Characterization of the alloreactivity and anti-leukemia reactivity of cord blood mononuclear cells.

It has been suggested that cord blood T cells may be less able to mediate GVHD than marrow-derived T cells due to their naive status. A decreased potential for GVHD may be advantageous for allogeneic transplant, but this benefit might be counteracted by loss of the GVHD associated graft-versus-leukemia (GVL) effect. The GVL potential of cord blood could be doubly compromised since cord blood NK cell activity is also decreased. To assess these issues we have performed extensive comparative functional and immunophenotypic evaluations of cord and adult mononuclear cells. We found a somewhat reduced alloproliferative, allostimulatory and allocytolytic capacity of cord blood mononuclear cells in bulk assays but not by limiting dilution assays. Immunophenotyping revealed no significant differences in the proportion of major lymphocyte subsets with the exception of the previously recognized predominance of CD45RA+ cells in both CD4 and CD8 cord blood T cells. Cord blood T cells expressed normal percentages of the cellular adhesion molecules, CD11a, CD18 and LFA-3; however, the antigen density of each of these molecules was less than that found on adult T cells. Fewer resting cord blood T cells expressed CD54, the ligand for LFA-1. Cord blood B cells and monocytes expressed normal levels of HLA-class I and HLA class II DR, DP and DQ antigens, suggesting that the decreased expression of cellular adhesion molecules or their receptors rather than a decrease in expression of HLA might have contributed to the lower alloreactivity of cord blood. Although the percentages of NK cells and NK cell subsets in adult and cord blood were similar our data confirmed that cord blood has very low NK lytic activity. In contrast, LAK activity was much more readily induced in cord blood compared with adult PBMC, a finding which could be explained in part by a higher frequency of LAK precursors and a more rapid expansion of NK cells in response to culture with medium containing of NK cells in response to culture with medium containing IL-2. Cord blood LAK cells were readily able to lyse fresh leukemia targets from patients with ALL, AML and CML. The data indicate that although the alloreactive potential of cord blood cells may be somewhat decreased, it is not absent and must be considered a factor in cord blood transplants. LAKp with the potential to lyse leukemia are present in increased numbers in cord blood and might contribute to the GVL effect of a cord blood transplant.

Analysis of reconstituting T cell receptor repertoires in bone marrow transplant recipients.

Allogeneic bone marrow transplantation (BMT) has become the treatment of choice for a variety of hematopoietic disorders. An important factor limiting the use of allogeneic BMT is delayed restoration of immune function. A quantitative understanding of immune reconstitution of the T cell compartment based on an efficient method of analysis would be of benefit. Distinct lineages of T cells which have resulted from previous and ongoing clonal expansion can be identified by their unique T cell receptors (TCR). Thus, TCR complexity can be used as a measure of repertoire complexity. Here we use a polymerase chain reaction-based approach which visualizes the size heterogeneity of the third complementarity determining region (CDR3) to study T cell reconstitution in adult bone marrow transplant recipients. We find that repertoire complexity, as determined by the number of bands of different length for each V family, reflects the general immune status of individuals tested. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. This approach provides a new tool in the analysis of reconstitution of alpha/beta T cell repertoires and it can be also be applied to B cells and gamma/delta T cells.

Inhibition of nitric oxide production is associated with enhanced weight loss, decreased survival, and impaired alloengraftment in mice undergoing graft-versus-host disease after bone marrow transplantation.

The pathophysiologic role of nitric oxide (NO) in graft-versus-host disease (GVHD) was investigated in a murine bone marrow (BM) transplantation model where donor and recipient were H-2-matched but differed at multiple minor histocompatibility antigens. Host AKR/J (H-2K) mice received lethal total body irradiation as pretransplant conditioning followed by transplantation of donor B10.BR (H-2K) BM cells with or without spleen cells as a source of GVH-reactive T cells. NO production, as assessed by serum nitrate and nitrite levels, was increased for up to 3 weeks posttransplant in animals undergoing both moderate and severe GVHD. Administration of NG-methyl-L-arginine (L-NMA), an inhibitor of nitric oxide synthase, to animals undergoing GVHD resulted in effective suppression of NO production when compared with saline-treated GVHD control animals. Suppression of NO production by L-NMA in GVHD animals was associated with enhanced weight loss early posttransplant and decreased overall survival. Histologic analysis of tissues from L-NMA-treated and saline-treated GVHD animals showed that early weight loss was not because of an exacerbation of GVHD, indicating that NO did not appear to play an immunosuppressive role in this experimental model. L-NMA-treated animals with enhanced weight loss were observed to have splenic atrophy, decreased extramedullary hematopoiesis, and a reduction in BM cellularity when compared with GVHD control mice that were weight-matched before transplant. Analysis of T-cell chimerism in the spleen showed that L-NMA treatment impaired donor T-cell repopulation. In vitro colony-forming unit (CFU) assays were performed to further assess the role of NO on BM progenitor cell growth. L-NMA added directly into culture had no effect on CFU-granulocyte/macrophage (CFU-GM) formation in normal murine BM. In contrast, total CFU-GM from L-NMA-treated animals were significantly reduced when compared with GVHD controls or BM control animals who did not develop GVHD. Collectively, these data indicate that inhibition of NO impairs hematopoietic reconstitution and support the premise that NO appears to play a novel role in the facilitation of alloengraftment posttransplant.

