donors included HLA-matched (N = 3) or mismatched (N = 8) related, or matched (N = 2) or mismatched (N = 3) unrelated donors. The primary graft donor was used in 6 of 16 cases. The grafts administered were unmodified peripheral blood stem cell transplantation (PBSCT) (N = 5) or bone marrow transplantation (BMT) (N = 3), TCD PBSCT (N = 8). All patients achieved engraftment at a median of 12 days and evaluable patients achieved complete donor chimerism. Six patients are alive with a median follow-up of 49 months, including 4/9 conditioned with Flu/CTX. In this series, outcome was statistically superior for younger patients (≤20 years). In summary, second HSCT using the combination of a fludarabine- and ATG-based, nonmyeloablative regimen and higher numbers of CD34+ progenitor cells has been associated with acceptable toxicity and allowed consistent engraftment with hematopoietic reconstitution in patients with previous graft failure.

BACKGROUND Antigen ganglioside GD2 is expressed abundantly on neuroblastoma (NB) cells. Anti-GD2 monoclonal antibody (MoAb) 3F8 kills NB cells by complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. Its utility in bone marrow (BM) purging is evaluated by a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to quantify the mRNA of GD2 synthase, the key enzyme in GD2 synthesis. METHODS From 1990 to 1993, 10 patients with relapsed/refractory Stage 4 NB participated in a pilot study. In these patients, MoAb 3F8 was used to purge tumor cells from harvested BM that had 5% or less tumor content by immunofluorescence (IF). Subsequently, 31 Stage 4 NB patients who underwent treatment on the N7 protocol (1994–1999) had their BM, which was in remission, purged by 3F8 before 131I-3F8 myeloablative radioimmunotherapy. GD2-positive tumor cells before and after purging were quantified by real-time quantitative RT-PCR of GD2 synthase. RESULTS GD2 positivity by IF was found before purging in six of eight patients in the pilot study. Five of six patients became negative postpurging. Of 31 patients on the N7 protocol, the more sensitive real-time quantitative RT-PCR detected GD2 synthase mRNA in the BM samples of 7 patients even though the prepurge BM samples were negative by histology and IF. Six of the seven BM samples became negative after 3F8 purging. Marker positivity before purging was statistically significant in predicting overall survival (P = 0.04), but not progression-free survival (P = 0.1). In vitro hematopoietic stem cell recovery and the median time to engraftment were acceptable. CONCLUSION Tumor cell depletion quantified by real-time RT-PCR demonstrated efficacy of MoAb 3F8 in BM purging. Cancer 2002;94:3042–8. © 2002 American Cancer Society. DOI 10.1002/cncr.10519

We have employed a new cytoreductive regimen to transplant two patients with Fanconi anaemia (FA), using T cell-depleted two HLA-allele disparate related peripheral blood stem cell transplants (PBSCTs). Patient 1, a 5-year-old male with FA and aplastic anaemia, initially received an HLA two-antigen mismatched unrelated cord blood transplant and failed to engraft. He received fludarabine (Flu) and cyclophosphamide (Cy), followed by a CD34(+) E-rosette(-) (CD34(+)E(-)), T cell-depleted, granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCT from his HLA B-DRB1 mismatched father. He received anti-thymocyte globulin (ATG), steroids, FK506 and G-CSF after transplant for rejection and graft-versus-host disease (GVHD) prophylaxis. The patient is now 23 months after SCT with no evidence of GVHD and with full haematopoietic and immune reconstitution. Patient 2, a 10-year-old boy with FA and myelodysplastic syndrome, received single-dose total body irradiation (SDTBI), Flu and Cy followed by a CD34(+)E(-), T-cell-depleted, G-CSF-mobilized PBSCT from his HLA B-DRB1 mismatched sister. He also received ATG, steroids, FK506 and G-CSF after transplant. The patient is now 12 months after SCT in complete remission with no evidence of GVHD. Absolute neutrophil counts (ANC) of > 1 x 10(9)/l were achieved on day 11 and day 10 post transplant respectively. Both patients are fully engrafted. In summary, we report two successful T-cell-depleted stem cell transplants from mismatched related donors for the treatment of Fanconi anaemia, using a fludarabine-based cytoreduction. Both patients experienced minimal toxicity, rapid engraftment and no GVHD.

Unrelated bone marrow transplantation (BMT) is often complicated by fatal opportunistic infections. To evaluate features unique to immune reconstitution after unrelated BMT, the lymphoid phenotype, in vitro function, and life-threatening opportunistic infections after unrelated and related T-cell–depleted (TCD) BMT were analyzed longitudinally and compared. The effects of posttransplant donor leukocyte infusions to treat or prevent cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infections on immune reconstitution were also analyzed. This study demonstrates that adult recipients of TCD unrelated BMTs experience prolonged and profound deficiencies of CD3+, CD4+, and CD8+ T-cell populations when compared with pediatric recipients of unrelated BMT and adults after related BMT (P < .01), that these adults have a significantly increased risk of life-threatening opportunistic infections, and that the rate of recovery of CD4 T cells correlates with the risk of developing these infections. Recovery of normal numbers of CD3+, CD8+, and CD4+ T-cell populations is similar in children after related or unrelated BMT. This study also demonstrates that adoptive immunotherapy with small numbers of unirradiated donor leukocytes can be associated with rapid restoration of CD3+, CD4+, and CD8+T-cell numbers, antigen-specific T-cell responses, and resolution of CMV- and EBV-associated disease after unrelated TCD BMT.

