Lymphocyte Colony Formation Research Articles

Effect of Tai Chi Chuan practice on immune system and pro-metastasis markers in early post-treatment breast cancer survivors

Purpose: To investigate the effect of Tai Chi Chuan (TCC) to improve immune system and decrease pro-metastasis markers in early post-treatment breast cancer survivors. Methods: 130 post-treatment breast cancer survivors were recruited and randomized 1:1 into TCC group and wait-list (control) group. The TCC group practiced for a 60-minutes session once per week, for a total of 52 weeks. 115 forms of Yang-style TCC were taught by a Tai Chi master. Blood samples were taken from each subject and complete blood count was performed. The expressions of NKG2D protein, P-selectin, and vascular endothelial growth factor (VEGF) in plasma were measured. Lymphocyte activity was measured by cell proliferation reagent and ATP assay. Images of lymphocyte colony formation were taken with an inverted microscope. Results: At 52 weeks, TCC group demonstrated a significantly higher WBC (p=0.001) , a significantly higher NKG2D value (p=0.001) and a significantly lower VEGF value (p=0.005) when compared to the wait-list group. However, there was a small, non-significant change for P-selectin values between the breast cancer survivor groups. After 72h incubation, TCC group had a significant increase in lymphocyte proliferation (p=0.001) and greater area of lymphocyte clusters or colonies (p=0.001). Conclusion: The practice of TCC could stimulate tumor immunosurveillance via NKG2D and activate the immune response. VEGF, a marker playing an important role in breast cancer and its metastases, was also reduced in those who practiced TCC. As an alternative for conventional exercise, post-treatment breast cancer survivors may select TCC in their rehabilitation program.

Lymphocyte colony formation by aleukemic sheep infected with bovine leukemia virus

The objective of this study was to investigate the effects of bovine leukemia virus (BLV) infection in sheep. A prospective study of the serologic, hematologic, and histologic changes of sheep infected with BLV was conducted. Antibodies to BLV were detectable in the sheep 3 weeks after exposure to blood from an infected cow and persisted during a 120 week examination period, whereas all control sheep remained seronegative. There were no statistically significant differences between the leucocyte counts, lymphocyte counts, and lymphocyte percentages of the infected and control sheep during the first 120 weeks of this study. However, one sheep did develop a leukopenia and lymphopenia 95 weeks after it became infected and died of histologically-confirmed lymphosarcoma 10 days later. A lymphocyte colony assay was used to study the effects of BLV infection on colony formation by sheep lymphocytes in vitro. There was no significant difference in the number of lymphocyte colonies formed by BLV infected and control sheep. Nor was there a significant difference in the number of colonies formed by lymphocytes from the BLV infected sheep, when the autologous sheep serum was replaced with either pooled serum from the infected sheep or with pooled serum from the control sheep. BLV infection in aleukemic sheep does not appear to have an adverse affect on colony formation by lymphocytes in vitro.

A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B- cell colony formation and an inhibitory effect of IL-1 alpha

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7- induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.

A Stimulatory Effect of Recombinant Murine Interleukin-7 (IL-7) on B-Cell Colony Formation and an Inhibitory Effect of IL-1α

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7-induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.

Effects of human interleukin 1 and human tumor necrosis factor on human T lymphocyte colony formation.

The growth of T lymphocyte colonies in agar is dependent on the cooperation of a number of different cell types and their products. Among these interacting cells are monocytes and/or macrophages. Monocytes are capable of producing both interleukin 1 (IL-1) and tumor necrosis factor (TNF). These two factors display a positive influence on T cell colony formation. Recombinant IL-1 and recombinant TNF were both shown to stimulate T cell colony growth in a dose-dependent manner, and this stimulation could be blocked by prior incubation with anti-IL-1 or anti-TNF, respectively. In addition, conditioned medium obtained from monocytes cultured in the presence of endotoxin also stimulated T cell colony formation. This stimulation by monocyte-conditioned medium was partially suppressed by incubation with anti-TNF or anti-IL-1, while incubation with both antibodies together displayed greater suppression. In conclusion, monocytes produce at least two factors, IL-1 and TNF, which can stimulate T cell colony formation by peripheral blood lymphocytes.

Human T lymphocyte colonies require IL2 and are inhibited by anti-Tac.

