Immunoreactivity of umbilical cord blood and post-partum maternal peripheral blood with regard to HLA-haploidentical transplantation.

Recent clinical reports have demonstrated that umbilical cord blood (CB) may be utilized as a source of transplantable hematopoietic stem cells when bone marrow is not available. However, it is not apparent that CB can be used to transplant partially HLA-matched siblings or matched, non-familial recipients. In this study the immunoreactivity of CB has been investigated within familial confines; 14 families were analyzed at the time of birth of their child and six of these families were reassessed at 6 months post-partum. In mixed lymphocyte reactions, CB was unable to significantly respond to stimulation with cells from either the mother or father. Furthermore, unlike adult peripheral blood, CB displayed depressed immune responses to alloantigen and T cell mitogen. At 6 months, the immune responses of the infant demonstrated normal development in terms of alloantigen and mitogen responses. However, at 6 months both the mother and the infant demonstrated a continued immune tolerance to one another. The data suggest that CB could be used in familial transplant situations when siblings are HLA-haploidentical if the donor/recipients are chosen based on the paternal haplotype. Furthermore, maternal bone marrow harvested during the 6 months immediately following delivery of a child also should be suitable as a stem cell graft in haploidentical situations.

Collection, separation and cryopreservation of umbilical cord blood for use in transplantation.

Bone marrow transplantation (BMT) is limited by the paucity of HLA-matched donors and the frequent occurrence of graft-versus-host disease (GVHD). Recent clinical reports have implied that the use of umbilical cord blood (UCB) may alleviate some of the problems associated with BMT. Banks of frozen UCB could make the problem of finding suitable stem cell donors easier and stem cell grafts would be more readily available. However, definitive experiments are needed to develop optimal methods for collection, separation and storage of cryopreserved UCB for extended periods of time. We have found that several simple techniques may be utilized to collect large volumes of UCB (up to 220 ml). Also, modification of a common density gradient separation method permits recovery of large quantities of UCB mononuclear cells. Finally, we have examined the effects of prolonged frozen storage on the ability to recover viable and functional UCB, particularly stem/progenitor cells. It was observed that storage of UCB in liquid nitrogen for as long as 7 years had minimal effects on cell viability, cellular composition of UCB and progenitor/stem cell capacity. Thus, the establishment of UCB banks for use in transplantation appears to be a feasible approach.

Human bone marrow and umbilical cord blood cells generate CD4+ and CD8+ single-positive T cells in murine fetal thymus organ culture.

Murine fetal thymus lobes isolated from both normal and scid/scid mice can be colonized by donor cells from either human bone marrow or human umbilical cord blood in vitro. Subsequent organ culture results in a transient production of a few CD4+ CD8+ (double-positive) cells and then the accumulation of CD4+ or CD8+ (single-positive) T cells. A significant number of immature T-cell intermediates (e.g., CD8low, CD3-/low cells) were present in early organ cultures, suggesting that these were progenitors of the mature CD3+/high single-positive T cells that dominated late cultures. Depletion of mature T cells from the donor-cell populations did not affect their ability to colonize thymus lobes. However, colonization depended on the presence of CD7+ progenitor T cells. Limiting dilution experiments using mature T-cell populations (human peripheral blood leukocytes, human bone marrow cells, and human umbilical cord blood cells) suggested that thymic organ culture supports the growth of progenitor T cells but does not support the growth of mature human T cells. Each of these donor populations produced single-positive populations with different CD4/CD8 ratios, suggesting that precursor cells from different sources differ qualitatively in their capacity to differentiate into T cells.

Analysis of the hairless mouse as a model for the effects of aging on the immune system.

Studies into the effects of aging on the immune system are hampered by the lack of a suitable animal model that is readily available and cost efficient. The mutant mouse, hairless (hr/hr genotype), has been shown to undergo an accelerated thymic involution with accompanying immunodeficiency. Thus, this strain of mouse has been proposed as a model for studying the interactions of aging and immune function. We have investigated the effects of homozygous hr gene expression over time on the immune function of these mice. It was observed that homozygous hr gene expression had minimal effects on peripheral lymphocyte subset compositions but did appear to result in changes in thymic differentiation. Further, hr/hr mice displayed decreased proliferative responses to IL2 and mitogen stimulation, although cytotoxic responses (both NK and T cell mediated) appeared normal. These defects appear to be attributable to T helper cell dysfunction. Each of the changes found in hr/hr mice were distinct from those seen with age-matched control mice. Thus, the hr/hr inbred strain of mouse does not appear to be a suitable model for use in analyzing the effects of aging on the immune system.