HLA-B44-directed cytotoxic T cells associated with acute graft-versus-host disease following unrelated bone marrow transplantation.

We describe the recipient of a marrow graft from an HLA-serologically identical unrelated donor from whom highly potent host-reactive CTL of donor origin were isolated in association with acute GVHD. Extensive sequence and biochemical analysis of the HLA complex of this donor and recipient revealed several disparities in class I and class II HLA with the potential to be recognized by T cells from the donor or the host. The donor-derived CTL exclusively recognized a class I HLA difference associated with HLA-B44. Nucleotide sequencing of donor and recipient cells revealed that the patient possessed the HLA-B*4402 allele recognized by IEF as B44.2 while the donor possessed HLA-B*4403 (IEF variant B44.1). These alleles differ at one amino acid residue located at position 156 in the alpha 2 domain. The donor-derived CTL were shown to be specific for B44.2 by blocking studies and by the lysis of five different B44.2+ unrelated cell lines, two of which were confirmed by sequencing to be homozygous for B*4402. A host-specific difference involving a HLA-DRB1 allele was not recognized by the CTL, neither did HLA differences unique to the donor HLA-B*4403 and HLA-DQ8 elicit a host response. These data show that certain HLA disparities may be tolerated at the same time that other disparities elicit a potent immunologic response. The chemical nature of the difference, its structural impact, as well as the conditions of transplant appear to influence the type of response which occurs.

Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status.

The analysis of the T cell repertoires involved in local or systemic immune responses is beginning to play an important role in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, alloimmunity including allograft rejection, and tumor immunity. Here we analyze circulating T cell repertoires by determining TCR beta-chain gene complexity using a modification of V beta family-specific PCR. This approach, called CDR3 size spectratyping, uses the size heterogeneity of the CDR3 as a further source of specificity in TCR analysis. It has been used here to analyze the complexity and stability of circulating T cell repertoires in normal adults, including bone marrow donors, and bone marrow transplant recipients. Normal spectratypes are both complex and stable. The repertoire complexity of marrow recipients correlates with their state of immune function. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. Spectratype analysis is applicable to other studies of specific repertoire skewing such as may be associated with immunodeficiency or found at sites of immune activity.

Effect of T-cell depletion as graft-versus-host disease prophylaxis on engraftment, relapse, and disease-free survival in unrelated marrow transplantation for chronic myelogenous leukemia.

Between January 1988 and March 1993, 48 patients received T-cell-depleted marrow grafts from unrelated donors as treatment for chronic myelogenous leukemia (CML). The median age of the population was 31.7 years (range 5.4 to 53) with 17 of 48 patients greater than 40 years of age. Twenty-seven patients were transplanted in chronic phase, 17 in accelerated phase, and 4 in blast crisis. All patients received a standardized preparative regimen of cyclophosphamide, high-dose cytosine arabinoside, methylprednisolone, and total body irradiation. Marrow grafts were depleted of mature T cells with the alpha beta T-cell receptor antibody T10B9 as graft-versus-host disease (GVHD) prophylaxis. All patients also received posttransplant cyclosporine therapy. Twenty-eight of 48 patients were mismatched with their donors for one or more HLA-A, B, DR, or DQ loci by either serology or high-resolution oligonucleotide genotyping. Nine of 28 were mismatched at multiple HLA loci. Durable engraftment was achieved in 94% (45/48) of patients. The actuarial probability of developing grades II to IV and grades III to IV acute GVHD were 39.6% (95% confidence interval (CI) 26.9 to 53.0) and 8.3% (95% CI 6.1 to 10.9) for the entire cohort. There was no difference in the incidence of grades II to IV acute GVHD between patients receiving matched (36.8%) or mismatched (41.4%) marrow grafts (P = .77). The actuarial probability of relapse at 2 years was 8.8% (95% CI 2.1 to 21.6) for the entire cohort and 18% (95% CI 4 to 41) for patients transplanted in either the accelerated or blast crisis phase (advanced disease). One cytogenetic relapse has occurred among patients transplanted in the chronic phase. The probability of disease-free survival at 2 years was 52% (95% CI 24 to 70) for patients transplanted in chronic phase and 46% (95% CI 25 to 73) for patients transplanted with advanced disease. No difference in disease-free survival was observed between patients receiving matched (49%) or mismatched (51%) marrow grafts (P = .90). This study shows that patients receiving unrelated T-cell-depleted marrow grafts for CML can achieve durable engraftment with a low incidence of severe GVHD and apparent preservation of graft-versus-leukemia reactivity. These data also suggest that T-cell depletion may allow patients who might otherwise experience unacceptable toxicity from GVHD-related complications caused by older age or increased HLA disparity to benefit from unrelated marrow grafts.

Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation: efficacy and toxicity of a defined T-cell dose.

Eight patients who had hematologic relapse of chronic myelogenous leukemia (CML) after undergoing allogeneic bone marrow transplantation (BMT) were treated with leukocyte infusions from the original bone marrow donors. All patients had previously received marrow grafts from HLA-identical siblings. Six patients were in the accelerated phase of their disease and two were in blast crisis. Each patient received a predetermined T-cell dose within a narrow range of 2.5 to 5.0 x 10(8) T cells/kg. Three patients also received short courses of therapy with alpha interferon to control elevated white blood cell counts within the first several weeks after leukocyte transfusions. Seven of eight evaluable patients developed graft-versus-host disease (GVHD) at a median of 32 days after the initial infusion. One patient had fatal GVHD. A second patient had grade 3 acute GVHD, which has responded to immunosuppressive therapy. The remaining patients all had mild grade I GVHD. Six patients continue to require modest doses of prednisone more than 6 months after infusion. Four patients developed marrow aplasia, which in three patients required marrow boosts from the original donors. Two of these three patients have normal hematopoietic function, whereas the third patient remains growth factor and transfusion dependent. Both patients treated in blast crisis have died, one from GVHD and one from disease progression. All six patients in the accelerated phase are alive and in cytogenetic remission at a median of 42 weeks after infusion. Five of these six patients are in molecular remission. This study demonstrates that leukocyte infusions that administered a defined T-cell dose can exert a profound graft-versus-leukemia effect and are an effective form of salvage immunotherapy in allogeneic marrow transplant recipients. This therapeutic approach appears to be a viable alternative to existing chemotherapeutic and immunomodulatory strategies for the treatment of relapsed CML.

Effect of GVHD on the recovery of NK cell activity and LAK precursors following BMT.

The mechanism by which GVHD augments the graft-versus-leukemia (GVL) effect of marrow transplants has not been ascertained. One possibility involves the secondary activation of natural killer (NK) cells by cytokines released during the GVHD process. To evaluate this possibility we have compared NK activity and lymphokine-activated killer cell precursor (LAKp) frequencies in serially sampled PBMC from recipients of unmanipulated autologous or allogeneic marrow with and without active GVHD. NK activity recovered rapidly after BMT and was elevated during episodes of acute GVHD. However, NK activity did not differ between recipients of autologous or allogeneic marrow without GVHD nor was NK activity increased in association with chronic GVHD. Endogenously-activated NK cells were detected only in recipients of allogeneic marrow but this did not correlate with GVHD status. In contrast to NK activity, LAKp frequencies fell below the control range during the first 8 weeks after BMT. By 9-14 weeks the median LAKp frequency was normal and did not differ between the three groups then or later after transplant. We conclude that acute GVHD may serve to increase the lytic activity of NK cells but does not result in increased LAKp. LAKp frequencies are below normal during the first two months after BMT, a finding not previously recognized from bulk culture LAK studies. The role of LAK effectors in GVL may involve more the degree of cellular activation rather than the number of cells activated.

Characterization of cord blood lymphocyte subpopulations.

