Null cell senescence and its potential significance to the immunobiology of aging.

The null cell compartments of human bone marrow and mouse spleen were arbitrarily divided into three subpopulations based upon the ability of cells to acquire T or B cell membrane markers when incubated with poly A:U or ubiquitin. There was an accumulation of T cell precursors with congenital absence of the thymus. In contrast, T cell precursors were reduced and there was an accumulation of uninduced null cells with old age. These observations suggest that there is an intrinsic defect of null cell differentiation with a drift towards more differentiated precursors in T cell differentiation with aging. This could result in a diminution in the range of responses by their progeny, mature T lymphocytes.

Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients.

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.

Vitamin D-mediated gene regulation in phenotypically defined human B cell subpopulations.

Isolation of distinct subpopulations of density-fractionated normal human B lymphocytes reveals that the requirements for up-regulation of the vitamin D receptor (VDR) and initiation of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3]-mediated genomic trans-activation are dependent upon the state of cellular activation. The kinetics of the response differ widely among these B cell subpopulations. However, these density-fractionated B cell subpopulations are phenotypically diverse and therefore are not representative of distinct stages of B cell maturation and differentiation. To examine the role of B cell differentiation on the induction and maintenance of biological receptivity to 1,25-(OH)2D3, we purified naive, germinal center, and memory B cells based on their expression of CD38 and CD44 surface antigens and surface Ig isotype. These phenotypically defined B cell subpopulations were all found to constitutively express VDR, and all exhibited similar activation requirements and kinetics for initiation of 1,25(OH)2D3-mediated genomic trans-activation. Taken together, these results suggest that defined stages of differentiation in normal B cells are not significant predicators of VDR expression or receptivity to 1,25-(OH)2D3. Rather, the degree of cellular activation, regardless of maturation stage, determines whether the effects of this immunoregulatory hormone will influence a mature B lymphocyte.

Plasma adenosine deaminase2: a marker for human immunodeficiency virus infection.

Plasma concentrations of the two isoenzymes of adenosine deaminase (ADA, E.C. 3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), were measured in a cohort of ambulatory patients infected with the human immunodeficiency virus (HIV) and controls. A sensitive isoenzyme-specific radioisotopic assay system was developed for these studies. Among 22 HIV-infected patients, plasma ADA2 was significantly elevated as compared with 16 control subjects (p less than 0.01) and 6 uninfected subjects having a risk factor for HIV infection (p less than 0.01). Plasma ADA2 was not associated with the stage of disease as defined by clinical status (p greater than 0.05) or helper (CD4) lymphocyte count (p greater than 0.05). Available evidence suggests that elevated plasma ADA2 could be a useful surrogate marker for HIV infection that occurs early in the disease process.

Establishment of stable human T-T hybridomas.

Human T-T hybridomas potentially provide an invaluable resource for a variety of immunoregulatory molecules that modulate the immune response. To date, success in this technology, using human cell populations, has been hampered by several problems associated with proliferative and functional instability of the hybrid cells. These forms of instability are the result of a multifactorial process, with 1 parameter of importance being the chromosome number of the malignant parent cell line used for fusion. The present studies describe the production of a stable human T-T hybridoma generated by fusing a near diploid (modal chromosome number of 48) aminopterin-sensitive T cell line, CEM TG E11, and lectin-stimulated human peripheral blood lymphocytes. The rapidly growing hybrid cells have been clonally selected for the production of a B cell growth factor. Hybridization was documented by the presence of HLA phenotypes reflecting the combined antigens of the fusion partners. Fusions with 4 other partners besides CEM TG E11, where the majority of the cells had modal chromosome numbers ranging from 78 to 94, were proliferatively unstable. To date, hybrid cells derived from the CEM TG E11 fusion have been doubling approximately every 48 h for greater than 12 months, and selected clones constitutively produce B cell growth factor.

Thymosin-like peptides as potential immunostimulants. Synthesis via the polymeric-reagent method.

