Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation.

The duration of immunodeficiency following marrow transplantation is not known. Questionnaires were used to study the infection rates in 72 patients surviving 20 to 30 years after marrow grafting. Furthermore, in 33 of the 72 patients and in 16 donors (siblings who originally donated the marrow) leukocyte subsets were assessed by flow cytometry. T-cell receptor excision circles (TRECs), markers of T cells generated de novo, were quantitated by real-time polymerase chain reaction. Immunoglobulin G(2) (IgG(2)) and antigen-specific IgG levels were determined by enzyme-linked immunosorbent assay. Infections diagnosed more than [corrected] 15 years after transplantation occurred rarely. The average rate was 0.07 infections per patient-year (one infection every 14 years), excluding respiratory tract infections, gastroenteritis, lip sores, and hepatitis C. The counts of circulating monocytes, natural killer cells, B cells, CD4 T cells, and CD8 T cells in the patients were not lower than in the donors. The counts of TREC(+) CD4 T cells in transplant recipients younger than age 18 years (at the time of transplantation) were not different from the counts in their donors. In contrast, the counts of TREC(+) CD4 T cells were lower in transplant recipients age 18 years or older, even in those with no history of clinical extensive chronic graft-versus-host disease, compared with their donors. The levels of total IgG(2) and specific IgG against Haemophilus influenzae and Streptococcus pneumoniae were similar in patients and donors. Overall, the immunity of patients surviving 20 to 30 years after transplantation is normal or near normal. Patients who received transplants in adulthood have a clinically insignificant deficiency of de novo-generated CD4 T cells, suggesting that in these patients the posttransplantation thymic insufficiency may not be fully reversible.

Hematopoietic responses to stress conditions in young dogs compared with elderly dogs.

Clinical observations show that older patients do not tolerate high-dose chemoradiotherapy as well as younger patients. It is unclear whether this is due to age-related differences in their responses to hematopoietic injury or to differential toxicities to other organs. In the present study, 6 young (0.5 years) and 6 elderly (8 years) dogs were challenged with 7 repeated nonlethal doses of 50 or 100 cGy total body irradiation (TBI) each (total 550 cGy), and 21 days of recombinant canine granulocyte-colony stimulating factor (rcG-CSF) after the last TBI dose. Recoveries of absolute neutrophil, platelet, and lymphocyte counts after each TBI dose, responses to rcG-CSF treatment, and telomere lengths in neutrophils were compared before and after the study. No differences were found in recoveries of neutrophils, platelets, or in responses to rcG-CSF among young and old dogs. In contrast, recoveries were suggestively worse in younger dogs. After rcG-CSF, platelet recoveries were poor in both groups compared with previous platelet recoveries (P <.01). Consequently, 2 old and 3 young dogs were euthanized because of persistent thrombocytopenia and bleeding. At the study's completion, marrow cellularities and peripheral blood counts of the remaining young and elderly dogs were equivalent. The telomere lengths in both groups were significantly reduced after the study versus beforehand (P =.03), but the median attritions of telomeres were not different. It was concluded that aging does not appear to affect hematopoietic cell recoveries after repeated low-dose TBI, suggesting that poor tolerance of radiochemotherapy regimens in older patients may be due to nonhematopoietic organ toxicities rather than age-related changes in hematopoietic stem cells reserves.

Ex vivo expansion of canine dendritic cells from CD34+ bone marrow progenitor cells.

BACKGROUND: The aims of this study were to ex vivo expand canine dendritic cells and determine their phenotype and functional characteristics. METHODS: CD34+-selected cells and CD34+-depleted canine bone marrow (BM) cells were cultured in Iscove's modified medium for 14 days. Cytokines added to the cultures included human granylocyte/macrophage colony-stimulating factor 5 ng/ml, hFlt3 ligand 200 ng/ml, and human tumor necrosis factor-alpha 10 ng/ml. Cultured cells and purified subpopulations were assessed for cell surface antigen expression, morphology, and function by flow cytometric analysis, electron microscopy, and an allogeneic mixed lymphocyte reaction at day 14. RESULTS: Two main cell populations were identified, DR++(bright)/CD14- and DR+(dim)/CD14+. Ex vivo expanded CD34+-selected cells showed increased allostimulatory activity compared to both cultured CD34+-depleted cells and mononuclear cells. In contrast, ex vivo expansion from CD34+-depleted cells was unsuccessful. After sorting cells from the ex vivo expanded CD34+-selected bone marrow to enrich for DR++/CD14- cells, a 42-fold increase (median) of allostimulatory activity was observed as compared with sorted DR+/CD14+ cells (P=0.02). CONCLUSIONS: Cells with dentric cell-like phenotypes and functions can be cultured from canine CD34+-selected bone marrow cells. Future studies will address the roles of these cells in engraftment, graft versus host reactions and graft-host tolerance in a canine hematogoietic stem cell transplantaton model.

Polyclonal hematopoiesis with variable telomere shortening in human long-term allogeneic marrow graft recipients.