Human T lymphocyte colony formation can be induced in a two-step culture system with phytohaemagglutinin (PHA) as the only exogenous growth factor. In this system the first step is the overnight pre-incubation of mononuclear cells in a liquid medium in the presence of PHA ('activation step'). The second step is the seeding of these PHA-activated cells into semi-solid agar, again in the presence of PHA, and their culture for 7 days ('proliferative step'). We have previously proposed that interleukin 2 (IL2) is produced during the second (proliferative) step and that this IL2 facilitates the production of colonies from PHA-activated colony-forming cells. Here we substantiate this proposal by demonstrating that both highly purified IL2 and recombinant IL2 stimulate T colony formation from PHA-treated cells. The monoclonal antibody anti-Tac, which is specifically directed against the IL2 receptor, dramatically inhibited colony formation when it was included during the agar culture (proliferative step). There was no effect when anti-Tac was included during the liquid pre-incubation culture (activation step). These results provide strong evidence that IL2 is the critical factor in the development of T lymphocyte colonies at the stage of conversion of PHA-activated cells into colonies.

Stimulation of human T lymphocyte colony formation by cefbuperazone and other beta-lactam antibiotics

Effects of β-lactam antibiotics (piperacillin, cefapirin, cefoperazone, cefbuperazone) on human T lymphocyte colony formation were investigated. Peripheral blood mononuclear cells from six normal volunteers were cultured with phytohemagglutinin-P (PHA) and each antibiotic in semi-solid agar for 7 days. These four antibiotics, especially cefbuperazone, increased the number of colonies of mononuclear cells from five volunteers.The effects of these antibiotics on PHA-induced and direct growth of the mononuclear cells were slight and variable among six volunteers. No relationship was observed between the effects on T lymphocyte colony formation and PHA-induced growth by four antibiotics.Cefbuperazone enhanced PHA-induced interleukin 2 production by mononuclear cells from two volunteers, in which enhanced colony formation by the drug was observed.These results suggest that cefbuperazone stimulates the immunological function of lymphocytes.

Immunoregulation of B lymphocyte colony formation by T cell subsets in patients with chronic lymphocytic leukemia

Normal B lymphocytes are activated, proliferate, and then differentiate into plasma cells and secrete immunoglobulin (Ig). We have reported that chronic lymphocytic leukemia (CLL) T4 cells help and CLL T8 cells lack suppressor effects on Ig synthesis by normal B cells (Blood 62:767, 1983). We have now explored the earlier phase, proliferation, using B cell colony formation; in semisolid media. B lymphocyte colonies from normal individuals and from patients with CLL were grown in 0.3% agarose overlayed with T cells or T cell subsets and the B cell mitogen staphylococcal protein A. Enriched T cells, OKT4 or OKT8, were obtained either by sheep erythrocyte rosettes or depletion of OKT8 or OKT4 cells by monoclonal antibody or complement, respectively. Twenty thousand B cells from normal subjects yielded 65 +/- 9, 64 +/- 7, and 19 +/- 6 colonies with autologous unfractionated T-, OKT4-, or OKT8- positive cells, respectively. This compared to 29 +/- 11, 81 +/- 11, and 15 +/- 4 colonies from patients with CLL with added autologous unfractionated T-, OKT4-, or OKT8-positive cells. To determine whether the fewer number of colonies in both normal subjects and patients with CLL with OKT8-positive cells was due to suppression or lack of help, the number of OKT4-positive cells was held constant, and OKT8-positive cells were added in increasing numbers. No suppression of colony formation could be demonstrated. Furthermore, the addition of increasing numbers of concanavalin A (Con A)-activated OKT8-positive cells did not suppress colony formation. These results suggest that the CLL T cell subsets behave in a functionally similar manner to normal T cell subsets, namely, (1) that normal and CLL B cell colony growth is helped by OKT4 cells; and (2) in contrast to immunoglobulin secretion by B cells, neither normal nor CLL OKT8 cells, unstimulated or activated by Con A, suppress B cell colony growth.

Immunoregulation of B Lymphocyte Colony Formation by T Cell Subsets in Patients With Chronic Lymphocytic Leukemia

Normal B lymphocytes are activated, proliferate, and then differentiate into plasma cells and secrete immunoglobulin (Ig). We have reported that chronic lymphocytic leukemia (CLL) T4 cells help and CLL T8 cells lack suppressor effects on Ig synthesis by normal B cells (Blood 62:767, 1983). We have now explored the earlier phase, proliferation, using B cell colony formation; in semisolid media. B lymphocyte colonies from normal individuals and from patients with CLL were grown in 0.3% agarose overlayed with T cells or T cell subsets and the B cell mitogen staphylococcal protein A. Enriched T cells, OKT4 or OKT8, were obtained either by sheep erythrocyte rosettes or depletion of OKT8 or OKT4 cells by monoclonal antibody or complement, respectively. Twenty thousand B cells from normal subjects yielded 65 +/- 9, 64 +/- 7, and 19 +/- 6 colonies with autologous unfractionated T-, OKT4-, or OKT8-positive cells, respectively. This compared to 29 +/- 11, 81 +/- 11, and 15 +/- 4 colonies from patients with CLL with added autologous unfractionated T-, OKT4-, or OKT8-positive cells. To determine whether the fewer number of colonies in both normal subjects and patients with CLL with OKT8-positive cells was due to suppression or lack of help, the number of OKT4-positive cells was held constant, and OKT8-positive cells were added in increasing numbers. No suppression of colony formation could be demonstrated. Furthermore, the addition of increasing numbers of concanavalin A (Con A)-activated OKT8-positive cells did not suppress colony formation. These results suggest that the CLL T cell subsets behave in a functionally similar manner to normal T cell subsets, namely, (1) that normal and CLL B cell colony growth is helped by OKT4 cells; and (2) in contrast to immunoglobulin secretion by B cells, neither normal nor CLL OKT8 cells, unstimulated or activated by Con A, suppress B cell colony growth.