Adoptive transfer of skin-selective autoimmunity induced by Skn alloantigenic disparities.

Two unlinked genes of the mouse, Skn-1 and Skn-2, each with alterative alleles, specify alternative cell-surface Skn alloantigens expressed only by epidermal and neural cells. C57BL/6 (B6) and A/J (A) strain mice differ at both Skn loci. Thus lethally irradiated B6 mice restored with (B6 x A)F1 hybrid hematopoietic cells [(B6 x A)/B6 chimeras] reject A strain (Skn-incompatible) skin grafts. Our studies were designed primarily to test the inference that (B6 x A)F1 lymphoid cells, after differentiating in B6 recipients, which lack the Skn alloantigens of A strain mice, may make an Skn-related, skin-selective autoimmune response when returned to their native (B6 x A)F1 habitat. Severe cutaneous lesions did, indeed, ensue after spleen cells of (B6 x A)/B6 chimeras were transferred to (B6 x A)F1 recipients, provided that three conditions were met--namely, (i) priming of the (B6 x A)/B6 chimeric donor by grafting and rejection of Skn-incompatible A strain skin grafts, (ii) stimulation of the recipient's skin as from shaving, at which sites the lesions were mainly located, and (iii) pretreatment of the (B6 x A)F1 recipients with cyclophosphamide or sublethal irradiation. Spleen cells of control female chimeras primed by grafting and rejection of H-Y (Skn-compatible) B6 male skin failed to incite the Skn-typical cutaneous lesions in (B6 x A)F1 recipients, indicating that these lesions were Skn-specific and not a nonspecific consequence of incompatible skin grafting per se. Normally compatible A strain skin grafts, but not Skn-compatible B6 skin grafts, were rejected by cyclophosphamide-treated (B6 x A)F1 recipients of (B6 x A)/B6 spleen cells from Skn-primed chimera donors. Treatment of primed chimeras' spleen cells with antiserum to H-2a (A strain) specifically abolished their capacity to adoptively incite the Skn-related autoimmune syndrome, confirming that the immune cells responsible are of (B6 x A)F1 origin and are not residual B6 derivatives. These findings add weight to the status of Skn systems as agents of tissue-selective histoincompatibility and, perhaps, of clinical disorders with a known or suspected autoimmune basis affecting the skin.

Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes.

Successful implementation of bone marrow transplantation for hematopoietic reconstitution is limited by the lack of suitably HLA-matched donors and by the occurrence of graft-versus-host disease that frequently accompanies this procedure. Recent clinical reports have implied that the use of umbilical cord blood as a source of transplantable stem cells may solve these problems. To date, definitive experiments have not been performed to assess the immunological potential of T cells found in umbilical cord blood, which could mediate graft-versus-host disease. In the present study we have observed that umbilical cord blood contains T lymphocytes that appear to be phenotypically immature. In addition, umbilical cord blood lymphocytes appeared to be functionally immature as shown by minimal responses to stimulation with interleukin 2, phytohemagglutinin, or alloantigens. Thus, umbilical cord blood may be more suitable for allogeneic transplantation than bone marrow in that these cord blood cells may not be as capable of mediating graft-versus-host disease.

Expression of urinary H-2 odortypes by infant mice.

The extended H-2 complex of genes in the mouse includes at least three loci that independently specify distinctive body odors, "odortypes," whose differential recognition influences mating choice and affects the maintenance of early pregnancy. A prime experimental method of identifying H-2 odortypes is the specially designed Y-maze in which mice are trained, by water deprivation and reward, to distinguish odors conducted to the arms of the maze from H-2-dissimilar mice or their urines. It is confirmed that H-2-dissimilar infant mice, unlike adult mice, are not distinguished by trained mice in the Y-maze. However, a previous conclusion that infant mice do not express H-2 odortypes is shown to be incorrect, because the urines of H-2-dissimilar infant mice, even at 1 day of age, were distinguished in the Y-maze. Thus urine, ingested by the mother, clearly could suffice for her to distinguish her own from other H-2-dissimilar pups. Further, urine would seem to be a unique source of H-2 odortypes. If, as we believe, H-2 odortypes represent mostly compound odors composed by H-2 genetic variation in the urinary output of odorous metabolites, as distinct from simple odors that depend on chemical differences of single odorants, then the kidney, which is not responsible for H-2 odortype specificity, may nevertheless impart a unique character to urinary odortypes by virtue of differential excretion/resorption processing of various constituent odorous metabolites. In that case, various organs and tissues, among which the hematopoietic/lymphoid system is known to contribute to H-2 odortype specificity, may exhibit tissue-specific varieties of H-2 odortypes, their products having not yet been subjected to renal processing.

