O'Reilly Rj

Graft rejection in recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia. Identification of host-derived antidonor allocytotoxic T lymphocytes.

Clinical trials with bone marrow depleted of donor T lymphocytes indicate that both the incidence and severity of graft-versus-host disease (GVHD) in patients undergoing bone marrow transplantation (BMT) for treatment of leukemia are greatly reduced. However, there has been a concurrent increase in the incidence of graft rejection, particularly among recipients of HLA-nonidentical marrow grafts. In order to investigate the nature of graft failure, peripheral blood mononuclear cells (PBMC) present at the time of graft failure have been characterized by phenotypic and functional analyses in 5 recipients of HLA-nonidentical marrow grafts. Rejection of HLA-nonidentical marrow grafts was associated with the emergence of host-derived T lymphocytes in all 5 patients. In 3 of these patients, the cells could be tested directly for cell-mediated cytotoxicity. Antidonor cytotoxicity was detected in each of these 3 patients. In one patient the target specificity of the cytotoxic lymphocytes was identified as the donor class I HLA antigen, HLA-B7. None of the patient PBMC mediated cytotoxicity against the natural killer cell target K562.

Long-term follow-up of patients treated with daclizumab for steroid-refractory acute graft-vs-host disease.

Daclizumab has been shown to have activity in acute GVHD, but appears to be associated with an increased risk of infection. To investigate further the long-term effects of daclizumab, we performed a retrospective review of 57 patients who underwent an allogeneic hematopoietic stem cell transplant from January 1993 through June 2000 and were treated with daclizumab for steroid-refractory acute GVHD. The median number of daclizumab doses given was 5 (range 1–22). GVHD was assessed at baseline, days 15, 29 and 43. By day 43, 54% patients had an improvement in their overall GVHD score, including 76% patients aged ⩽18. Opportunistic infections developed in 95% patients. Forty-three patients (75%) died following treatment with daclizumab. The causes of death included active GVHD and infection (79%), active GVHD (5%), chronic GVHD (2%) and relapse (14%). Patients with grade 3–4 GVHD had a significantly shorter median survival than patients with grade 1–2 GVHD (2.0 vs 5.1 months, P=0.001). Daclizumab has no infusion-related toxicity, is active in steroid-refractory GVHD, especially among pediatric patients, but is associated with significant morbidity and mortality due to infectious complications. Careful patient selection and aggressive prophylaxis against viral and fungal infections are recommended.

Quantitation of T lymphocytes in human bone marrow by a limiting dilution assay

A limiting-dilution microculture assay (LDMA) for quantitation of T lymphocytes in human bone marrow is described. Phytohemagglutinin (PHA)-responsive T cells are maintained in interleukin 2 (IL-2)-containing medium with feeder cells in a total volume of 20 μl. After 16 days of culture, each well is scored by microscopic examination as positive or negative based on the presence or absence of cell growth. A limiting dilution analysis of the relationship between the number of cells seeded per well and the fraction of wells without growth demonstrate that the data are consistant with single-hit kinetics. Minimum chi square statistics were used to establish the line of best fit to calculate the T lymphocyte frequency in a sample. This method for enumeration of T cells was applied to untreated samples of bone marrow, soybean-agglutinin-negative (SBA-) marrow, and soybean-agglutinin-negative marrow cells subjected to a single sheep red blood cell (SRBC) rosette (SBA-E-) or double SRBC rosette (SBA-E-E-) depletion. It was demonstrated that the LDMA can detect as few as 4.3x105 T cells in a total of 109 bone marrow mononuclear cells. The assay system also allows for a comparison of T lymphocytes in the untreated marrow with the T-cell-depleted marrow samples. The mean number of T cells in untreated marrow was 1x109 and in T-cell-depleted samples 4.3x105. This corresponds to a 3.5 log or 99.96% reduction in total T cell number by the SBA-E-rosette technique. The phenotypic analysis of single positive wells as well as pooled cells from all positive wells indicate that at least 95% of the wells scored microscopically as positive for T cell growth did in fact contain T cells. The assay requires only 1x106 mononuclear cells for complete analysis and, therefore, compares favorably with previously published methods.

Final height in pediatric patients after hyperfractionated total body irradiation and stem cell transplantation.

Impaired linear growth has been shown to occur in individuals treated during childhood with single-dose and fractionated total body irradiation (TBI) before stem cell transplantation. Our objective was to describe the final heights attained and patient/treatment factors correlating with final height in a cohort of childhood cancer survivors treated with hyperfractionated TBI (total dose 1375 or 1500u2009cGy). Thirty individuals (18 men) were included in the study. The mean final height standard deviation score (s.d.s.) was −1.9±0.2, significantly lower than height s.d.s. at TBI (−0.2±0.2, P<0.001). Final height s.d.s. was significantly correlated with age at diagnosis, age at TBI and target height (P=0.04, P<0.001, P<0.001, respectively). Treatment with growth hormone (GH) (n=7) maintained mean height s.d.s. at −2.0 from the onset of GH therapy until attainment of final height. The mean final sitting height s.d.s. was −2.2±0.2 (n=16), significantly shorter than mean final standing height s.d.s. (P<0.01). In conclusion, treatment with hyperfractionated TBI is associated with a reduction in standing height and an even greater reduction in sitting height. Final height after hyperfractionated TBI was similar to that reported after fractionated TBI.