Heterogeneous accessory cell requirement for human peripheral blood T lymphocyte activation by PHA into IL-2-responsive colony-forming cells

Mitogen-driven T cell proliferation in liquid culture requires accessory cells that cooperate in interleukin 2 production. We have investigated the accessory cell requirement for human lymphocyte colony formation under PHA stimulation. Semisolid medium limits cell-to-cell contact emphasizing the role of cooperating cells both in growth factor production and in triggering events. Culturing at high T cell density demonstrates that accessory cells can be substituted for colony formation by exogenous IL-2. Culturing at low T cell density in the presence of IL-2 also demonstrates that accessory cells are required for activation of a subset of progenitors into IL-2 responsive colony-forming cells. Consequently, T colony progenitors, contained in the E-rosetting cell fraction of peripheral blood, are heterogeneous in their triggering signals: a minor subset is directly inducible by PHA, and a major subset is inducible by PHA in the presence of accessory cells. We found that monocytes and some leukemic B cells support effective accessory function in both colony growth factor production and colony progenitor sensitization.

The effects of cyclosporin and hydrocortisone on human T lymphocyte colony formation

The immunosuppressive activities of two compounds, cyclosporin A and hydrocortisone, were evaluated using a human T lymphocyte colony assay. Both compounds produced a dose-related reduction in colony formation. With cyclosporin, concentrations of 1.0–100 μg/ml virtually abolished PHA-induced lymphocyte growth; as little as 0.01 μg/ml decreased clonal growth by 48%. Suppression was observed as late as 4 days after the addition of PHA. The addition of exogenous IL-2 did not completely restore clonal growth to normal. Similarly, hydrocortisone, in concentrations of 0.4 μg/ml, produced a 96% inhibition in clonal growth. Partial suppression could also be obtained if the drug was added as late as 4 days after PHA. Exogenous IL-2 completely reversed the inhibitory effects of hydrocortisone. By contrast, IL-1 was able to only partially restore growth potential. Parallel studies using TPA indicated that both immunosup-pressants inhibited responses to this mitogen. However, in plates containing both TPA and PHA, hydrocortisone failed to impair clonal growth.

Use of the T lymphocyte colony-forming assay to study human lymphocyte interactions.

The role of various mononuclear cell subpopulations necessary for T lymphocyte colony formation was investigated. Neither T cell-enriched nor T cell-depleted populations formed colonies as readily as did unfractionated cells. Addition of small numbers of the T cell-depleted pool greatly enhanced the capacity of the T cell-enriched pool to form colonies. Populations depleted of cells bearing the OKT4 phenotype were deficient in their colony-forming capacity. This assay may prove useful in evaluating patients with subtle abnormalities of mononuclear cell function.

In Vitro B Lymphocyte Colony Formation in Normal Subjects and Patients with Rheumatoid Arthritis

In Vitro B Lymphocyte Colony Formation in Normal Subjects and Patients with Rheumatoid Arthritis

Dependency of human T lymphocyte colony formation on soluble factors produced by accessory or tumor cells.

When plated in agar, PHA-stimulated Ficoll-Hypaque separated human mononuclear cells readily form discrete T lymphocyte colonies. Separation of these mononuclear cells on Sephadex G-10 columns yields enriched populations of T lymphocytes (greater than 95% E rosette-positive); these purified cells do not respond to this nonspecific mitogen by undergoing clonal expansion. Colony growth, however, can be restored by the addition of irradiated adherent cells. T cell responses can also be reconstituted by incubating the Sephadex-nonadherent fraction with irradiated cultured human tumor cells of monocytic (U937 and HL60) and B lymphocytic (Raji and Daudi cells) lines, but not from a T cell clone (MOLT-4) or erythroleukemic (K562) cells. Growth can also be induced in cultures containing T cells by adding cells from patients with either acute myeloblastic or B cell chronic lymphocytic leukemia or by the addition of B cell-enriched, monocyte-depleted normal mononuclear cells. Soluble factors prepared from Sephadex-adherent cells or active tumor cell lines are effective in promoting T cell colony formation. These results indicate that accessory cells are required for PHA responses of cloned human T cells. This function can be supplied by both monocytic and B lymphocytic tumor cell lines and is due to the release of soluble factors that act, in conjunction with the mitogen, to activate responsive lymphocytes.