Expression of type IV collagenase correlates with the invasion of human lymphoblastoid cell lines and pathogenesis in SCID mice.

An in vitro model, called the Membrane Invasion Culture System (MICS), was used to study the invasive potential of an Epstein-Barr virus (EBV) positive lymphoblastoid cell line (LCL), an EBV-negative Burkitt lymphoma (BL) cell line of American origin and an EBV-positive BL of African origin. MICS measured the ability of these cell lines to invade reconstituted basement membrane-coated filters, which correlated with their tumorigenic and metastatic capabilities in a SCID mouse model. Furthermore, the significantly greater invasive behaviour of the EBV-positive LCL was directly correlated with the cells' ability to express and secrete human type IV collagenase (72 kDa), an important metalloproteinase responsible for the degradation of collagen IV in basement membranes. The data suggest that MICS and the SCID mouse are useful tests of tumorigenicity in lymphoid cells, with measurable effects in both systems related to human type IV collagenase activity. Both models allow further exploration of malignant phenotypes associated with EBV transformation of lymphoid tissues.

Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation.

This paper reviews our recent laboratory and clinical studies demonstrating the efficacious use of human umbilical cord blood for HLA-matched allogeneic sibling stem/progenitor cell transplantation in cases of Fanconi's anemia. Future implications and potential problems are discussed with regards to (a) the possibility of maternal cell contamination, (b) the broadness of applicability with regards to other diseases that might be transplanted, and whether such transplants are feasible in adults, as well as in children, and (c) the immunological reactivity of cord blood cells, and whether these cells can be used to cross histocompatibility barriers more easily than that of bone marrow from adults.

Regulation of alternative splicing in the generation of isoforms of the mouse Ly-5 (CD45) glycoprotein.

Alternative splicing generates various Ly-5 glycoprotein isoforms of the cell surface that typify different cell lineages and stages of hematopoietic differentiation in the mouse; exons 4-6 are incorporated to generate a B-cell isoform (B220) and excluded from a T-cell isoform (T200), the other coding exons (3 and 7-33) being shared. As a first step to understanding the mechanisms regulating Ly-5 alternative splicing, and thus determining Ly-5 isoforms, a minigene representing exons 3-7 was transfected into Ly-5-expressor T cells and B cells and into nonexpressor L cells for comparison of splicing patterns. We conclude that all the information required for faithful splice-site selection according to cell type is contained within the resulting pre-mRNA. The splicing pattern manifested by nonexpressor L cells may represent a default and nonregulated type. We postulate trans-acting factor(s) to account for the selection of appropriate exons, and we provide support for this interpretation from analysis of fused hybrid T-B cells, which exhibited B-cell specific Ly-5 transcripts. Splicing patterns were well conserved despite substantial disruption of constructs. However, extensive deletion analyses suggested that cis sequences flanking and within exon 6 affect the exclusion of that exon in T cells.

Further data on the selective expression of Ly-5 isoforms.

Ly-5 (CD45) glycoproteins of the mouse, expressed by all or most hematopoietic cell lineages and specified by a single Ly-5 gene, range in size from isoform T200 of T cells (the smallest), in which exons 4, 5, and 6 are not represented, to isoform B220 of B cells (the largest), in which all three of these optional exons are represented. The main purpose of the present study, utilizing the polymerase chain reaction (PCR), was to ascertain whether known isoforms of intermediate size are generated by single or dual usage of optional exons 4, 5, and 6. Transcripts representing all eight isoforms predictable from varied use of three exons were observed among a diverse panel of nine B-cell tumors in culture, but there was no evident concordance with known contrasting differential features that distinguish members of the B-cell tumor panel. No two B tumors exhibited the same variety of transcripts and the relative quantities of transcripts expressed varied greatly from tumor to tumor. Cloning of B-cell tumors did not alter their distinctive transcript patterns. Separation methods (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE) did not suffice to segregate all corresponding expressed isoforms but did establish that transcripts representing usage of a single optional exon and of two optional exons were actually translated, which supports a provisional inference that all eight isoforms exist. The considerable diversity of B-cell transcript phenotypes was not seen among seven T-cell leukemias, two cytolytic T-cell lines, and three Th 1 helper T-cell lines, all of which displayed a uniform phenotype comprising major expression of the T200 transcript (no optional exon) and minor expression of a transcript employing exon 5. However, a panel of five cloned Th2 T-cell lines, which represent a second and functionally different branch of the helper/inducer T-cell category, exhibited a characteristic transcript pattern which distinguished them from a panel of three Th1 T-cell lines. The major transcript in the Th2 lines was also T200, but the Th2 lines showed higher representation of transcripts containing optional exons. A single Th2 clone expressed an unusual transcript suggesting a potential isoform not compounded simply by varied inclusion of the three identified optional exons. After activation of the helper T-cell lines with concanavalin A (Con A), expression of transcripts containing optional exons appeared to decrease.