Thirty-one consecutive patients with acute myelogenous leukemia (AML) in first complete remission and 8 with AML in second complete remission received T cell–depleted allogeneic bone marrow transplants from HLA-identical sibling donors. Patients received myeloablative cytoreduction consisting of hyperfractionated total body irradiation, thiotepa, and cyclophosphamide. Those patients at risk for immune-mediated graft rejection received additional immune suppression with antithymocyte globulin and methylprednisolone in the early peritransplant period. Patients with AML who underwent allogeneic T-cell–depleted bone marrow transplantations (BMT) in first or second remission have achieved respective disease-free survival (DFS) probabilities of 77% (median follow-up at approximately 56 months) and 50% (median follow-up at approximately 48 months). Ten of 31 patients transplanted in first remission were ≥ 40 years old and have attained a DFS at 4 years of 70%. For patients with AML transplanted in first or second remission, the respective cause-specific probabilities of relapse were 3.2% or 12.5%, and those of nonleukemic mortality were 19.4% or 37.5%. There were no cases of immune-mediated graft rejection and no cases of grade II to IV acute graft-versus-host disease (GVHD). All survivors enjoy Karnofsky performance scores (KPS) of 100%, except 2 patients with KPS of 80% to 90%. T-cell–depleted allogeneic BMT can provide durable DFS together with an excellent performance status in the majority of patients with de novo AML. In addition, GVHD is not an obligatory correlate of the graft-versus-leukemia benefit or freedom from relapse afforded by allogeneic BMT administered as postremission therapy for AML. This study provides a basis for prospective comparison with other postremission therapies considered standard in the management of patients with this disease.

We examined the ability of patient-derived human leukemic blasts to generate leukemic growth and dissemination in severe combined immunodeficiency (SCID) mice by subcutaneous inoculation without conditioning treatment or administration of growth-promoting cytokines. Additionally, we correlated the growth pattern with the clinical outcome of patients from whom the leukemic cells were derived. The leukemias displayed three distinct growth patterns, ie, either aggressive, indolent, or no tumor growth. Leukemic cells from 6 of 13 patients with acute myeloid leukemia (AML), 4 of 7 T-cell acute lymphoblastic leukemia (T-ALL), and 11 of 16 patients with B-lineage ALL grew as subcutaneous tumors, with a significant number subsequently disseminating into distant organs in SCID mice. Patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SCID mice had a relatively poor clinical outcome, whereas patients with AML and T- or B-lineage ALL whose leukemic blasts grew indolently or whose cells failed to induce growth had a more favorable clinical course. Our study has shown that the subcutaneous inoculation of patient-derived human leukemic cells in SCID mice can engraft and grow as subcutaneous tumors with subsequent dissemination to distant organs in a manner analogous to their pattern of growth in humans. Additionally, these data suggest a clinical correlation to the growth and dissemination of some leukemic subtypes that may represent not only an additional prognosticator for patient outcome, but also a vehicle for the study of the biologic behavior of human leukemias and the development of novel therapeutic strategies.

Infusions of large numbers (> 10(8)/kg) of donor leukocytes can induce remissions in patients with chronic myeloid leukemia (CML) who relapse after marrow transplantation. We wanted to determine if substantially lower numbers of donor leukocytes could induce remissions and, if so, whether this would reduce the 90% incidence of graft-versus-host disease (GVHD) associated with this therapy. Twenty-two patients with relapsed CML were studied: 2 in molecular relapse, 6 in cytogenetic relapse, 10 in chronic phase, and 4 in accelerated phase. Each patient received escalating doses of donor leukocytes at 4- to 33-week intervals. Leukocyte doses were calculated as T cells per kilogram of recipient weight. There were 8 dose levels between 1 x 10(5) and 5 x 10(8). Lineage-specific chimerism and residual leukemia detection were assessed using sensitive polymerase chain reaction (PCR) methodologies. Nineteen of the 22 patients achieved remission. Remissions were achieved at the following T-cell doses: 1 x 10(7) (n = 8), 5 x 10(7) (n = 4), 1 x 10(8) (n = 3), and 5 x 10(8) (n = 4). To date, 15 of the 17 evaluable patients have become BCR-ABL negative by PCR. The incidence of GVHD was correlated with the dose of T cells administered. Only 1 of the 8 patients who achieved remission at a T-cell dose of 1 x 10(7)/kg developed GVHD, whereas this complication developed in 8 of the 11 responders who received a T-cell dose of > or = 5 x 10(7)/kg. Three patients died in remission, 1 secondary to marrow aplasia, 1 of respiratory failure and 1 of complications of chronic GVHD. Sixteen patients who were mixed T-cell chimeras before treatment became full donor T-cell chimeras at the time of remission. Donor leukocytes with a T-cell content as low as 1 x 10(7)/kg can result in complete donor chimerism together with a potent graft-versus-leukemia (GVL) effect. The dose of donor leukocytes or T cells used may be important in determining both the GVL response and the incidence of GVHD. In many patients, this potent GVL effect can occur in the absence of clinical GVHD.