This paper reports our attempt at designing new immunostimulating peptides which are chemically related to the bioactive peptides thymosin alpha 1 and thymopentin. Three peptides were synthesized, Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr (3), Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (2), and Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (1), each of which contains the thymopentin sequence and portions of the bioactive sequence of thymosin alpha 1. Peptides 1-3 were assembled from selected blocked fragments that were synthesized by the polymeric-reagent method, using PHBT (polystyrene-bound 1-hydroxybenzotriazole) as the activating polymer. The ability of peptides 1-3 to enhance the activation (RNA synthesis) and proliferation (DNA synthesis) of human T lymphocytes was determined. In comparison to thymosin alpha 1, thymosin alpha 1 (15-28), and thymopentin, peptides 1-3 did not show significant enhancement of these processes.

Paclitaxel-induced apoptosis in Jurkat, a leukemic T cell line, is enhanced by ceramide.

We hypothesized that the lipid second messenger, ceramide, and microtubule-directed chemotherapeutic agents might engage converging pathways in inducing apoptosis. Our studies demonstrated that simultaneous treatment of Jurkat cells with paclitaxel and ceramide enhanced paclitaxel-induced cell growth inhibition. Cell cycle analysis indicated a significant increase in the hypodiploid population over that observed with paclitaxel treatment alone. Morphologic evaluation and a TUNEL assay confirmed a dramatic increase in apoptosis in Jurkat cells treated with the combination of these two agents. This is the first demonstration that paclitaxel and ceramide interact in a supra-additive manner to decrease leukemic T-cell growth, suggesting a possible application of paclitaxel and ceramide in combination therapy.

Antitumor effects of polyvalent and monovalent vaccines coupled with interleukin-2 in a metastatic melanoma model.

We have previously reported that preimmunization of mice with formalinized extracellular antigens (fECA) derived from melanoma cells, in combination with interleukin 2 (IL-2) treatment and surgical resection, decreased subsequent tumor growth and increased survival of mice in a new model for spontaneous metastasis of melanoma. In this study, we have modified the sequence of tumor growth and therapy to more closely mimic the clinical situation. Mice were challenged subcutaneously in the tail with 5 x 10(5) B16 F10 melanoma cells and, by day 21, all of them had developed localized melanoma tumors. The primary tumor-bearing tails of control and experimental animals were then resected distal to the base of the tail, and therapy of the mice was initiated the following day. Groups of mice received different polyvalent and monovalent murine melanoma vaccines (including native or formalin treated extracellular antigens, intact melanoma cells, or purified B700 antigen), with or without concomitant low doses of IL-2. The results demonstrate that the vaccine therapies elicited significant increases in survival of the mice, accompanied by reductions in the size of lymph nodes and in the number of pulmonary metastases. These effects, particularly with the intact melanoma cell vaccine, could be improved even further with concomitant IL-2 treatment.

Intrasplenic vaccination against experimental melanoma.

The immunodominant component of a formalinized extracellular antigen (fECA) vaccine prepared from B16 F10 melanoma cells is the melanoma-associated antigen B700. We now demonstrate that a single prophylactic intrasplenic inoculation of B700 antigen (1-10 mu g) stimulates the production of antibodies which have antiproliferative effects on B16 F10 melanoma cells in vitro. In addition, potential cytotoxic effects of splenocytes from B700 antigen inoculated mice were evaluated for two cellular immune effector functions, natural killer (NK) cell activity and lymphokine activated killer (LAK) cell activity; both activities were increased following B700 antigen inoculation. Intrasplenic injection of B700 antigen elicited an increase in the expression of the CD25 surface antigen (IL-2 R alpha) by T lymphocytes and up-regulated the expression of IL-2 R alpha mRNA. Thus both humoral and cellular cytotoxic immune responses might play roles in the decreased growth of primary tumors in B700 antigen inoculated mice and in the higher survival rate in this group of animals.

Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line.