Donor-derived hematopoiesis was assessed in 17 patients who received allogeneic marrow grafts from HLA-matched siblings between 1971 and 1980. Complete blood counts were normal or near normal in all patients except one. Chimerism analyses, using either dual-color XY-chromosome fluorescence in situ hybridization (FISH) or analysis of variable number tandem repeat loci, indicated that 15 out of 16 patients had greater than 97% donor-derived hematopoiesis, whereas 1 patient had indeterminate chimerism. All 12 recipients of grafts from female donors exhibited polyclonal hematopoiesis by X-linked clonal analysis with the use of molecular probes. Of the 17 recipients, 9 exhibited a less than 1.0-kilobase shortening of granulocyte telomere length compared with their respective donors, according to terminal restriction fragment analysis or flow-FISH with a fluorescein-labeled peptide nucleic acid probe. These data suggest that under standard transplantation conditions, the stem cell proliferative potential is not compromised during hematopoietic reconstitution. (Blood. 2000;96:3991-3994)

An anti-LFA-1 monoclonal antibody (LDA-8) induces cellular aggregation of human lymphoblastoid cell lines and peripheral blood lymphocytes.

The human leukocyte-function-associated antigen-1 (LFA-1) plays a key role in intercellular adhesion interactions of the immune response. A monoclonal antibody (mab), designated LDA-8, is described that recognizes LFA-1. In contrast to nearly all other anti-LFA-1 mabs, which inhibit cellular aggregation, LDA-8 induces cell-cell aggregation. The LDA-8 mab was generated by immunizing mice with membrane fragments from the Jurkat T-cell line. The LDA-8 mab stained peripheral blood mononuclear cells (PBMC), monocyte-depleted peripheral blood lymphocytes, purified monocytes, and a number of T and B tumor cell lines. The LDA-8 mab induced aggregation of PBMC from normal donors, as well as of cells from T-cell lines (MOLT4 and CEM). Control mabs directed against HLA class 1 or CD4 did not induce aggregation. Aggregation was concentration- and time-dependent. EDTA added to the cultures 1 hour prior to or together with the LDA-8 mab did not inhibit LDA-8-induced aggregate formation. Anti LFA-1 alpha-chain mab added to the cells 1 hour prior to LDA-8 mab, or together with the LDA-8 mab, also did not inhibit LDA-8-induced aggregation. In contrast, anti-LFA-1 beta-chain mab, added to the cells together with or 1 hour prior to the LDA-8 mab, significantly inhibited LDA-8-induced aggregate formation. The LDA-8 mab immunoprecipitated two polypeptide chains of 110 kDa and 160 kDa under non-reducing conditions and of 92 kDa and 162 kDa under reducing conditions, from cells of the MOLT-4 or CEM T-cell lines or phytohemagglutinin (PHA)-stimulated PBMC. The molecular mass of these polypeptides was identical to that of polypeptides immunoprecipitated by the anti-LFA-1 TS1.22 mab, suggesting that the LDA-8 mab and the anti-LFA-1 mab recognize the same molecule. This was confirmed by sequential immunoprecipitation. The LDA-8 mab recognizes a unique epitope on LFA-1 and induces cell aggregation that is blocked by mabs recognizing the beta-chain, but not the alpha-chain of the LDA-1 molecule.

Selective gamma-chain T-cell receptor gene rearrangements in a patient with Omenn's syndrome: absence of V-II subgroup (V gamma 9) transcripts.

Only gamma-chain T-cell receptor transcripts utilizing V-1 subgroup gene segments were found in peripheral blood lymphocytes from a patient with Omenn's syndrome. gamma-Chain T-cell receptor transcripts utilizing the V gamma 9 (V-II subgroup) gene segment were absent in peripheral blood lymphocytes from this patient. V gamma 9 J gamma 1.2 C gamma 1 rearrangements are those primarily found in peripheral blood lymphocytes (70 to 85%) from normal donors.

Preferential rearrangements of the V gamma I subgroup of the gamma-chain of the T-cell antigen receptor to J gamma 2C gamma 2 gene segments in peripheral blood lymphocyte transcripts from normal donors.

To investigate whether there are preferential VJC gene rearrangements of the gamma-chain of the human T-cell antigen receptor (TCR), we amplified and sequenced gamma-chain TCR transcripts from peripheral blood lymphocytes from adult normal donors. cDNA was synthesized from total RNA and amplified by the polymerase chain reaction (PCR) using 5' primers specific for either the V gamma I or the V gamma II subgroups of the gamma-chain of the TCR. The amplified cDNAs were then cloned and sequenced. The majority (approximately 83%) of the cDNAs employing V-I subgroup gene segments rearranged to J gamma 2 (J gamma 2.1 or J gamma 2.3) C gamma 2 gene segments. This was in contrast to the predominant rearrangement of the V gamma II subgroup (V gamma 9) to J gamma 1.2C gamma 1. The remaining 13% of the cDNAs employing V gamma I subgroup gene segments rearranged to J gamma 1.1C gamma 1 or J gamma 1.3C gamma 1. There was significant N diversity as well as imprecise joining at the VJ junction. gamma delta TCR utilizing the C gamma 1 gene segment are disulfide-linked, whereas those utilizing the C gamma 2 gene segment are non-disulfide-linked. These results demonstrate that peripheral blood gamma-chain transcripts exhibit preferential rearrangements of V gamma I subgroup gene segments to J gamma 2(2.1,2.3)C gamma 2 gene segments. By contrast, V gamma II subgroup (V gamma 9) transcripts exhibit rearrangements to J gamma 1.2C gamma 1.