Item does not contain fulltext BACKGROUND: The effect of intensified platelet inhibition for patients with unstable angina or myocardial infarction without ST-segment elevation who do not undergo revascularization has not been delineated. METHODS: In this double-blind, randomized trial, in a primary analysis involving 7243 patients under the age of 75 years receiving aspirin, we evaluated up to 30 months of treatment with prasugrel (10 mg daily) versus clopidogrel (75 mg daily). In a secondary analysis involving 2083 patients 75 years of age or older, we evaluated 5 mg of prasugrel versus 75 mg of clopidogrel. RESULTS: At a median follow-up of 17 months, the primary end point of death from cardiovascular causes, myocardial infarction, or stroke among patients under the age of 75 years occurred in 13.9% of the prasugrel group and 16.0% of the clopidogrel group (hazard ratio in the prasugrel group, 0.91; 95% confidence interval [CI], 0.79 to 1.05; P=0.21). Similar results were observed in the overall population. The prespecified analysis of multiple recurrent ischemic events (all components of the primary end point) suggested a lower risk for prasugrel among patients under the age of 75 years (hazard ratio, 0.85; 95% CI, 0.72 to 1.00; P=0.04). Rates of severe and intracranial bleeding were similar in the two groups in all age groups. There was no significant between-group difference in the frequency of nonhemorrhagic serious adverse events, except for a higher frequency of heart failure in the clopidogrel group. CONCLUSIONS: Among patients with unstable angina or myocardial infarction without ST-segment elevation, prasugrel did not significantly reduce the frequency of the primary end point, as compared with clopidogrel, and similar risks of bleeding were observed. (Funded by Eli Lilly and Daiichi Sankyo; TRILOGY ACS ClinicalTrials.gov number, NCT00699998.).

Graft-vs-host disease (GVHD) in allogeneic hematopoietic transplantation can be abrogated by T-cell depletion (TCD) of the graft. Researchers have sought the optimal TCD procedure, which would alter the activity, number, and/or subpopulation profile of T cells to acceptable levels, while retaining sufficient engraftment potential of the harvested hematopoietic stem cells. The techniques that have successfully survived the translation from research studies into practical clinical application may be analyzed by their effectiveness, efficiency, ease of application, and cost. The predominant techniques rely on either physical separation of the T cell (binding to erythrocytes, lectins, centrifugation) or reaction with monoclonal antibodies (immunomagnetic, panning, complement-mediated cytotoxicity, immunotoxins). Comparative trials between the various techniques are few, making comparisons difficult. However, all of the techniques, whatever their relative advantages and disadvantages, must meet the same challenges.

Significant amounts of information are currently available within the database generated by the responses to the Bone Marrow Processing Survey. As additional Surveys are returned, the data will continue to be entered into a Lotus spreadsheet, until a more sophisticated database with a programmed interface becomes available. The readership is encouraged to enter or edit the databank by returning a completed or amended Survey to the Society. Copies of the form are available from the Society or can be found in the first issue of the Journal of Hematotherapy.

Aspergillus fumigatus is an environmental fungus that causes life-threatening infections in neutropenic patients. In the absence of intact innate immunity, inhaled A. fumigatus spores (conidia) germinate in the lung, forming hyphae that invade blood vessels and disseminate to other tissues. Although macrophages and neutrophils are postulated to provide defense against invasive fungal infection, animal models and human studies suggest that circulating monocytes also contribute to antifungal immunity. Although human monocyte subsets, defined as either CD14+CD16− or CD14+CD16+, have been extensively characterized, their respective roles during fungal infection remain undefined. We isolated CD14+CD16− and CD14+CD16+ monocytes from healthy allogeneic hematopoietic stem cell transplantation donors and compared their ability to phagocytose and inhibit A. fumigatus conidia. Both monocyte subsets efficiently phagocytose conidia, but only CD14+CD16− monocytes inhibit conidial germination yet secrete little TNF. In contrast CD14+CD16+ do not inhibit conidial germination and secrete large amounts of TNF. Although CD14+CD16− and CD14+CD16+ monocytes differ in their response to dormant conidia, responses are similar if conidia are already germinated at the time of monocyte uptake. Our study demonstrates that functional CD14+CD16− and CD14+CD16+ monocytes can be isolated from allogeneic hematopoietic stem cell transplantation donors and that these subsets differ in their response to A. fumigatus conidia.

This study reports the development of an assay, the Pre-colony-forming unit (CFU) assay, which detects human hematopoietic precursors. The Pre- CFU assay is based on the observation that precursors to CFU- granulocyte-macrophage (CFU-GM) that are undetectable in clonogenic assays differentiate into CFU-GM preferentially following treatment in suspension culture with recombinant human interleukin-1 alpha (rhIL-1 alpha) combined with rhIL-3. Using the Pre-CFU assay, hematopoietic precursors were detected in human bone marrow depleted of CFU-GM progenitors and differentiated hematopoietic elements via 4- hydroperoxycyclophosphamide treatment coupled with selection for CD34+ cells (4-HCresistant/CD34+ marrow). Additionally, the Pre-CFU assay detected recovery of hematopoiesis substantially earlier than the CFU- GM assay in primates following myeloablation with 5-fluorouracil. The Pre-CFU assay was used to asses purification of a phenotypically defined hematopoietic precursor population, the lin-CD34+ population. The lin-CD34+ population lacks detectable surface markers for T-cell, B- cell, natural killer cell, and myeloid lineage, possesses the CD34 antigen, is devoid of CFU-GM progenitors, and yields Pre-CFU assay values comparable with 4-HCresistant/CD34+ marrow. Using a combination of phenotypic analysis and Pre-CFU assay analysis, the action of rhIL-1 alpha plus rhIL-3 treatment on lin-CD34+ cells was further characterized. The data indicate that rhIL-1 alpha plus rhIL-3 treatment induces proliferation and differentiation of early hematopoietic precursors into progenitors and terminally differentiated cells, without inducing a significant expansion of the precursor population itself.