Human umbilical cord blood: a clinically useful source of transplantable hematopoietic stem/progenitor cells.

This is a review and discussion of studies leading to the first use of human umbilical cord blood, material usually discarded, for the provision of stem/progenitor cells for clinical hematopoietic reconstitution. This prospect arose as a result of extensive studies of the harvesting and cryopreservation of cord blood and of its numerical content of progenitor cells demonstrable in vitro. A male patient with Fanconi anemia (FA) was conditioned with a modified regimen of cyclophosphamide and irradiation that accommodates the abnormally high sensitivity to these agents that is characteristic of FA. Cryopreserved cord blood had been retrieved at birth from a female sibling known from prenatal testing to be unaffected by FA and to be human leukocyte antigen (HLA)-compatible with the prospective sibling recipient. After conditioning and therapeutic infusion of thawed cord blood, successful hematopoietic reconstitution was indicated by the general health of the patient, who had previously required supportive transfusions, by satisfactory hematological criteria and by counts of hematopoietic progenitor cells of various types in the bone marrow. Complete engraftment of the myeloid system with donor cells was evident from cytogenetics, ABO typing, study of DNA polymorphisms, and normal cellular resistance to cytotoxic agents that reveal the fragility of FA cells; the blood contained a residuum of host lymphocytes exhibiting chromosomal damage, but the trend has been towards eliminating these damaged cells. This implies that cord blood from a single individual should provide sufficient reconstituting cells for effective hematopoietic repopulation of an autologous or an HLA-compatible allogeneic recipient.

Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells.

The purpose of this study was to evaluate human umbilical cord blood as an alternative to bone marrow in the provision of transplantable stem/progenitor cells for hematopoietic reconstitution. Although no direct quantitative assay for human hematopoietic repopulating cells is at present available, the granulocyte-macrophage progenitor cell (CFU-GM) assay has been used with success as a valid indicator of engrafting capability. We examined greater than 100 collections of human umbilical cord blood for their content of nucleated cells and granulocyte-macrophage, erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, in many cases both before and after cryopreservation. First it was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy after transport of cord blood by overnight air freight carriage from a remote obstetrical service. Granulocyte-macrophage progenitor cells from cord blood so received responded normally to stimulation by purified recombinant preparations of granulocyte-macrophage, granulocyte, and macrophage colony-stimulating factors and interleukin 3. The salient finding, based on analysis of 101 cord blood collections, is that the numbers of progenitor cells present in the low-density (less than 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for major histocompatibility complex-matched allogeneic hematopoietic reconstitution.

Organization of the Ly-5 gene.

A single Ly-5 gene is known to generate a variety of transmembrane glycoprotein isoforms that distinguish various cell lineages and stages of differentiation within the hematopoietic developmental compartment of the mouse. Systems homologous to Ly-5 are known in rats and in humans. The complete exon-intron organization of the Ly-5 gene is described in this report. The Ly-5 gene occupies about 120 kilobases of chromosome 1 and comprises 34 exons, of which 32 (Ex-3 to Ex-34) are protein coding. Ex-1, Ex-2, and parts of Ex-3 and Ex-34 are untranslated. In all cDNA clones examined, either Ex-1 or Ex-2 was represented, but not both, implying that Ex-1 and Ex-2 in Ly-5 mRNA may be mutually exclusive. Primer extension and S1 nuclease protection mapping were used to identify initiation (cap) sites for transcription. The finding of putative cap sites for Ex-1 and Ex-2, and of corresponding TATA-like sequences, suggests the presence of two promoters. In both Ex-1+ and Ex-2+ cDNA clones the next exon is Ex-3, which has a translation-initiating codon. The intron between Ex-3 and Ex-4 is unusually long, about 50 kilobases. Evidence is given that Ex-5, like Ex-6 and Ex-7 (studied previously), is another alternative exon that is selectively programmed, alone or together with Ex-6 or Ex-7 or both, to generate actual or potential Ly-5 isoforms by alternative splicing.

