Nancy H. Collins

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.

Evaluation of HLA‐Haplotype Disparate Parental Marrow Grafts Depleted of T Lymphocytes by Differential Agglutination with a Soybean Lectin and E‐Rosette Depletion for the Treatment of Severe Combined Immunodeficiency

Abstract. The factors that impact upon successful bone marrow transplantation leading to immunologic reconstitution in severe combined immune deficiency (SCID), Wiskott‐Aldrich syndrome, and in other lethal congenital immunodeficiencies are reviewed. Evidence is presented that graft‐versus‐host disease (GVHD) can be abrogated by the depletion of T cells, even from histoincompatible marrow grafts. However, graft resistance or restricted immune reconstitution has been observed with significant frequency. The bases for T cell reconstitution and limitations in B cell humoral immune recovery in the postgrafting period are reviewed, together with emerging evidence that pretransplant cytoreduction might obviate some of these problems.

Distinct Responses of Human Monocyte Subsets to Aspergillus fumigatus Conidia

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.

Transplantation in Remission Improves the Disease-Free Survival of Patients with Advanced Myelodysplastic Syndromes Treated with Myeloablative T Cell-Depleted Stem Cell Transplants from HLA-Identical Siblings

From 1985 to 2004, 49 patients with advanced myelodysplastic syndromes (MDS) (> or =5% blasts) or acute myeloid leukemia (AML) transformed from MDS underwent T cell depleted bone marrow or peripheral blood hematopoietic stem cell transplantation (HSCT) from HLA-identical siblings following conditioning with a myeloablative regimen that included total body irradiation (44 patients) or busulfan (5 patients). Thirty-six patients received chemotherapy (3 low dose and 33 induction doses) before conditioning, and 13 patients did not receive any chemotherapy. Prior to transplantation, 22 of the 36 treated patients were in hematologic remission; 4 were in a second refractory cytopenia phase (26 responders); 8 had failed to achieve remission; and 2 of the responders had progression or relapse of their MDS (10 failures). No post-transplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD) was given. The median age was 48 yrs (range 13-61). Forty-five of the 49 patients engrafted; 2 had primary graft failure; and 2 died before engraftment. Only 3 patients developed acute GVHD (aGVHD) (grades I and III) and 1 chronic GVHD (cGVHD). At 3 yrs post-transplantation, the overall survival (OS) was 54% in the responders; 31% in the untreated group; and 0% in the failure group (P=.0004). The disease free survival (DFS) was 50%, 15% and 0% in each group respectively (P=.0008). In multivariate analysis, disease status before cytoreduction remained highly correlated with DFS (P<.001). The cumulative incidence (CI) of relapse at 2-yrs post-transplantation for the responders was 23%; for the untreated group was 38%; and for the failures was 50%. The CI of non-relapse mortality at 2-yrs post-transplantation, for the responders was 23%; for the untreated group was 38%; and for the failures was 40%. All survivors achieved a Karnofsky Performance Status (KPS) of > or =90. These results indicate that patients with advanced MDS who achieve and remain in remission or a second refractory cytopenia phase with chemotherapy before conditioning can achieve successful long-term remissions following a myeloablative T cell depleted allogeneic HSCT.

Stem cell transplantation for the treatment of Fanconi anaemia using a fludarabine‐based cytoreductive regimen and T‐cell‐depleted related HLA‐mismatched peripheral blood stem cell grafts

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 × 109/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.

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.

Carbohydrate differences in human high molecular weight antigens of B- and T-cell lines