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182 780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner—responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

Patients who have become split lymphoid chimeras (T cells of donor origin, B cells and monocytes of host origin) following transplantation of HLA-haploidentical marrow for the treatment of severe combined immunodeficiency disease provide a unique model for the study of tolerance. One such patient, UPN 345, was transplanted with maternal marrow and was found to have antidonor proliferative reactivity without detectable donor-directed cytotoxicity when tested at 18, 23, and 66 mos following bone marrow transplantation. In bulk culture, the proliferation to donor cells could be blocked by monoclonal antibodies to HLA-DR and -DQ. Nine clones with antidonor reactivity were established by limiting dilution techniques from a mixed lymphocyte culture between engrafted T cells and irradiated donor E rosette-negative cells. All of the clones were of maternal donor origin, and all were CD3+CD4+CD8-. The clones were tested for proliferative and cytotoxic activity toward donor, host, and paternal B-lymphoblastoid cell lines (B-LCL). Six clones proliferated strongly to maternal B-LCL but not to host B-LCL. Six clones were found to exclusively lyse maternal B-LCL. Four of the clones had both antidonor cytotoxic and antidonor proliferative reactivity. Monoclonal antibody blocking studies were performed on five of the six clones with cytotoxic activity. The antidonor cytotoxicity was not inhibited by monoclonal antibodies to class I determinants; however, three clones were inhibited in the presence of monoclonal antibody to DR, one clone was inhibited by anti-DQ monoclonal antibody, and one clone was inhibited by anti-DP monoclonal antibody. The cytotoxicity of all five clones was inhibited by monoclonal antibody to CD4. These data indicate that antidonor reactivity may also include a cytotoxic component which is not apparent in bulk cultures and which, based on our limiting dilution studies, is probably controlled by regulatory cells. Both the antidonor cytotoxicity and the antidonor proliferation appear to be directed primarily toward donor HLA class II antigens that are not shared with the patient.

We have investigated the role of interleukin-2 (IL2) as a differentiation factor for human marrow-derived NK cell progenitors and have assessed the effects of interleukin-1 (IL1) on this activity. The effects of these cytokines on early NK cell precursors was determined by testing marrow which had been depleted of mature cells and of CD2+ cells by treatment with soybean agglutinin and sheep erythrocytes (SBA−E−BM). The cytolytic activities of the SBA−E−BM were tested in 51Cr release assays following 7–8 days of liquid culture. K562 targets were used to assess NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity were measurable in marrow incubated in medium without cytokines, or in medium containing IL1 alone. In contrast, culture in medium containing IL2 resulted in a dose-dependent development of lytic activity. NK and LAK activities could be differentiated by the percentage of cultures in which the activity developed, the dose of IL2 required, the time kinetics of induction, and the effect of depletion of residual cells with NK phenotype prior to culture. The most lytically active effectors of both activities, however, were CD56+. Immunofluorescence analyses before and after culture with IL2 revealed that Leu19+ (CD56) cells increased from

Antithymocyte globulin (ATG) has been used in allogeneic stem-cell transplantation to prevent graft rejection and graft-versus-host disease (GvHD). Its use, however, has been associated with delayed T-cell reconstitution and prolonged susceptibility to opportunistic infections (OIs) especially in patients undergoing T cell–depleted (TCD) transplantation. Recently, a prospective trial was conducted in 52 adult patients (median age, 47 years) with various hematologic malignancies undergoing TCD transplantation from HLA-matched related donors without the use of ATG. The cytoreductive regimen consisted of hyperfractionated total body irradiation (HFTBI), thiotepa, and fludarabine. The preferred source of the graft was peripheral blood stem cells (PBSCs). No additional graft rejection or GvHD prophylaxis was given. All evaluable patients engrafted without any immune-mediated graft rejections. Disease-free survival (DFS) at 3 years was 61% in all patients, and 70% in patients with standard-risk disease. Acute GvHD was limited to grade 2 in 8% and chronic GvHD in 9% of patients. Life-threatening OIs occurred in 3 of 52 patients and was fatal in 1. This study demonstrates durable engraftment with a low incidence of GvHD despite the lack of ATG, as well as the curative potential of this regimen.

s / Biol Blood Marrow Transplant 19 (2013) S313eS341 S314 lines: Raji-2R and Raji-4RH (P< .001). However, there was no significant difference against CD20control cells: RS4;11 and Jurkat. Consistently, intracellular CD107a degranulation was enhanced in CAR+ exPBNK compared to CARexPBNK in response to CD20+ Ramos. MIC expressionwas significantly increased in Ramos (P< .05) and in NALM-6 (P < .001) after romidepsin treatment. CD20 expression was significantly increased in NALM-6 (P < .001) after romidepsin treatment. CAR+ exPBNK in vitro cytotoxicity was significantly enhanced against romidepsin treated Ramos (P< .02) and NALM-6 (P< .01) compared to untreated targets. Conclusion: Anti-CD20 CAR expression in exPBNK cells results in significant and specific in vitro cytotoxicity against CD20+ B-L/L. Romidepsin increases MICA/B and CD20 expression in B-L/L. CAR exPBNK significantly enhanced cytotoxicity against romidepsin treated B-L/L. Future directions include examining the combination effect of CAR exPBNK and Romidepsin against B-L/L in xenograft mice.

