Michail Papamichail

Immunoregulatory effects of fraction 5 thymus peptides. I. Thymosin α1 enhances while thymosin β4 suppresses the human autologous and allogeneic mixed lymphocyte reaction

Thymosin alpha 1 and thymosin beta 4, two peptides isolated from preparations of calf thymus fraction 5, were tested in the human mixed lymphocyte reaction (MLR). Thymosin alpha 1 was found capable of enhancing both the allogeneic and autologous MLR. On the contrary, thymosin beta 4 suppressed MLR proliferative responses. Study of the responses of the T cell subpopulations revealed that T4+ (helper/inducer) cells but not T8+ (suppressor/cytotoxic) are responsible for the enhanced, proliferative response to allo- and autoantigens in the presence of thymosin alpha 1. Both the autologous and the allogeneic proliferative responses of either T4+ cells or T8+ cells were not influenced by the addition of thymosin beta 4 in the cultures. However, when T4+ and T8+ subsets were cocultured, thymosin beta 4 was capable of activating T8+ cells to suppress the allogeneic and the autologous proliferative response of T4+ cells. These studies show that thymosin fraction 5 peptides exert immunoregulatory effects on the human MLR proliferative responses in vitro.

Large-scale expansion of CD3(+)CD56(+) lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants.

Abstract. We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56+ natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3+CD56+ cells (NKT cells) that expand dramatically under these conditions. CD3+CD56+ cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3+CD56+ cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3+CD56+ cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3+CD56+ cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), gamma-interferon (IFN-γ) and perforin. CD3+CD56+ cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3+CD56+ cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.

Mistletoe lectin I-induced effects on human cytotoxic lymphocytes. I. Synergism with IL-2 in the induction of enhanced LAK cytotoxicity.

This report demonstrates that in vitro activation of human cells with the beta-galactoside-specific lectin from mistletoe (ML-I) or interleukin-2 (IL-2) results in different patterns of activation and function of cytotoxic cells. It is now well established that natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity is mainly mediated by resting (NK) and IL-2-activated (LAK) CD56-positive (+) cells respectively. Culture of peripheral blood lymphocytes (PBL) for 3 days with ML-I led to expansion and activation of T cells which demonstrated NK- and LAK-like cytotoxicity. T lymphocyte subset analysis revealed that in total PBL, ML-I preferentially stimulated and expanded CD8+ T cells which mediated the cytotoxic effect. Incubation of highly purified CD8+ T cells alone with ML-I did not lead to induction of cytotoxicity, which required the presence of both CD4+ and CD14+ (monocytes) cells, suggesting that ML-I does not exert a direct effect on CD8+ T cells. Activation of PBL with both ML-I and IL-2 resulted in simultaneous induction of T and CD56+ cell-mediated NK and LAK cytotoxicity. These data suggest that treatment with ML-I and IL-2 might provide an approach to induce maximum cytotoxicity against tumors and to recruit both T and NK cells for tumor therapy.

Enhancement of human T lymphocyte functions by prothymosin α. I. Augmentation of mixed lymphocyte culture reactions and soluble protein-induced proliferative responses

Prothymosin alpha (ProT alpha), a 115-amino-acid thymic polypeptide, was tested for its effect on soluble antigen, allo- and auto-antigen-induced human T-cell proliferation. ProT alpha enhanced the secondary T-cell proliferative response to ovalbumin (OVA)- and keyhole limpet haemocyanin (KLH)-pulsed antigen-presenting cells (peripheral blood monocytes). Maximum enhancement (20-fold for OVA and 23-fold for KLH) occurred when suboptimal concentrations of either OVA or KLH were employed. Subset depletion experiments showed that the helper/inducer T-cell subpopulation was responsible for the observed enhancement. In the mixed lymphocyte reaction (MLR), ProT alpha enhanced autoantigen- (autoMLR; 9- to 14-fold) as well as the alloantigen- (alloMLR; 8- to 10-fold) induced T-cell proliferation when suboptimal ratios of the participating cells were used. Preincubation of the stimulating (autologous or allogeneic monocytes) with ProT alpha induced significantly higher T-cell proliferation in both primary and secondary MLR responses as compared to that induced by non-treated monocytes. In contrast, T lymphocytes pre-incubated with ProT alpha did not show enhanced proliferative activity when tested subsequently in the MLR. Suboptimal numbers of T cells exhibited high proliferative activity when pre-incubated with ProT alpha in the presence of autologous monocytes. These studies suggest that ProT alpha potentiates T-cell proliferative responses not directly, but via monocytes which are included in the cultures either as antigen-presenting cells or accessory and/or stimulator cells. The importance of ProT alpha in pathologically occurring defective cellular immune response systems discussed.

