Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.

We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the 9D7 anti-P13K mAb from peripheral blood lymphocytes from a patient with mild CVI expanded in culture with rIL-2 and polyclonal activators. Using the same 9D7 anti-P13K mAb and immunoblotting analysis, we identified a 35 kDa gamma-chain polypeptide under reducing conditions expressed on purified L3T4- Lyt2- BALB/c mouse thymocytes. This gamma-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.     

Cycling of peripheral blood and marrow lymphocytes in cyclic neutropenia.

A 16-month-old male patient with cyclic neutropenia was found to have cyclic fluctuations of monocytes, lymphocytes, platelets, and eosinophils in the peripheral blood. Changes in lymphocyte counts were not obviously related to B, T, or natural killer cells. All classes of immunoglobulins were elevated throughout the cycle. Studies of the marrow morphology revealed remarkable cyclic oscillations of lymphoid as well as myeloid lineage cells. Granulocyte-macrophage progenitors (CFU-c) cycled and were virtually absent 1 wk prior to the neutropenic nadir. The cyclic changes in marrow lymphoid cell numbers were primarily due to changes in numbers of surface immunoglobulin negative (sIg-), cytoplasmic Ig+ (cIg+) pre-B cells. Pre-B cell numbers cycled from normal to extraordinarily elevated values with the same periodicity but reciprocal to the neutrophil cycle. We propose that the primary defect in cyclic neutropenia may either be a periodic failure of an early myeloid differentiation factor or a blunted response of early myeloid precursors to a common hemopoietic growth factor. This may lead to periodic fluctuations in the production or delivery of growth factors (or factor) that influence early stages of differentiation of other hemopoietic cells, including pre-B cells. The essential periodic deficiency is consequently reflected in deficient production of CFU-c accompanied by excessive production or accumulation of pre-B cells (and probably other hemopoietic precursors) in the marrow.     

Functional T cells in athymic nude mice.

After passage of spleen cells from nu/nu mice over a nylon wool column, concanavalin A-responsive cells can be detected in the presence of 2-mercaptoethanol, and specific cytotoxic T lymphocytes can be generated without exposure to interleukin 2 (IL-2). The spleen cells of the nu/nu mice born of nu/nu parents and nursed by nu/nu mothers had significantly fewer Thy-1+ T cells and a lesser capacity to generate cytotoxic T lymphocytes than did the conventionally bred nu/nu mice. Nonetheless, such cells were clearly present. IL-2 may act to cause these post-thymic T cells to proliferate. Therefore, it seems inappropriate to consider IL-2 as an inducer of the differentiation of T cells in the absence of thymic influence on the basis of the capacity of IL-2 to induce the appearance of a T-lymphocyte population in nu/nu mice.     

A human soluble suppressor factor affecting lymphocyte responses in vitro.

A soluble suppressor factor (SSF) has been demonstrated in the supernatant of normal human peripheral blood lymphocyte cultures that exhibits suppressive activity toward the proliferative response of normal lymphocytes to concanavalin A or alloantigens in mixed lymphocyte culture (MLC) or toward pokeweed mitogen-stimulated immunoglobulin synthesis and secretion in vitro. Suppression of the proliferative response in MLC reached maximal levels when added SSF-containing supernatant approximated 20% by volume of the culture medium. Suppression in the MLC was found to act at the proliferative stage. SSF acts independently of cytotoxicity and is stable at 56 degrees C for 30 min but is inactivated at higher temperatures. Addition of SSF to the MLC as late as day 4 after initiation of the culture results in suppression of transformation. This factor(s) may regulate the magnitude of several immune responses in humans.     

Selective growth of a population of human basophil cells in vitro.

An initially homogeneous population of basophilic polymorphonuclear leukocytes was derived from human fetal liver cells grown in culture for 5-7 days. The cells were characterized as basophils by their morphology, histologic staining characteristics, and histamine content, and by the presence of IgE receptors on their surface. In this culture system the basophils were viable for up to 10 days.     

The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells

Interferons (IFNs) have been shown to have significant effects on hematopoietic cell growth. Previous studies defining these effects have utilized mouse and human alpha-, beta-, and gamma-IFN isolated from supernatants of stimulated cells. Despite purification, the possible presence of other lymphokines and soluble factors remains a concern. In this study, the effects of gene-cloned alpha- and gamma-IFN on colony- forming units of granulocyte/macrophage (CFU-GM) progenitors cultured from the peripheral blood of normal volunteers were examined. In addition, blast cell colonies from one patient with acute myelogenous leukemia (AML) were studied. The growth of normal CFU-GM and AML blast cell colonies was inhibited in a dose-dependent manner by gamma- and alpha-IFN. gamma-IFN was ten to 100 times more potent than alpha-IFN in that this species of IFN reduced colony formation by greater than 50% at concentrations of less than 15 antiviral U/mL. The effects of gamma- IFN were neutralized by a monoclonal antibody specific for gamma-IFN. These in vitro studies indicate that human gamma-IFN may be an important modulator of myelopoiesis. Although these data indicate a possible efficacy of gamma-IFN in the treatment of AML, the in vitro results should be considered for their in vivo significance.     

Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML

Relapse after autologous bone marrow transplantation for chronic myelogenous leukemia (CML) can be due either to the persistence of leukemia cells in systemic tissues following preparative therapy, or due to the persistence of leukemia cells in the autologous marrow used to restore marrow function after intensive therapy. To help distinguish between these two possible causes of relapse, we used safety-modified retroviruses, which contain the bacterial resistance gene NEO, to mark autologous marrow cells that had been collected from patients early in the phase of hematopoietic recovery after in vivo chemotherapy. The cells were then subjected to ex vivo CD34 selection following collection and 30% of the bone marrow were exposed to a safety-modified virus. This marrow was infused after delivery of systemic therapy, which consisted of total body irradiation (1,020 cGy), cyclophosphamide (120 mg/kg), and VP-16 (750 mg/m2). RT PCR assays specific for the bacterial NEO mRNA, which was coded for by the virus, and the bcr-abl mRNA showed that in two evaluable CML patients transplanted with marked cells, sufficient numbers of leukemia cells remained in the infused marrow to contribute to systemic relapse. In addition, both normal and leukemic cells positive for the retroviral transgenome persisted in the systemic circulation of the patients for at least 280 days posttransplant showing that the infused marrow was responsible for the return of hematopoiesis following the preparative therapy. This observation shows that it is possible to use a replication-incompetent safety-modified retrovirus in order to introduce DNA sequences into the hematopoietic cells of patients undergoing autologous bone marrow transplantation. Moreover, this data suggested that additional fractionation procedures will be necessary to reduce the probability of relapse after bone marrow transplantation in at least the advanced stages of the disease in CML patients undergoing autologous bone marrow transplantation procedures.     

Growth of a pure population of mouse mast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes.

A pure population of mast cells was obtained after 14 days of culturing mouse bone marrow cells in the presence of medium derived from concanavalin A-stimulated mouse spleen cells. The cells were characterized as mast cells by their morphologic appearance and histologic staining, by their histamine content (450 ng per 10(6) cells) and by the demonstration of IgE receptors on their surface (150,000--440,000 receptor sites per cell). The histamine content and the number of IgE receptors remained constant for at least 7 wk of culture. These mast cells could be passively sensitized to mice hybridoma IgE. They then released 43% of their histamine content upon incubation with anti-mouse hybridoma IgE.     

Influence of dietary restriction on immunologic function and renal disease in (NZB × NZW) F1 mice

In (NZB x NZW)F(1) (B/W) mice, moderate caloric intake [10 kcal (41.8 kJ) per day] from the time of weaning was associated with maintenance of lower body weight, greater capacity of spleen cells to be stimulated with T-cell mitogens, and better preserved capacity to generate cytotoxic cells in response to in vitro and in vivo stimulation with allogeneic tumor cells. Plaque-forming cell response to sheep erythrocytes was also well maintained in animals on the restricted diets when sensitization was accomplished either in vitro or in vivo. Spontaneous suppressor cell activity against plaque-forming cells that developed in controls did not appear in the mice on the restricted diet. Significantly less circulating antibody to native DNA was present in the blood of mice 10 months of age when their dietary intake had been restricted. Histological analysis revealed that the development of renal disease and the deposition of gamma globulin in the glomerular capillaries was markedly inhibited in the mice on restricted diets. Dietary restriction from the time of weaning thus appears to prolong significantly the life of autoimmunity-prone (NZB x NZW)F(1) male and female mice and to alter lymphoid cell immune function, thereby decreasing the autoimmune processes and immunological assault associated with progressive renal disease in these animals.     

Influence of dietary energy restriction on the numbers and proportions of Ly-1+ B lymphocytes in autoimmunity-prone mice.

Chronic energy-intake restriction (CEIR) has been shown to increase life-span, delay diseases expression, and inhibit immunological perturbations in all strains of autoimmunity-prone mice studied, including NZB, (NZB x NZW)F1, MRL/Mp-lpr/lpr, BXSB, and kd/kd mice. In (NZB x NZB)F1 mice, increased percentages and increased absolute numbers of Ly-1+ B lymphocytes in spleen, mesenteric lymph nodes, thymus, and bone marrow as revealed by two-color immunofluorescence analysis were greatly reduced by CEIR with a diet high in carbohydrate and low in fat. This influence on a possibly crucial lymphocyte subpopulation was associated with delayed onset of disease and with greatly prolonged life-span. In the present investigation, the percentages and absolute number of Ly-1+ B lymphocytes in the spleen, peritoneal exudate, and peripheral blood were found to be increased in each autoimmunity-prone strain studied. CEIR decreased the absolute and relative numbers of the Ly-1+ B lymphocytes in mice of each of the autoimmunity-prone strains and returned the number and proportions of Ly-1+ B cells close to levels present in the same locations in genetically long-lived C57BL/6, DBA/2, or BALB/c mice fed a standard commercial diet ad libitum.