The development of an in vivo model for the study of CML would be of significant importance in studying its biological behavior and developing novel therapeutic strategies. We examined the ability of human leukemic cells derived from patients in either chronic (CP), accelerated (AP) or blast phase (BP) CML to grow and disseminate in CB17-SCID mice by subcutaneous (s.c.) inoculation without conditioning treatment or administration of cytokines. Additionally, samples derived from patients with CP-CML were injected s.c. into CB17-SCID mice treated with anti-Asialo GM1 (an anti-NK cell antibody) and NOD-SCID mice (absent NK cell activity) to study the potential role of NK cell-mediated anti-leukemic activity in preventing the propagation of CP-CML cells. We observed a significant differential growth pattern of CML cells in the mice such that BP-CML grew rapidly as s.c. tumors and disseminated, while AP-CML or CP-CML cells grew temporarily as small nodules that spontaneously regressed and did not disseminate. This differential growth pattern suggests possible important biological differences. Furthermore, no significant difference in s.c. growth or dissemination of CP-CML samples derived from newly diagnosed patients in untreated CB17-SCID mice and CB-17 SCID mice treated with Anti-Asialo GM1 and NOD-SCID mice occurred, suggesting that factors other than NK cell anti-leukemic activity may be important.

s / Biol Blood Marrow Transplant 19 (2013) S313eS341 S314 lines: Raji-2R and Raji-4RH (P< .001). However, there was no significant difference against CD20control cells: RS4;11 and Jurkat. Consistently, intracellular CD107a degranulation was enhanced in CAR+ exPBNK compared to CARexPBNK in response to CD20+ Ramos. MIC expressionwas significantly increased in Ramos (P< .05) and in NALM-6 (P < .001) after romidepsin treatment. CD20 expression was significantly increased in NALM-6 (P < .001) after romidepsin treatment. CAR+ exPBNK in vitro cytotoxicity was significantly enhanced against romidepsin treated Ramos (P< .02) and NALM-6 (P< .01) compared to untreated targets. Conclusion: Anti-CD20 CAR expression in exPBNK cells results in significant and specific in vitro cytotoxicity against CD20+ B-L/L. Romidepsin increases MICA/B and CD20 expression in B-L/L. CAR exPBNK significantly enhanced cytotoxicity against romidepsin treated B-L/L. Future directions include examining the combination effect of CAR exPBNK and Romidepsin against B-L/L in xenograft mice.

We examined the effect of graft composition parameters including total white blood cell (WBC), myeloid cell dose (CD34+, CD34+/CD38+) lymphoid cell dose (CD3+, CD4+, CD8+), and activated lymphoid cells expressing activation antigens (CD25, CD69, HLA-DR) in 123 bone marrow (BM) and 198 peripheral blood progenitor cell (PBPC) transplants from unrelated donors (URD) between July 2003-March 2005. Most recipients (85% BM, 94% PBPC) had a hematologic malignancy. Samples were shipped from 33 participating centers for analysis at a central laboratory. Most BM grafts (67%) were processed: 24% T-cell depleted, 26% red blood cell (RBC) depleted, 12% plasma depleted and 5% mononuclear cell concentrated. Fewer PBPC grafts (29%) were processed: 19% plasma removal, 9% CD34+ selection and 1% RBC depletion. All samples had acceptable viability (≥70%). CD34 recovery was high for BM grafts after plasma or RBC depletion (84% and 62%, respectively) but low after T-cell depletion (26%). CD34 recovery was high for plasma depleted PBPC (83%). Plasma or RBC depletion had minimal effect on lymphoid cell content of either BM or PBPC, while T- cell depletion or CD34 selection resulted in 3–4 log10 depletion of CD3+ and HLA-DR+ lymphoid cells. We did not observe differences in transplant outcomes among those receiving processed vs. non-processed BM or PBPC grafts. The overall or subset cellular composition of BM grafts was not associated with the likelihood of hematopoietic recovery, acute and chronic graft-versus-host disease (GVHD) or 100-day survival. After adjusting for other clinically significant factors, risks of overall mortality were significantly lower when BM grafts contained ≥2.36 × 106/kg CD25 cells (relative risk [RR] 0.55, p=0.048) and ≥8.20 × 104/kg CD 34+/38+ (RR 0.58, p=0.043); no other subsets were associated with overall survival. With PBPC grafts, day-28 neutrophil recovery was higher with grafts containing ≥3.30 × 106/kg CD69 cells (97% vs. 89%, p=0.036). Platelet recovery by day-90 was higher with PBPC grafts containing: ≥5.44 × 106/kg CD34+ cells (87% vs. 74%, p=0.027), ≥2.20 × 108/kg CD3+ cells (87% vs. 74%, p=0.028) and ≥8.15 × 107/kg CD8+ cells (88% vs. 73%, p=0.011). The likelihood of acute GVHD was not associated with the composition of PBPC grafts, but chronic GVHD was higher with PBPC grafts containing ≥2.20 × 108/kg CD3+ cells (RR 1.66, p=0.029). Early mortality rates were lower with PBPC grafts containing ≥8.15 × 107/kg CD 8+ cells (RR 0.47, p=0.025) and overall mortality rates were lower with PBPC grafts containing ≥5.44 × 106/kg CD 34+ cells (RR 0.58, p=0.003) and ≥8.15 × 107/kg CD 8+ cells (RR 0.67, p=0.029). These data suggest that adequate CD25 and CD34+/38+ are necessary for optimal survival after URD BM and adequate CD34+ and CD8+ after PBPC transplantation. Further analysis of the ideal graft composition associated with transplant outcome will be needed to plan graft engineering manipulations to improve patients’ survival and could be particularly relevant for PBPC transplants which are associated with higher chronic GVHD.