PURPOSE: Paclitaxel, a chemotherapeutic agent used in the treatment of recalcitrant ovarian and breast as well as other neoplasms, is being investigated for the treatment of squamous cell carcinoma of the head and neck. Our previous studies have demonstrated that exogenously added ceramide enhances apoptosis in paclitaxel-exposed human leukemic cells. In this study, we showed that exogenous ceramide augmented paclitaxel-induced apoptosis in Tu138 cells in vitro when added simultaneously in combination with the paclitaxel. METHODS: The combined cytotoxic effects of paclitaxel and ceramide exposure against Tu138 cells were assessed by an MTT dye assay, cell cycle analysis, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, and isobologram analysis for synergistic activity. RESULTS: The MTT dye assay results indicated augmentation of time- and concentration-dependent paclitaxel-mediated cell cytotoxicity by simultaneous ceramide treatment. Paclitaxel treatment of Tu138 cells also resulted in an accumulation of cells in the G2-M phase of the cell cycle. This paclitaxel-mediated G2-M phase accumulation decreased significantly with the addition of ceramide, indicating that combined paclitaxel/ceramide treatment resulted in the elimination of Tu138 cells from the S and/or G2-M phases of the cell cycle. Furthermore, ceramide enhancement of paclitaxel-mediated apoptosis was also detected by the TUNEL assay. CONCLUSION: Our results suggest that paclitaxel/ceramide combination therapy may be an attractive alternative to conventional methods of chemotherapy for head and neck cancer, and should be further explored.

Taxol pretreatment of tumor targets amplifies natural killer cell mediated lysis.

Taxol is known to polymerize and stabilize microtubules and thereby alter many cellular functions. Our studies examined the effects of taxol pretreatment of tumor targets and cytotoxic effector cells in an effort to determine whether such treatment would result in increased tumor cell lysis without affecting cytotoxic cell function. Our studies demonstrated that taxol concentrations of 6-30 ng/ml which induced approximately 50% growth inhibition and > or = 50% block in the G2/M phase of the K562 cell targets did not have any significant effect on the functional ability of NK cells to lyse K562 cells. Pretreatment of K562 cells with taxol (6 and 30 ng/ml) resulted in an increase in K562 cell lysis by NK cells (or NK cells stimulated with 100 units/ml of rIL-2) in 7 out of 9 donors. The amplification of NK cell-mediated lysis of tumor targets due to taxol pretreatment may provide a combination therapeutic approach which includes taxol treatment followed by rIL-2 stimulation of the immune killer cell function.

Contribution of serum inhibitory factors and immune cellular defects to the depressed cell-mediated immunity in patients with head and neck cancer.

The immune system of patients with head and neck cancer is frequently depressed. Serum inhibitory factors and immune cell dysfunction are known contributors to this depression, but their relative roles are unclear. We have examined these factors to determine whether a common pathway is involved. Is the defect an unresponding "switched-off cell" or is it a remedial defect responsive to the removal of serum inhibitory factors and/or to lymphokine restoration? Immune tests were performed in 66 patients with high-stage head and neck cancer. Serum inhibitory factors were measured by incubation of heat-inactivated serum (10%) with phytohemagglutinin (PHA)-stimulated lymphocytes or natural killer (NK) cells using the K562 assay. Lymphokine-activated killer (LAK) cell cytotoxicity was measured (in the presence/absence of serum) using chromium 51-labeled Raji tumor cells cultured 5 days with interleukin-2 (IL-2) (100 or 1,000 U/mL) and/or interferon-alpha (INF-alpha) (100 U/mL). IL-2 receptors, CD25 or p55 (low affinity) and p75 (high affinity), were measured by flow cytometry through fluorescence-activated cell sorter analysis. Serum inhibitory factors were detected in more than 50% of the patients. Head and neck cancer sera significantly inhibiting the normal lymphocyte response to PHA (11 of 22 patients), as well as significantly inhibiting the NK response of normal lymphocytes and the functional expression of the IL-2 receptor. LAK cell function at low-dose IL-2 was depressed in 45% of the patients (9 of 20) and was restored by increased IL-2 (1,000 U/mL) or a combination of IL-2 and INF-alpha. Twenty-five percent of the patients were unresponsive to maximum lymphokine stimulation. Half of the patients had depressed expression of the low-affinity IL-2 receptor (CD25). The cause of immune depression in patients with head and neck cancer is multifactorial and is related to serum inhibitory factors, as well as to inherent cellular defects. Based on these data, we would suggest a therapeutic approach in selected patients that includes the removal of serum inhibitory factors by plasmapheresis and restoration of cellular defects by combined IL-2 with or without INF-alpha.