Prothymosin α Restores the Depressed Autologous and Allogeneic Mixed Lymphocyte Responses in Patients with Systemic Lupus Erythematosus

AbstractSUMMARY Systemic lupus erythematosus (SLE) is characterized by a variety of profound T-cell abnormalities among which are decreased autologous and allogeneic mixed lymphocyte reactions (auto-MLR and allo-MLR, respectively). In a group of 10 patients with SLE, the mean auto-MLR and allo-MLR responses, tested by tritiated thymidine incorporation, were significantly decreased. If optimal doses of highly purified prothymosin a (ProT α) were present during the auto- of allo-MLR, the T-cell proliferative responses of SLE patients were increased to normal levels. ProT α had more pronouced enhancing effect in patients than in normal individuals. Among patients, ProT α was more effective in those who had active disease and low proliferative responses. These results demonstrate that ProT α can fully restore the deficient T-cell proliferative responses in auto- and allo-MLR in patients with SLE. ProT α, or a certain peptidic fragment of it, could prove potentially useful in the treatment of SLE.

Enhancement of Human T Lymphocyte Function by Prothymosin α: Incremed Production of Interleukin-2 and Expression of Interleukin-2 Receptors in Normal Human Peripheral Blood t Lymphocytes

The in vitro incubation of phytohemagglutinin (PHA)- or alloantigen-stimulated peripheral blood T cells with prothymosin alpha (ProT alpha) resulted in a marked and reproducible increase in the production of interleukin-2 (IL-2). Incubation of T cells with ProT alpha, in the absence of PHA or alloantigen, failed to induce any production of IL-2. ProT alpha by itself did not exert any IL-2 activity. Finally, ProT alpha was shown to increase the expression of IL-2 receptors on phytohemagglutinin- or alloantigen-activated T cells. These data provide the basis for understanding the in vitro immunoenhancing effects of ProT alpha in cellular immune systems.

Decreased expression of HLA-DR antigens on monocytes in patients with multiple sclerosis

Immunofluorescence, cell binding assays and enzyme immunoassays were used to investigate the expression of class II major histocompatibility antigens on peripheral blood monocytes in 67 patients with multiple sclerosis. Monocytes from patients with active disease expressed fewer HLA-DR molecules on their surface than normal monocytes; furthermore the percentage of cells which exhibited detectable amounts of surface HLA-DR antigens was decreased in patients with active multiple sclerosis. During the inactive stage of the disease both deficiencies were milder, probably representing secondary pathogenetic phenomena. Quantitation of monocyte surface HLA-DR antigen expression could be valuable in assessing the clinical disease activity. The demonstration of a molecular defect in patients with multiple sclerosis will improve our understanding of the pathogenesis of the disease.

Prothymosin-α enhances HLA-DR antigen expression on monocytes from patients with multiple sclerosis

Abstract Monocytes from patients with multiple sclerosis (MS) express decreased numbers of class II major histocompatibility complex (MHC) antigens in peripheral blood and are poor stimulators in the autologous mixed lymphocyte reaction (autoMLR). We assessed the effect of prothymosin-α (ProTα) on the expression of MHC class II antigens by monocytes. Immediately after isolation, monocytes were analyzed for MHC class II antigen expression using a radiolabelled monoclonal antibody specific for a monomorphic determinant on HLA-DR antigens. After incubation with ProTα we observed significant increases in HLA-DR antigens on MS monocytes (1.5- to 4-fold increase compared to freshly isolated monocytes). Kinetic analysis revealed that enhancement peaked after 2 days of incubation with ProTα. The increase in HLA-DR antigen on MS monocytes resulted in the restoration of the deficient autoMLR in MS patients. This is the first demonstration suggesting a link between HLA-DR antigen expression and cellular immune defects in MS. The significance of low autoMLR responses for T suppressor levels in MS patients is discussed.