Central reference flow cytometry laboratories are used in multicenter trials to eliminate inter laboratory variation in testing. Yet, factors that materially affect the quality of data such as graft processing, sample composition, packaging, and shipment have been little studied. The Baylor Center for Cell and Gene Therapy (BCGT) was the central reference laboratory for the NMDP IRB-2002-0068 multicenter study on the correlation of graft composition on clinical outcome. Samples of processed or non-processed grafts of 489 bone marrow (BM) and 696 peripheral blood progenitor cell (PBPC) grafts from July 2003-March 2004 were shipped to BCGT using 2 types of shipping containers with frozen gel packs to keep samples cool during transit. On arrival samples were assessed for temperature of gel packs, sample integrity (clumps, clots requiring filtration), viability by flow cytometry using 7AAD, and immunophenotype (CD34+, CD3+, and CD3+ T-cell subsets, CD25, CD69, HLA-DR). The FDA “standard” of 70% viability was used to define acceptable sample viability. Integrity and viability of BM and PBPC samples were not influenced by the method of cell processing: non-processed, plasma depletion, mononuclear cell concentration or T-cell depletion. BM samples were not affected by either temperature or time to evaluation. Viability of PBPC samples was adversely affected by both gel pack temperature and time from collection to evaluation. A 55% mean viability was observed with gel packs arriving thawed-warm vs 89% with frozen gel packs. Additionally, the samples that arrived >48hr after collection also had mean viability less than the 70% standard value. Viability of samples was inversely correlated with gel pack temperature on arrival and time from collection to evaluation. Therefore, the quality of FACS data and by implication, the graft utility was largely influenced by cell type, temperature in transit, and the time from collection to evaluation. Thus, while the use of a central reference laboratory can produce reliable data on cells following a variety of processing methods, certain factors must be optimized in order to collect data that accurately reflects the samples under study. Shipping standards to maintain graft viability and reliability of laboratory assessments of graft composition must be established and validated against the success of transplantation outcomes.

Systemic light-chain (AL) amyloidosis is both a protein deposition disorder and a monoclonal plasma cell dyscrasia. Effective treatment depends on reduction of the clonal plasma cells that produce the toxic light chains. Although high-dose melphalan with autologous stem cell transplant (MEL SCT) is effective for AL patients, the mobilization and collection of peripheral blood stem cells with G-CSF can be problematic despite their lack of prior chemotherapy and minimal marrow infiltration with plasma cells. In addition, unusual toxicities of G-CSF mobilization such as hypotension have been noted in AL patients. As new therapies show efficacy in AL, the role of stem cell mobilization and MEL SCT will likely be re-evaluated, increasing the importance of understanding the variability in stem cell mobilization. Recent work has shown that the mechanism of G-CSF mobilization likely involves the sympathetic nervous system (Cell2006;124:407). Interestingly, amyloid is well known to cause autonomic neuropathy. In addition, involvement of the heart in amyloidosis has also been shown to cause cardiac adrenergic denervation. If G-CSF mobilization depends upon the sympathetic nervous system, then the type of organ involvement could predict the yield of stem cell collection in AL patients mobilized with G-CSF. With these concerns in mind, we retrospectively assessed the results of stem cell mobilization and collection in all patients with AL mobilized with G-CSF (6ug/kg bid) for 4 days, with collections beginning on day 5 with a collection target of 10x106 CD34+ cells per kg in up to 4 leukaphereses. At initial evaluation, we assessed patients using standard criteria to identify the dominant organ of involvement (Am J Hematol2005;79:319) and to determine candidacy for risk-adapted MEL SCT (Blood2002;99:4276). Over a 7-year period 105 patients with AL were mobilized and collected as described, including 40 with kidney (K), 21 with liver/GI or soft-tissue (L/GI/ST), 31 with cardiac (H) and 13 with peripheral nervous system (PNS) involvement. We recorded the number of leukaphereses per patient and the dose of CD34+ cells/kg collected per leukapheresis. There were no significant differences in the number of leukaphereses per group. The K, L/GI/ST and H groups had a median of 2, and the PNS group of 3, collections. There were significant differences in CD34+ cells/kg collected based on dominant organ involvement: patients with H or PNS involvement had significantly fewer CD34+ cells/kg collected (see figure below; K vs H p=0.03, K vs PNS p=0.02, L/GI/ST vs H p=0.07, L/GI/ST vs PNS p=0.04; two tailed Mann-Whitney). Moreover, 15/44 H+PNS patients failed to collect at least 4x106 CD34+cells/kg compared to 7/61 K+L/GI/ST patients (p Figure Figure