A group of high molecular weight cell-surface glycoproteins characteristic of hematopoietic cells has been described in mice (Trowbridge et al. 1975), rats (Fabre and Williams 1977), and humans (Omary et al. 1980, Dalchau et al. 1980). Different subsets of lymphocytes express different forms of these antigens (Trowbridge 1978, Hoessli and Vassalli 1980). These families of glycoproteins have been analyzed biochemically (Trowbridge et al. 1975, Trowbridge et al. 1976, Andersson and Gahmberg 1978) and by the precipitation of specific components with xenogeneic antisera (Fabre and Williams 1977, Hoessli and Vassalli 1980, Andersson and Metzger 1978, Niaudet and Greaves 1980) and with mouse monoclonal antibodies (Trowbridge 1978, Omary et al. 1980 Dalchau et al. 1980, Dalchau and Fabre 1981, Coffman and Weissman 198l). In these studies, the major high molecular-weight glycoproteins of B-lymphocytes and B-cell lines were found to be larger than the ~corresponding components of T-lymphocytes and T-cell lines (Trowbridge 1978, Sunderland et al. 1979, Omary et al. 1980, Hoessli and Vassalli 1980, Dunlap et al. 1980, Coffman and Weissman 1981, Berger et al. 1980). The high and low molecular weight species are, however, considered to be related, as antibodies to o n e population react with the other. Also, peptide maps generated from immunoprecipitated glycoproteins from Band T-cells have been shown to be very similar or identical (Omary et al. 1980, Dunlap et al. 1980). In general, however, the high molecular weight forms (M r = 200 000-220 000) were considered to be characteristic of B-cells whereas the lower molecular weight species (Mr=170000-200000) predominated in T-cells. In this communication, we analyze the high molecular-weight glycoproteins of human hematopoietic cells using a new mouse monoclonal antibody* (moAb 4C). MoAb 4C was produced by the hybridoma method of K6hler and Milstein (1975) after the immunization of BALB/c mice with phytohemagglutinin-stimulated

Characterization of B cells in severe combined immunodeficiency disease

The circulating lymphoid cells of eight consecutive untreated infants with severe combined immunodeficiency disease (SCID) with B cells were analyzed for surface marker expression and function. The B cells of these children expressed sIg, HLA-DR, CD19 (B4), CD20 (B1), CD21 (B2), Leu-8, and lacked expression of CD10 (CALLA), as do normal peripheral blood B lymphocytes. SCID B cells also expressed antigens that are normally absent or present on only a minor subset of circulating adult B lymphocytes, including CD1c (M241), CD38 (OKT10), CD23 (PL13), with or without concomitant CD5 (Leu-1) expression. The B cells of these children were capable of proliferating in vitro when stimulated with Staphylococcus aureus Cowan I. However, in the presence of pokeweed mitogen, S. aureus Cowan I, and normal T cells, the sIg+ cells of these children produced only IgM. Studies performed on normal B cells obtained from cord blood, young children, and adults reveal that whereas cord blood B cells are predominantly CD1c, CD38, and CD23 positive, B-cell expression of these antigens decreases with age. Cord blood B cells, similar to SCID B cells, produce only IgM when stimulated in vitro with pokeweed mitogen and S. aureus Cowan I. Based on these observations, we hypothesize that SCID B cells represent a population of B cells present during normal B-cell ontogeny which becomes a minor subset when an individual develops full immunologic competence.

The Effect of the Composition of Unrelated Donor Bone Marrow and Peripheral Blood Progenitor Cell Grafts on Transplantation Outcomes

To test the hypothesis that the outcome of hematopoietic stem cell (HSC) grafts is at least partially determined by the cellular composition of the graft, the National Marrow Donor Program (NMDP) analyzed the correlation of cellular phenotypes of unrelated grafts with graft outcome. Samples from 94 bone marrow (BM) and 181 peripheral blood progenitor cell (PBPC) grafts for transplantations at 40 U.S. transplant centers between 2003 and 2005 were analyzed at a single immunophenotyping reference laboratory. Samples were shipped from transplant centers upon receipt of graft. Graft cellular composition included analysis of leukocyte total cell numbers, and subsets of myeloid [CD34(+), CD34(+) CD38(-)], lymphoid [CD3(+), CD3(+) CD4(+), CD3(+) CD8(+)], and activated lymphoid cells [CD3(+) CD25(+), CD3(+) CD69(+), CD3(+) HLA-DR(+)] coexpressing CD3(+). There was substantial variability in the cellular composition of BM and PBPC grafts before and after graft processing by red blood cell (RBC) removal or plasma depletion in preparation for transplant. With BM grafts, cellular composition was not associated with hematopoietic recovery, graft-versus-host disease (GVHD), or survival. With PBPC grafts, survival rates were higher with CD34(+)>5 x 10(6)/kg, 59% compared to 34% with CD34(+)< or =5 x 10(6)/kg at 1 year. Platelet recovery was higher with PBPC containing CD3(+) CD8(+) >8 x 10(7)/kg. Neutrophil recovery or GVHD could not be predicted by any cellular subsets of PBPC grafts. Although survival was superior with PBPC grafts containing >5 x 10(6) CD34(+)/kg, an optimal graft mix of myeloid, lymphoid, and activated lymphoid subsets was not identified.

Quantitation of GD2 synthase mRNA by real-time reverse transcription-polymerase chain reaction: Utility in bone marrow purging of neuroblastoma by anti-GD2 antibody 3F8

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.