Human cord blood is an attractive alternative to marrow-derived stem cells for transplantation. Experiences with cord blood transplants suggest that graft-versus-host disease (GvHD) may be less readily induced, even in the face of HLA differences. However, this decreased potential for GvHD might also abrogate the graft-versus-leukemia (GvL) effects of the transplant. The GvL potential might be doubly compromised since cord blood NK activity is also decreased. We have compared alloreactivity, NK cell activity and lymphokine-activated killer cell (LAK) activity of cord blood mononuclear cells with adult mononuclear cells. We find a reduced (but not absent) alloproliferative, allostimulatory and allocytotoxic capacity of cord blood mononuclear cells. Phenotyping revealed no significant differences in the proportion of T cells in cord-versus-adult blood, but cord blood T cells were nearly all of the naive CD45RA subset. Expression of LFA-1 alpha and LFA-1 beta was normal on resting cord T cells; however, they expressed significantly less ICAM-1 (CD54) than did adult PBMC. Cord blood B cells and monocytes expressed normal levels of HLA Class II. Although no differences were found in NK cell percentages or subsets in resting cord blood, cord blood NK activity was very low. However, LAK activity was much more readily induced in cord blood as compared to adult PBMC, which could be explained in part by a higher frequency of LAK precursors (LAKp). Cord blood LAK cells were readily able to lyse fresh leukemia targets from patients with ALL, AML, and CML.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant interleukin 3 induces interleukin 2 receptor expression on early myeloid cells in normal human bone marrow.

Human interleukin 3 (IL-3) is a multipotential cytokine that supports the growth of early hematopoietic progenitors and promotes their response to other, later-acting cytokines. We found that IL-3 was able to induce the expression of interleukin 2 (IL-2) receptor (IL-2R) (CD25) on a subset of early myeloid cells in normal human bone marrow that had been first depleted of mature hematopoietic cells and E-rosette-positive T cells by treatment with soybean lectin and sheep erythrocytes (SBA-E-BM). Immunofluorescence analysis revealed that the CD25+ cells were contained almost entirely within the lymphoblastoid gate of the IL-3-cultured marrow. CD25 was undetectable on freshly isolated marrow and less than 10% CD25+ cells could be detected following liquid culture at 37 degrees C in the presence of 10% human serum, 10% fetal calf serum, or under serum-free conditions. Addition of IL-3 (100 U/ml) significantly increased the expression of CD25 to 37%, 31%, and 24%, respectively. CD25 could also be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), but no IL-2R was detectable following exposure to granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1), interleukin 4 (IL-4), or IL-2. Expression of CD25 was dependent on the dose of IL-3 or GM-CSF added and was maximal within 24 h of exposure. Two-color immunofluorescence analysis demonstrated that CD25 was not expressed by cells of lymphoid lineage or by mature monocytes, but rather was present on cells that coexpressed CD13, CD33, CD34, MY8, and HLA-DR, and that lacked CD14 or CD11b, thus placing the CD25+ cells at or near the myeloblast stage of differentiation. An identical phenotype was found for CD25+ cells induced by GM-CSF. Cycloheximide completely inhibited the IL-3-induced expression of CD25, indicating the necessity for protein synthesis, and although most of the CD25+ cells were in G0/G1 phase, 25% of the cells were in S or G2M phase, indicating that receptor expression was not cell-cycle dependent. The p75 chain of IL-2R was not detected on the CD25+ cells. IL-3 was also found to directly induce CD25 in greater than 46% of SBA-E-BM enriched for CD34+ cells by panning. Consistent with the expression of only p55 IL-2R, the functional activity of IL-2 on enriched CD34+ cells exposed to IL-3 could not be demonstrated in either granulocyte-macrophage colony-forming unit (CFU-GM) assays or proliferation assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolonged remission of accelerated phase Philadelphia chromosome negative chronic myeloid leukemia following autologous recovery of normal hematopoietic elements after busulfan/cyclophosphamide and allogeneic marrow transplantation.

We describe here a patient with accelerated phase Philadelphia chromosome (Ph1) negative chronic myelogenous leukemia without BCR gene rearrangement, who received an allogeneic bone marrow transplant following a conditioning regimen consisting of busulfan (BU) and cyclophosphamide (CY). Hematopoiesis was restored following splenectomy performed 1 month post-transplant. There were no distinguishing cytogenetic differences between donor and host. Five years post-transplant the patient relapsed with the original disease. Restriction fragment length polymorphism (RFLP) studies performed at that time exhibited host specific DNA markers suggesting recurrent leukemia of host origin. RFLP analysis of the cells cryopreserved immediately post-transplant also revealed all cells to be of host origin. This patient experienced 5 years of remission with autologous hematopoietic recovery from an aggressive myeloproliferative disorder after high dose BU and CY without engraftment of donor hematopoietic cells.