Curative strategies for hematologic malignancies include alloHSCT, which has become a treatment option for older pts, as well as those with more extensive prior therapy and comorbidities. This has stimulated research on the development of less toxic but comparably effective approaches to transplantation at the level of cytoreduction, alternate graft sources, graft manipulation, and GvHD prophylaxis. To this end, between 7/2001 and 12/2005, we administered TCD HSCTs, derived from HLA-M or HLA-MM URDs, to 36 adult pts as treatment for a variety of hematologic malignancies on a clinical trial. The conditioning regimen was designed to reduce regimen related toxicity while preserving adequate immunosuppression to allow for engraftment. The TCD grafts allowed transplantation across HLA disparate pt-donor pairs without the use of additional renal and hepatotoxic GvHD prophylaxis. The conditioning regimen consisted of hyperfractionated total body irradiation (HFTBI) (1375 cGy), thiotepa (5mg/kg) x 2d, fludarabine (25mg/m2) x 5d and antithymocyte globulin (ATG) x 2d. HLA typing was performed by DNA SSOP analyses for A,B,C, DRB1, and DQB1, and donors were matched at ≥8 of 10 alleles. Pts received TCD-PBSC (n=29) or TCD-BM (n=7) from HLA-M (21 pairs) or HLA-MM (15 pairs) URDs. PBSCs were TCD by Isolex 300i CD34+ selection followed by sheep E-rosette depletion, and BMs were depleted with soybean agglutination and sheep E-rosette depletion. The median age was 40.6 (range 18–63)yrs; 10 pts ≥ 50 yrs. Diseases included AML and ALL CR1 (only standard or high risk), AML and ALL CR2, ALL ≥ CR3, acute biphenotypic leukemia, CML in CP, MDS, T-PLL. The median followup is 22 (range 6–55) mos. All evaluable pts engrafted neutrophils, 31 of 35 evaluable pts engrafted platelets. Four pts died of complications prior to platelet engraftment, including one pt with late graft failure. The 100d non-relapse mortality was 20% with most (>50%) deaths due to infection. The incidence of acute (a) grade II–III and chronic (c) GvHD was low for the entire group of 36 pts at 11% and 28%, respectively, when compared to that of unmodified transplantation. The incidence of aGvHD was 14% and 7%, and cGvHD (majority - limited) was 26% and 30% for HLA-M and HLA-MM transplant pairs, respectively. Estimated 3 yr DFS is 60% for both HLA-M and HLA-MM transplants, and 83% for standard risk and 41% for high risk disease pts. Only one pt has relapsed. These results indicate that a transplantation strategy using HFTBI, thiotepa, fludarabine and ATG followed by TCD PBSC or BM, but without posttransplant immunomodulating agents, is well-tolerated in an older patient group (median age 40 yrs) even with HLA-MM URDs. Although this approach appears to provide an antileukemic effect for acute leukemia pts transplanted in remission of acute leukemia, this will need to be confirmed with longer followup.

Between 05/98 and 06/04, 15 consecutive patients with FA received hematopoietic stem cell transplants (SCT) from alternative donors at our Center. There were 7 males and 8 females aged 5 to 24 years (median 11.5). Hematologic diagnoses included aplastic anemia (AA) (N=5), myelodysplastic syndrome (MDS) in RAEB (N=4), RAEBT (N=1) or acute myelogenous leukemia (AML) (N=5). High risk features included: Age > 20 years (n=4), prior multiple transfusions (n=11), prior androgen treatment (n=12), prior infections (n=10), or advanced MDS or AML (n=9). Eight pts had related mismatched donors transplants with respective matching at 3/6 (6/10), 4/6 (6/10), 4/6 (7/10) (n=2)), 5/6 (8/10) (n=3) and 5/6 (9/10) HLA-antigens. Seven pts had unrelated donors transplants with respective matching at 5/6 (7/10), 5/6 (8/10) (n=2), 5/6 (9/10) and 6/6 (10/10) (n=3) HLA-antigens. Cytoreduction included single dose total body irradiation (SDTBI) (450 cGy), fludarabine (Flu) (30 mg/m2 x 5) and cyclophosphamide (Cy) (10 mg/Kg x 4). Immunosuppression included rabbit anti-thymocyte globulin (Thymoglobulin) and tacrolimus for all patients. Grafts were G-CSF mobilized CD34+ and E-rosette negative (E-) peripheral blood stem cell transplants for 12 pts and soybean agglutinin negative (SBA-) and E-rosette negative marrow transplants for 3 pts. Cell doses of the grafts were 1.5 – 29.6 x 106 CD34 cells/Kg and 0 – 26 x 103 CD3 cells/Kg. As evidenced by RFLP or FISH, all 15 evaluable pts were fully engrafted and complete chimeras. Fourteen pts were evaluable for graft-versus-host disease (GvHD). GvHD of the skin and of the gut was suspected in two pts but resolved completely prior to immunosuppressive treatment. With a median follow-up of 2.5 years (range 0.2–6), 13 of 15 pts are alive and 11 of 15 are alive disease-free. There were two deaths: one pt died from sepsis/ARDS at 2 months post SCT and one pt from pneumonitis/ARDS and EBV-infection 6 months post SCT. Three pts relapsed (MDS-RAEB x 1 – AML x 2): One pt relapsed 7 months post transplant, received a 2nd transplant from the same donor following busulfan and Flu and is alive, disease-free 18 months post SCT, while the other two pts are awaiting a second SCT. In summary, this cytoreductive regimen used with T-cell depleted stem cell transplants from unrelated or HLA-mismatched related donors for the treatment of high risk patients with Fanconi anemia, results in rapid hematopoietic engraftment and lymphohematopoietic reconstitution with minimal GVHD and a high disease-free survival.