Structural features of Ly-5 glycoproteins of the mouse and counterparts in other mammals.

The Ly-5 system of the mouse defines a set of transmembrane glycoprotein isoforms (T200, B220, etc) that hallmark various lineages and stages of hematopoietic differentiation. These isoforms are the products of a single Ly-5 gene comprising 34 exons, 32 of them (Exs-3-34) protein-coding and three (Exs-5-7) selectively represented in different isoforms (e.g., all three in isoform B220 but none in isoform T200). Probable structural features of Ly-5 glycoproteins, largely inferred from Ly-5 gene composition, are presented and compared with the rat L-CA and human LCA/T200 systems, which are phylogenetic counterparts of Ly-5 as an index of the extent and nature of structural conservation. The outer (N-terminal) region of the Ly-5 T200 isoform comprises three broadly similar domains (Exs-4, 8, 9) with salient features that jointly favor free interaction with the aqueous environment and are shared by the L-CA and human LCA/T200 systems despite an overall interspecies protein sequence similarity in this region of only about 50%. In the larger B220 isoform this region includes epitopes dictated by the selective exons Exs-5, 6, 7, these being more conserved than the shared exons Exs-4, 8, 9 and no doubt sustaining the differential functions of the respective isoforms. Comparison of the genomic sequences of Ex-5 in the Ly-5 and human systems suggests that a shift in splice donor site accounts for an extra 23 amino acids in the human Ex-5-coding domain, which is the only salient structural difference between the mouse Ly-5 and human systems. The inner extracellular region (Exs-10-16) includes subregions of high variability, but again there are shared salient interspecies similarities such as sites and numbers of Cys residues that imply a conserved, tightly-folded conformation, in contrast to the more open conformation predicted for the outer extracellular region. The transmembrane region (Ex-17) is highly conserved, as is the very large cytoplasmic region (Exs-17-34) which may interact with the plasma membrane but probably does not traverse it.

Alternative use of 5' exons in the specification of Ly-5 isoforms distinguishing hematopoietic cell lineages.

Previous inferences that Ly-5 glycoprotein isoforms of murine hematopoietic cells are generated by alternative splicing of primary transcripts of a single Ly-5 gene are supported by the present study. A cDNA library was prepared from B cells by extension from primer representing a known T-cell cDNA sequence. Three different Ly-5 clones from this library included sequences missing in T-cell cDNA clones. From the constitution of cDNA clones and of the Ly-5 gene, and from S1 nuclease mapping, it is concluded that at least two exons, provisionally numbered Ex-6(B) and Ex-7(B), in the 5'-proximal region are mainly represented in mRNA of the B-cell lines examined but not of the T-cell lines examined. Also, exons 1 and 2 appear to be used alternatively in different species of B-cell mRNA and probably also in different species of T-cell mRNA.

Sequences of Ly-5 cDNA: isoform-related diversity of Ly-5 mRNA.

The Ly-5 system of the mouse is expressed exclusively by hematopoietic cells and comprises a series of glycoprotein isoforms that typify different hematopoietic cell lineages. The 200-kDa isoform of T cells and the 220-kDa isoform of B cells are known to differ in peptide composition. The complete 1152 amino acid sequence of the 200-kDa isoform protein deduced from cDNA sequence appears to comprise a leader sequence of some 30 residues, an external N-terminal domain of 370 residues, a probably single transmembrane domain of 22 residues, and an unusually large cytoplasmic domain of 730 residues. Both the external and cytoplasmic domains include regions of internal homology suggestive of evolution from a smaller ancestral gene. RNA transfer blotting has previously shown that B-cell mRNA for Ly-5 is larger than T-cell mRNA. S1 nuclease protection mapping with Ly-5 cDNA probes suggests that this difference can be ascribed to interpolation of an extra B-cell sequence located at the 5' end of B-cell mRNA, probably immediately following the leader sequence. From restriction mapping of overlapping Ly-5 genomic clones spanning 60 kilobases it is concluded that Ly-5 isoforms are generated by differential processing of transcripts of a single gene, rather than from a family of linked Ly-5 genes.