Cord blood transplants: potential utility and potential limitations.

Cord blood has been utilized successfully for the hematopoietic reconstitution of children with lethal disorders of hematopoiesis, as an alternative to marrow derived stem cells. The majority of the transplants performed to date have utilized umbilical cord blood from HLA-identical siblings, however, much of the interest in this modality is due to its potential as a source of readily available unrelated stem cells. Cord blood offers intriguing theoretical advantages over the use of unrelated bone marrow, but additionally suffers from several limitations as well. This review is intended to highlight a number of these issues, rather than to serve as a detailed review of the clinical experience with cord blood transplantation to date.

Characterization of the in vitro sensitivity of human lymphoid and hematopoietic progenitors to L-leucyl-L-leucine methyl ester.

L-leucyl-L-leucine methyl ester (LLME) is a lysosomotropic agent that in microMolar concentrations has been found to be selectively toxic to human and murine precursor and effector cytotoxic cells, irrespective of their surface membrane phenotype. We describe a new method of synthesis of LLME and evaluate the effects of this preparation on human lymphoid and hematopoietic progenitor cells. The new method of synthesis did not change the previously characterized activities of LLME. Consistent with previous observations, NK effectors, LAK precursors and effectors, and allospecific CTL (aCTL) effectors were completely ablated by treatment with 50-250 microM LLME, while the activities of helper T cells and B cells were preserved after treatments of up to 1000 microM LLME. The effects of LLME treatment on human marrow-derived erythroid, myeloid, and monocyte progenitors have not been previously described. We found that the growth of each of these committed precursors was reduced or eliminated following treatment with 100-250 microM LLME. Admixture of LLME-treated marrow with marrow depleted of T cells and other mature cellular elements resulted in increased growth of myeloid and erythroid colonies suggesting that cells that could provide colony-enhancing activities were preserved. In contrast to previous studies in humans, we found a minority of individuals to have aCTL precursors that were partially resistant to LLME. PBL from 10 of 15 individuals tested showed nearly complete ablation of aCTL precursors following treatment with 375 microM LLME. The remaining 5 individuals demonstrated significant aCTL precursor activity after identical treatment. The resistance to LLME was restricted to aCTL precursors, and neither increasing the dose of LLME nor prolonging the time of treatment completely overcame the resistance. The pattern of susceptibility (sensitive versus resistant) was found to be independent of the degree or type of HLA disparity between responder and stimulator. LLME-treated cultures with and without CTL activity contained a predominance of CD4+ T cells. However, in the subjects tested LLME-resistant aCTL was shown to be CD8+. In vitro priming of aCTL precursors from sensitive individuals did not consistently result in the development of resistance to LLME. These data indicate that further studies are needed to evaluate the effects of LLME on human stem cells and to determine the potential role of resistant aCTL precursors in GvHD prior to application of this technique as a form of selective T cell depletion in humans.

B-cell differentiation following autologous, conventional, or T-cell depleted bone marrow transplantation: a recapitulation of normal B-cell ontogeny.

The circulating lymphocytes of 88 consecutive patients following autologous, conventional, or T-cell depleted bone marrow transplantation were serially analyzed for B-cell surface antigen expression and function. In the majority of patients, except for those who developed chronic graft-versus-host disease, the number of circulating CD20+ B cell normalized by the fourth posttransplant month. The earliest detectable B cells normally expressed HLA-DR, CD19, surface immunoglobulin (slg), CD21, Leu-8, and lacked expression of CD10 (CALLA). In addition, the circulating B cells expressed CD1c, CD38, CD5, and CD23 for the first year following transplant, antigens that are normally expressed on a small percentage of circulating B cells in normal adults, but highly expressed on cord blood B cells. Similar to cord blood B cells, patient B cells isolated during the first year following transplant, proliferated normally to Staphylococcus aureus Cowan strain I (SAC), and produced IgM, but minimal or no IgG when stimulated with pokeweed mitogen and SAC, unlike normal adult B cells that produce both. The similar phenotype and function of posttransplant and cord blood B cells, and their similar rate of decline in patients and normal children adds further evidence to support the hypothesis that B-cell differentiation posttransplant is recapitulating normal B-cell ontogeny.

Cytotoxic and proliferative T-cell clones with antidonor reactivity from a patient transplanted for severe combined immunodeficiency disease.

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.