Sensitization of asthmatic children to common environmental allergens according to their residence.

The medical records of 974 asthmatic children aged 1-14 years (mean 8.7 +/- 3.9 years) who had been evaluated with skin prick tests (SPT) in two referral Children's Hospitals in Athens from 1975 to 1987 were analyzed. The children were grouped according to their residence into groups from urban area (UR), rural area (RU), and coastals (CO). The prevalence of positive SPT and the sensitizing allergens according to the residential area and the family atopic history were considered. It was found that 662/974 (68%) children had positive SPT with 63.6%, 70.7%, and 80.4% in UR, RU, and CO respectively. There was a significant difference in the prevalence of positive SPT between UR and CO. A positive family atopic history was more often accompanied by positive SPT in UR only. Sensitization to grass pollens was noted with higher prevalence in UR. The house dust mite Dermatophagoides pteronyssinus sensitization was more prevalent in CO. Our results support the notion that the environment can influence the prevalence of sensitization to common environmental antigens, the kind of sensitizing allergen, and the expressiveness of the genetic factor with regard to development of atopic asthma.

Novel immunologic strategies in ovarian carcinoma.

The purpose of our study was to develop new biologic systems for the treatment or diagnosis of patients with ovarian carcinoma through expansion of T-cell lines from the tumor-infiltrating lymphocytes of patients with ovarian carcinoma in low-dose recombinant interleukin-2 in sufficient numbers for treatment and human monoclonal antibodies that recognize cell-surface tumor-associated antigen determinants on ovarian carcinoma cells. Technologic advances in tumor immunology and new data presented in relation to ovarian carcinoma were used to develop T-cell lines for the treatment of advanced ovarian carcinoma patients. Logarithmic expansion of T-cell lines was performed in a hollow-fiber bioreactor, and a pilot clinical trial was initiated to treat ovarian carcinoma patients with intraperitoneal tumor-infiltrating lymphocytes plus low-dose recombinant interleukin-2. Human hybridomas were produced by fusion of regional lymph node B cells with a heteromyeloma cell line SPATZ 4. Two ovarian carcinoma patients have been treated with tumor-infiltrating lymphocytes expanded to 1 x 10(10) to 1 x 10(11) with manageable side effects and evidence of biologic activity. Human monoclonal antibodies have been developed that recognize tumor-associated antigen determinants. Recombinant interleukin-2-expanded tumor-infiltrating lymphocytes and human monoclonal antibodies recognize different molecular entities on tumor cells and act by different mechanisms. These approaches may be complementary to one another in future treatment strategies for ovarian carcinoma.

Gamma/delta T cell antigen receptors expressed on tumor-infiltrating lymphocytes from patients with solid tumors.

The expression of gamma/delta T cell antigen receptors (TcR) in T cell lines or clones derived from tumor-infiltrating lymphocytes (TIL) from patients with solid tumors was investigated. gamma/delta TcR T cell lines were derived from TIL from patients with Wilms tumor, sarcoma or metastatic melanoma by stimulation with autologous tumor cells alone and recombinant interleukin 2 and they exhibited nonspecific cytotoxicity against autologous and allogeneic tumor cells, or cells of the K562 or the MEL21 tumor cell lines. Two T cell lines were derived from a patient with Wilms tumor. One of them expressed a non-disulfide-linked gamma/delta TcR using the 60-kDa gamma chain, whereas, the other expressed a disulfide-linked gamma/delta TcR. A T cell line was derived from a patient with sarcoma and expressed a disulfide-linked gamma/delta TcR, whereas, a T cell line derived from a patient with melanoma expressed a non-disulfide-linked gamma chain of 62 kDa. Several T cell clones were developed from patients with metastatic melanoma or Wilms tumor and expressed either disulfide- or non-disulfide-linked gamma/delta TcR. Northern analysis of RNA from certain of these clones revealed a full-length gamma chain transcript, whereas, the alpha or beta chain transcripts were either absent or truncated. These T cell clones exhibited nonspecific cytotoxicity. Both disulfide- and non-disulfide-linked TIL T cell lines and clones expressed the delta TCS1 determinant. gamma/delta TcR+ cells in freshly prepared TIL from these patients were present in low proportions (less than 5%) and their delta TCS1/delta 1 ratios were within the range observed in the peripheral blood of normal donors. These results demonstrate that both disulfide- and non-disulfide-linked gamma/delta TcR are expressed on T cell lines and clones derived from TIL from solid tumors. Non-disulfide-linked gamma/delta TcR using the 56-66-kDa gamma chain are frequently found on TIL-derived T cell lines and clones. These 56-66-kDa gamma chains are rarely expressed on T cell lines or clones derived from peripheral blood lymphocytes of normal donors.