Distribution of HLA genotypes in families of patients with acute leukemia. Implications for transplantation.

The HLA genotypes of members of 104 families of patients with acute myelogeneous leukemia (AML), 89 families of patients with acute lymphocytic leukemia (ALL), and 40 control families were analyzed. The results indicate that the HLA genotype of both ALL and AML leukemic patients is found more frequently than the expected 25% among their siblings; the increase for AML patients is statistically significant. This suggests that an HLA-linked factor(s) conferring susceptibility to leukemia may also be associated with increased gametic survival. The chance of finding an HLA-matched sibling for bone marrow transplantation is increased.

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.

Hyperfractionated total lymphoid irradiation and cyclophosphamide for preparation of previously transfused patients undergoing HLA-identical marrow transplantation for severe aplastic anemia

PURPOSEnTo assess the immunosuppressive capacity of hyperfractionated total lymphoid irradiation and cyclophosphamide for transplantation of unmodified allogeneic marrow in sensitized aplastic anemia patients.nnnMETHODS AND MATERIALSnFrom February 1983 to September 1990, 23 multiply transfused aplastic anemia patients underwent unmodified bone marrow transplantation from HLA genotypically identical sibling donors following preparation with 6 Gy hyperfractionated total lymphoid irradiation and 160 mg/kg cyclophosphamide. Graft-versus-host disease prophylaxis included steroids in one patient, methotrexate in four, cyclosporine in seven, and methotrexate/cyclosporine in 12. There were 17 males and 6 females with a median age of 13 (range: 2.5-32).nnnRESULTSnOne patient died early before engraftment of bacterial sepsis. Twenty-two patients were evaluable for engraftment. Three experienced graft failure including one primary, and two late graft failures associated with cyclosporine withdrawal. Acute graft-versus-host disease occurred in 7/22 (> or = grade II in 6), and chronic graft-versus-host disease in 3/17 patients. Except for a patient who received total body irradiation for a second transplant, no patient in this series developed interstitial pneumonia. Fifteen patients are alive with follow-up of 38-125 months (median 68). The overall actuarial survival at 5 years is 69%, at 8 years it is 60%, with one late death. The survival of adult patients was similar to that of younger patients (> or = 16 years old: 63%, < 16 years old: 55%). The development of acute graft-versus-host disease adversely influenced survival (88% with Grade 0-I, 17% with grade II-IV; p = 0.002). No hypothyroidism or secondary malignancies have been documented in this series.nnnCONCLUSIONnPretransplant immunosuppression with 6 Gy of hyperfractionated total lymphoid irradiation and 160 mg/kg CY reduces but does not eliminate the incidence of graft failure in sensitized aplastic anemia patients. The dose and the mode of administration of total lymphoid irradiation in this trial may be associated with a lower incidence of late side effects. Survival is comparable to that obtained using preparative regimens without radiation.

In vitro differentiation of human marrow T cell precursors by thymic factors in severe combined immunodeficiency.

Marrow cells from 16 patients with severe combined immunodeficiency diseases (SCID) were examined for the presence of T cell precursors which could be induced to express surface markers and functions of T lymphocytes after exposure in vitro to thymic extracts or peptides of thymic origin (thymopoietin and thymopoietin32-36 (TP-5). Marrow cells from 14 patients studied before transplantation revealed three patterns of response. In five patients, inducible T cell precursors were not detected. In six patients, precursors were detected which could be induced to express a human T lymphocyte antigen (HTLA) but acquired little or no ability to rosette with sheep erythrocytes (SRBCs). Induction of HTLA-positive and E rosette-positive lymphocytes was normal in only two patients, both of whom were engrafted with maternal lymphocytes as an apparent result of an intrauterine infusion. Induction of mixed leukocyte reaction (MLR) responses following exposure to thymic hormones was also observed in one of these two patients. In no case could significant responses to mitogens be elicited. Following transplantation, marrow cells from fully engrafted, immunologically reconstituted patients could be induced to bear HTLA, to form E rosettes, and to respond to mitogens and allogeneic cells following exposure to thymic extracts, thymopoietin, or TP-5. Thus, most patients with SCID manifest differentiative abnormalities intrinsic to lymphoid precursors which are corrected following engraftment of functioning allogeneic lymphoid precursors from normal donors.