Multiple infusions of CD20-targeted T cells and low-dose IL-2 after SCT for high-risk non-Hodgkin's lymphoma: a pilot study.

A pilot phase I clinical trial involving 15 infusions of anti-CD3 × anti-CD20 bispecific Ab (CD20Bi)-armed anti-CD3-activated T cells (aATC) and low-dose IL-2 was conducted in three non-Hodgkin's lymphoma (NHL) patients (two high-risk and one refractory) after autologous SCT. The feasibility of T-cell expansion, safety of aATC infusions, cytotoxic immune responses and trafficking of aATC were evaluated. Three NHL patients received 15 infusions of 5 × 10(9) aATC (three infusions/week for 3 weeks and one infusion/week for 6 weeks) between days 1 and 65 after SCT with IL-2. There were no dose-limiting toxicities. Chills, fever, hypotension and malaise were the common side effects. Engraftment was delayed in one patient with a low stem cell dose. CD20Bi aATC infusions induced specific cytotoxicity directed at lymphoma targets. Endogenous peripheral blood mononuclear cells from two patients mediated anti-lymphoma cytotoxicity above preSCT background (P<0.001). (111)In labeled aATC trafficked to the lungs at 1 h and accumulated in the liver and bone marrow after 24 h. aATC infusions given over 69 days in combination with IL-2 were safe, did not inhibit engraftment, and induced endogenous cytotoxic responses directed at lymphoma targets.

Low expression of Abelson interactor-1 is linked to acquired drug resistance in Bcr-Abl-induced leukemia.

The basis for persistence of leukemic stem cells in the bone marrow microenvironment remains poorly understood. We present evidence that signaling cross-talk between α4 integrin and Abelson interactor-1 (Abi-1) is involved in the acquisition of an anchorage-dependent phenotype and drug resistance in Bcr-Abl-positive leukemia cells. Comparison of Abi-1 (ABI-1) and α4 integrin (ITGA4) gene expression in relapsing Bcr-Abl-positive CD34+progenitor cells demonstrated a reduction in Abi-1 and an increase in α4 integrin mRNA in the absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and α4 integrin expression, as well as linkage to elevated phospho-Akt and phospho-Erk signaling, was confirmed in imatinib mesylate -resistant leukemic cells. These results indicate that the α4-Abi-1 signaling pathway may mediate acquisition of the drug-resistant phenotype of leukemic cells.

Changes in the compositions of the null cell compartment with murine autoimmunity.

The subpopulations that comprise the null cell compartment were examined sequentially in various strains of autoimmune-prone mice. Different patterns emerged that were consistent within strains but differed from strain to strain. Abnormalities appear earlier in life in short-lived mice, such as male BXSB and MRL/l mice, than in relatively long-lived strains, such as female BXSB and NZB mice. The accumulation of T cells in MRL/l mice was accompanied by null cell changes that contrasted with those that developed in AKR/J mice after their spleens were infiltrated with leukemic T cells. It would seem that lymphocyte perturbations with murine autoimmunity also involve their precursor cells and that these precursor cell changes vary in different strains, perhaps in relation to different genetic factors.

Modulation of cytokine production by carnitine.

The ability of carnitine congeners to modulate cytokine production by human peripheral blood mononuclear cells (PBMC) was investigated. Modulation of cytokine production by PBMC of young (30 years of age or younger) and old (70 years of age or older) normal donors was first compared. The PBMC were collected over Ficoll-Hypaque and incubated in the presence of various concentrations of acetyl L-carnitine for 24 h. Subsequently the supernatants were collected and examined for cytokine production. The presence of cytokines in tissue culture supernatants was examined by ELISA. The cytokines measured included IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, TNFalpha, GM-CSF, and IFNgamma. The results showed that at 50 mug/ml of acetyl L-carnitine the most significant response was obtained for TNFalpha. In this regard four of five young donors responded, but only one of five old donors responded. More recently these studies were expanded to examine the ability of L-carnitine to modulate cytokine production at higher doses, 200 and 400 mug/ml, in young donors. The results of these studies showed that in addition to TNFalpha, significant production of IL-1beta and IL-6 was observed. These preliminary studies provide evidence that carnitine may modulate immune functions through the production of selected cytokines.