Studies of T-Lymphocytes in Preleukemic Disorders and Acute Nonlymphocytic Leukemia: In Vitro Radiosensitivity, Mitogenic Responsiveness, Colony Formation, and Enumeration of Lymphocytic Subpopulations

Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and lymphocyte colony formation (CFU-L) from whole blood were measured following in vitro x-irradiation. Lymphocytes from patients with myelodysplastic disorders, acute nonlymphocytic leukemia, and patients at increased risk for leukemia because of their primary disease and/or cytotoxic therapy were found to be significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals. Cloning efficiencies and mitogenic responsiveness of patient lymphocytes were significantly depressed as compared to normal values. Using monoclonal antibodies to specific surface markers, quantitative abnormalities in lymphocytic subpopulations from myelodysplastic patients also were observed. These findings are suggestive of a defect at the T-cell level that may directly or indirectly affect hematopoiesis.

Studies of T-lymphocytes in preleukemic disorders and acute nonlymphocytic leukemia: in vitro radiosensitivity, mitogenic responsiveness, colony formation, and enumeration of lymphocytic subpopulations

Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and lymphocyte colony formation (CFU-L) from whole blood were measured following in vitro x-irradiation. Lymphocytes from patients with myelodysplastic disorders, acute nonlymphocytic leukemia, and patients at increased risk for leukemia because of their primary disease and/or cytotoxic therapy were found to be significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals. Cloning efficiencies and mitogenic responsiveness of patient lymphocytes were significantly depressed as compared to normal values. Using monoclonal antibodies to specific surface markers, quantitative abnormalities in lymphocytic subpopulations from myelodysplastic patients also were observed. These findings are suggestive of a defect at the T-cell level that may directly or indirectly affect hematopoiesis.

Opposing effects of 12‐o‐tetradecanoylphorbol 13‐acetate on human myeloid and lymphoid cell proliferation

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on human hematopoietic cells has been investigated. It was found that 1-10 ng/ml of TPA totally abrogated erythroid and granulocytic colony growth and, simultaneously in the presence of PHA, stimulated T-lymphocyte colony formation. TPA concentrations insufficient to inhibit myeloid colony growth also failed to stimulate lymphocyte colony formation. Optimal culture conditions for the growth of these colonies required the presence of TPA, PHA, and leukocyte-conditioned medium in the cultures. Cells within the colonies were 80-90% E-rosette positive and by monoclonal antibody characterization contained 45-66% OKT3-positive cells. Colony-forming cells were found in both E-rosette-positive and-negative fractions. Although by cell surface marker characterization the cells within the colonies had properties of T-cells, the exact relationship of cells forming colonies under these conditions to those detected in other T-cell colony assays remain to be determined.

Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture

Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal t lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.

Impairment of T Lymphocyte Colony Formation in Hodgkin’s Disease: Effect of Soluble Inhibitory Factors on Normal T Lymphocyte Colony Formation Potential

PHA-induced T lymphocyte colonies from peripheral blood mononuclear cells of untreated Hodgkin's disease (HD) patients and normal healthy donors were assayed by one-step stimulation in microagar capillary cultures. A significant depression in the T cell colony formation was observed in HD patients in comparison with normal healthy donors. Clinical staging of the disease had no influence on this abnormality. Preincubation of HD lymphocytes for 24 h in tissue culture medium did not produce any appreciable recovery in the colony formation potential. However, in the presence of 24-hour culture supernatants of HD mononuclear cells, there was significant inhibition in the colony formation by lymphocytes obtained from normal healthy donors. Significance of these observations is discussed.

Cell-cell cooperation in lymphocyte colony formation: studies in human allogeneic marrow transplantation.

The pace of restoration of phytohemagglutinin- (PHA) stimulated lymphocyte colony-forming response was investigated in 50 bone marrow transplant (BMT) recipients (49 allogeneic, 1 syngeneic) studied on 86 occasions. On the majority of occasions (28 of 48), patients studied early (3 to 8 wk) after BMT failed to make detectable lymphocyte colonies, whereas patients studied late (greater than 6 mo) after BMT displayed responses comparable to those of healthy adult volunteers. The sequential study of 10 of the 15 patients with positive lymphocyte colony-forming responses early after BMT revealed that this response is short-lived. The results of recipient donor mixing experiments and experiments involving the addition of the lymphokine interleukin 2 (IL 2) to recipient cells indicated that lymphocytes obtained from unresponsive recipients evaluated early after BMT can be driven to colony formation, and suggest that the major limiting component in such subjects is functional T help.