Mechanism of Action of Prothynosin α in the Human Autologous Mixed Lymphocyte Response

AbstractProthymosin α(Prota), an immunologically active polypeptide derived initially from rat thymus, and now pig thymus, was tested for its effect on autoantigen-induced human T cell proliferation in vitro. Pig ProTa was found to enhance the autologous mixed lymphocyte response (auto-NLR). Optimum enhancement was achieved at doses which varied among different donors. Treatment of the stimulatory monocytes with ProTa resulted in considerably higher auto-MLR responses as compared to those with non treated monocytes. ProTa was without effect on T lymphocytes. In contrast, T lymphocytes exhibited enhanced proliferative activity when treated with ProTa in the environment of autologous monocytes. Horeover, supernatants from cultures of monocytes incubated with ProTa(ProTa-sup) were also shown to enhance the human auto-NLR either after addition in cultures or after preincubation with responder T lymphocytes. In addition, ProTa-sup did not demonstrate any detectable inter-leukin I (IL 1) or interleukin 2 (1L 2)...

Granulocyte-macrophage colony-stimulating factor improves immunological parameters in patients with refractory solid tumours receiving second-line chemotherapy: Correlation with clinical responses

In this report, we studied the immunorestorative properties of subcutaneously administered granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with refractory solid tumours receiving second-line chemotherapy. Such patients exhibit abnormal immune responses in vivo and in vitro and, therefore, it was of interest to examine the effect of GM-CSF-induced immunomodulation on clinical response. We examined patients with primary malignant carcinomas (head and neck, n = 10; urogenital tract, n = 17; penis n = 6; colorectal, n = 8) who were treated with carboplatin (JM8), 300 ng/m2 on days 1 and 22, leucovorin (LV), 200 mg/m2 plus 5-fluoracil (5-FU), 500 mg/m2 on days 8, 15 and 29 and four cycles of daily injections with placebo or GM-CSF, 300 micrograms/day on days 3-6, 10-13, 17-20 and 24-27. Peripheral blood was collected from the patients one day after the end of each of the four-cycle injections with placebo or GM-CSF, namely on days 7, 14, 21 and 28. Peripheral blood mononuclear cells (PBMC) were tested in the autologous mixed lymphocyte reaction (AMLR) and for natural killer (NK) or lymphokine-activated killer (LAK) cell activity. Cytokine levels in serum were measured by immunoenzymatic (ELISA) assay. A total of 21 patients received a four-cycle regimen with GM-CSF (Group 1) and 20 were similarly treated with placebo (Group 2). All received standard chemotherapy as outlined above. Before GM-CSF treatment, all patients exhibited increased serum levels of interleukin-1 (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), IL-6 and prostaglandin E2 (PGE2) and decreased serum levels of IL-2. Cellular immune responses (AMLR, NK- and LAK-cytotoxicity) were also low in all patients. Five patients from Group 1 had a PR (partial response), 2 patients had CR (complete response), and 14 patients had stable disease. Seven patients from Group 2 showed progressive disease, 3 had a PR and 10 had stable disease. All immune parameters were significantly improved during treatment in Group 1 but remained unchanged or even deteriorated in Group 2. Administration of GM-CSF during treatment of cancer patients with conventional chemotherapeutic drugs results in a marked potentiation of deficient cellular immune responses in vitro and a change towards normalisation of cytokine serum levels. The results reported herein support the use of GM-CSF as immunopotentiator during chemotherapy, but more patients must be studied before definite conclusions can be drawn.