Twenty five patients have been enrolled on this trial to date. There were 14 males and 11 females aged 0.6–54 years. Patients’ diagnoses and stage included: NHL in CR2 or refractory (n=2), AML (n=7), including 5 pts with secondary AML, ALL > CR3 (n=4), CML in CP2 (N=1) and high risk MDS (n=11) including 5 pts with secondary MDS. Eight pts had a matched related donor, 14 pts an unrelated donor and 3 pts a mismatched related donor. Cytoreduction consisted of busulfan (Bu) (0.8–1 mg/Kg/dose x 10 doses), melphalan (Mel) (70 mg/Kg/day x 2) and fludarabine (Flu) (25 mg/m2/day x 5). Graft rejection prophylaxis included rabbit ATG (Thymoglobulin) (2.5 mg/Kg/day x 2). Four pts tolerated only one of two doses of the ATG, 2 pts received equine ATG and one pt Alemtuzumab. Twenty one pts received G-CSF mobilized peripheral blood stem cell transplants that were T-cell depleted by CD34 selection and E-rosetting while the other four pts received Soybean agglutinin E-rosette depleted marrow grafts. Cell doses were 1.3–20.5 x 106 CD34 cells/Kg. and 0 -100 x 103 CD3 cells/Kg. Engraftment occurred in 24 pts. One pt suffered a graft failure; This pt had initial low busulfan levels, and received bone marrow derived stem cells with a low cell dose from a 5/6 HLA-matched unrelated donor. Acute graft-versus-host disease occurred in four pts: grade 1 (n=2) and grade 2 (n=2) and no pts developed any grade 3-4 severe GvHD. Two patients were diagnosed with chronic GvHD: localized (n=1) and extensive (n=1). Two patients developed sepsis early post BMT, with secondary multi organ failure and early mortality, while for the rest of the patients, regimen-related toxicity was acceptable. Relapse occurred in 9 pts. Mortality included 7 pts from relapse, two pts from sepsis and multi-organ failure, 3 pts from infections, and one pt from unknown causes. The overall survival (OS) and disease-free survival (DFS) at 2 yrs for the entire patient cohort were respectively 44% and 42 %; The DFS was 50% for patients with secondary MDS or AML. In summary, the cytoreduction with Bu Mel and Flu allowed consistent engraftment of T-cell depleted grafts and was associated with acceptable outcome for patients with secondary MDS or AML.

The aim of this study was to compare the extent of in vitro T cell depletion and recovery of hematopoietic progenitor cells achieved with five methods of T cell depletion. Bone marrow samples from the same source were treated with monoclonal antibody Campath-1 (CP1) and human complement, XomaZyme-H65 (anti-T cell ricin A chain immunotoxin), or soybean agglutinin (SBA) alone or in combination with sheep erythrocytes (EAET) or a cocktail of immunomagnetic beads (B) directly coated with anti-CD2, anti-CD3, or anti-CD8 monoclonal antibodies. Residual T cells were enumerated by limiting dilution analysis, EAET rosetting, and proliferative responses to phytohemagglutinin. The results of this study demonstrated the following reductions in BM T cells as detected by limiting dilution analysis (mean % control): SBA+B (99.9%), SBA+EAET (99.8%), CP1+C' (99.4%), anti-T cell ricin A chain immunotoxin (99.0%), and SBA alone (94.2%). Neither PHA response nor enumeration of residual EAET rosettes provided discriminating differences in the degree of T cell depletion by treatment method when T cell reductions exceeded 99.0% by LDA. These results demonstrate the ability of CP1+C', XomaZyme-H65, and SBA plus sheep erythrocyte or magnetic bead depletion to achieve a greater than 99% reduction of BM T cells and the importance of limiting dilution analysis in defining differences in T cell numbers when depletion exceeded 99%.

Early clinical trials using T lymphocyte-depleted human marrow for transplantation have reported that such grafts reduce, to varying degrees, both the incidence and the severity of graft-v-host disease (GVHD). However, to date, no clear estimates have been made as to what degree of T cell depletion is necessary to prevent GVHD in every case. To address this problem, we used a limiting dilution assay (LDA) to quantitate residual clonable T lymphocytes in human T cell-depleted bone marrow in 31 HLA-identical transplants for leukemia. The number of phytohemagglutinin -interleukin 2-responsive T lymphocytes determined by LDA and expressed as T cell per kilogram recipient weight was found to correlate with the subsequent development of GVHD: no patients who received less than 1 X 10(5) T cell per kilogram developed GVHD (N = 24). Of the seven patients who received 1 X 10(5) to 4.4 X 10(5) T cell per kilogram, four patients developed grade I or II skin GVHD. This study thus provides a quantitative estimate of the number of T lymphocytes necessary to initiate clinically detectable GVHD in an HLA- identical host.

The attributes of the test grafts with which putatively tolerant rats are challenged influence their immune response. Lewis (Lew) rats inoculated at birth with Lew/BN F1 hybrid bone marrow cells accept large skin allografts more readily than small allografts, and F1 hybrid skin grafts survive better than BN transplants. The results indicate that the survivals of these major histocompatibility complex-incompatible grafts are determined by the same factors that operate when only weak histoincompatibilities prevail.