Update and future perspectives of a thymic biological response modifier (Thymomodulin).

Thymomodulin (Ellem Industria Farmaceutica spa, Milan, Italy) is a calf thymus acid lysate with immunomodulating activities. It is composed of several peptides with a molecular weight range of 1-10kD. Extensive studies in animal systems showed that Thymomodulin exhibited no, or very little toxicity even when used at high doses. Studies done in vitro and in vivo demonstrated that Thymomodulin is a biologically active compound which regulates the maturation of human and murine pre T lymphocytes, as well as modulate the functions of apparently mature human and animal B and T lymphocytes. It was observed that Thymomodulin can promote myelopoiesis as demonstrated by an increase of granulocyte-macrophage colonies in agar. Although additional studies to examine its target cell lineage are required, it appears that Thymomodulin exhibits specificity toward T cells. Therefore, enhancement of other cell lineage functions by Thymomodulin may be indirect, and mainly due to its effect on T cells. Of major importance is to note that Thymomodulin is prepared in a manner which allows it to maintain its biological activity when administered orally.

Thymomodulin: biological properties and clinical applications.

Thymomodulin (Ellem Industria Farmaceutica s.p.a., Milan, Italy) is a calf thymus acid lysate derivative, composed of several peptides with a molecular weight range of 1-10 kD. Thymomodulin did not exhibit any mutagenic effect. Furthermore, thymomodulin used in animal studies showed no toxicity even when used at high concentrations. Of major significance are the observations in murine and human systems that thymomodulin remains active when administered orally. In vitro and in vivo administered thymomodulin was able to induce the maturation of T-lymphocytes. Additionally, studies in vitro showed that this thymic derivative can enhance the functions of mature T-lymphocytes with cascading effects on B-cell and macrophage functions. Extensive human clinical trials with thymomodulin showed that this agent can improve the clinical symptoms observed with various disease processes, including infections, allergies and malignancies, and can improve immunological functions during ageing.

Lymphokines and monokines as regulators of human lymphoproliferation.

The development of a competent immunoregulatory response in the face of an antigenic challenge is modulated by soluble proteins of relatively low molecular mass. Lymphokines and monokines, secreted by cells of T lineage and cells of the monocyte/microphage series, respectively, function in a bimodal amplification network that results in the proliferation and differentiation of the immunoregulatory cells. Interleukin 1 is typically assayed by its effect on thymocytes or by its ability to promote the T cell-dependent release of interleukin 2. Interleukin 2 is routinely measured by its ability to support the long-term growth of cultured T cells, whereas B cell growth factor is measured by its ability to support the long-term growth of cultured B lymphocytes. The availability of homogeneous purified factors and the subsequent availability of monoclonal antibodies against these reagents should allow for the development of rapid quantitative assays for these analytes in diverse biological fluids. In addition, large quantities of purified reagents will promote studies to determine therapeutic efficacy in several immunodeficiency syndromes.

Association of an interleukin abnormality with the T cell defect in Hodgkin's disease.

The cellular immune defect in untreated Hodgkin's disease (HD) has long been recognized. This defect appears to be responsible for at least some of the morbidity and ultimately the mortality associated with the disease. In recent years, many studies have shown that the T cell component of the immune response is the apparent site where the defect in HD exists and where the immunoregulatory abnormalities that may account for the deficit are observed. The discovery of the lymphokines and monokines, comprising the human interleukin system, has elucidated some aspects of the regulatory control of the functional pathways involved in T lymphocyte activation and proliferation. The interleukin system can therefore provide the framework to dissect immunodeficiency states, such as that seen in HD. The present study indicates that HD patients' interleukin 1 (IL1) response appears to be normal, as is their T cell proliferative response to exogenous IL2. Interleukin 2 production by HD patients' peripheral blood mononuclear cells, however, is decreased when compared with age/sex-matched controls. The inability to generate IL2 after appropriate stimulation may reflect either a primary cellular defect or a regulatory defect, such as excessive immunosuppression, giving rise to the characteristic T cell hyporesponsiveness seen in HD.