Victor I. Seledtsov

Antiproliferative Effect of Bone Marrow Cells on Leukemic Cells

When normal murine bone marrow (BM) cells were cultured with either L1210 lymphoma cells or P815 mastocytoma cells for 24 h, considerable tumor growth suppression without substantial tumor cell lysis was found. Under the same conditions, normal spleen cells also demonstrated the antitumor cytostatic activity, but not as significant as that characteristic of BM cells, whereas both normal thymus and lymph node cells had not any suppressive effect on tumor cell proliferation. The comparable cytostatic effects occurred in both syngeneic and allogeneic BM-tumor cell combinations. The cytostatic BM-effectors were distinct from T and B lymphocytes or mature macrophages. After being separated on a discontinuous Percoll density gradient, the cells active in suppressing tumor growth were recovered predominantly in 1.075 and 1.060 density fractions. The cytostatic BM effectors, at least in their part, were resistant to x-irradiation up to 2000 rad included. Collectively these results suggest that normal BM, being deficient in cell-mediated antitumor cytolytic activity, has a significant leukemia growth inhibitory potential; and that cytostatic BM effectors are similar in their characteristics to natural suppressor cells.

Characterization of Erythroid Cell-Derived Natural Suppressor Activity

Nucleated erythroid cells (NEC) have been previously reported to the capable of suppressing antibody-mediated primary (IgM) and secondary (IgG) immune responses to thymus-dependent antigens. In the present study we indicated that NEC, separated from the spleens of mice following phenylhydrazine treatment were able to suppress directly the proliferative response of preactivated B cells to lipopolysaccharide (LPS) in vitro. While being active in suppressing B cell blastogenesis, NEC, however, failed to reduce both cell proliferation and cytotoxic T lymphocyte (CTL) generation in an allogeneic mixed lymphocyte culture (MLC). NEC also lacked a significant effect on interleukin (IL)-2 production and utilization by concanavalin A (Con A)-activated T lymphocytes. The NEC-derived suppression of B cell proliferation was, at least in part, mediated by soluble molecules. The specific blockade of transforming growth factor (TGF)-beta synthesis with antisense oligodeoxynucleotides (OD) binding TGF-beta mRNA, as well as the neutralization of TGF-beta activity with anti-TGF-beta antibodies (Ab), resulted in a detectable diminished ability of the NEC-conditioned medium (CM) to suppress B cell blastogenesis. Taken together, the results suggest that: 1) NEC may suppress directly B cell responses, while not affecting T cell ones; 2) NEC may mediate their natural suppressor (NS) activity partially through releasing TGF-beta.

A novel dual NO-donating oxime and c-Jun N-terminal kinase inhibitor protects against cerebral ischemia-reperfusion injury in mice.

The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30min) with subsequent reperfusion (48h). Mice were treated with IQ-1S (25mg/kg) suspended in 10% solutol or with vehicle alone 30min before and 24h after middle cerebral artery (MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30min of MCAO provoked by a filament and during the first 30min of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48h of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury.

A POSSIBLE ROLE OF PRE-EXISTING IGM/IGG ANTIBODIES IN DETERMINING IMMUNE RESPONSE TYPE

On the basis of the data indicating the existence of two types of immuno‐protection, namely macrophage‐mediated and mast cell‐basophil‐mediated, it is argued that by reacting with potential pathogens, pre‐existing IgM and IgG antibodies (both natural and induced by environmental microflora) might promote involvement of macrophages in the presentation process, favouring the generation of pathogen‐specific T helper 1 (Th 1), hut not Th2, responses. Alternatively, the failure of these antibodies to effectively recognize pathogens might be associated with active involvement of pathogen‐specific B cells in presenting Ag and, as a consequence, with the predominant development of Th2. rather than Th 1, responses.

Direct effects of interleukin-7 on the function of human T cells in vitro

CD3+ T lymphocytes were isolated by positive magnetic separation from the peripheral blood of healthy donors. In the absence of any additional activating stimuli, interleukin-7 (IL-7) was shown to augment the levels of T cells expressing CD25 activation marker both in CD4-positive and in CD4-negative effector memory (CD45RA-CD197-) T cell subsets, as well as in terminally differentiated (CD45RA+CD197-) T cells, without significantly affecting the activation status of naive (CD45RA+CD197+) and central memory (CD45RA-CD197+) T cells. In addition, IL-7 noticeably enhanced the production of IL-2, interferon-γ (IFN-γ), and IL-10, but not IL-4, in T cells. The direct effects of IL-7 on T cell activation induced in vitro by MACSiBead™ particles coated with CD2, CD3, and CD28 antibodies (Abs) were also investigated. Upon cell activation, IL-7 significantly augmented the levels of CD25+ T cells in naive (CD45RA+CD197+), central memory (CD45RA-CD197+), and effector memory (CD45RA-CD197-) T-cell compartments. In addition, IL-7 facilitated activation of CD4- (but not CD4+) terminally differentiated effector (CD45RA+CD197-) T cells. Finally, IL-7 was found to upregulate the production of IL-2, IFN-γ, IL-4, and IL-10 by activated T cells. In conclusion, we speculate that IL-7 is capable of enhancing functional T cell activity without causing significant functional inbalance between various T cell subsets.

Direct effects of interleukin-8 on growth and functional activity of T lymphocytes

Abstract CD3+ T‐lymphocytes were isolated from the normal donors by positive magnetic separation. Activation of the T cells with particles conjugated with antibodies to CD3, CD28 and CD2 molecules led to a marked increase in T‐cell production of interleukine‐8 (IL‐8). We present evidence that IL‐8 receptor &agr;‐chain (CXCR1, CD181) is expressed on the cell surface of 13.3% T cells. Activation of T‐lymphocytes resulted in significant enhancement of CD181+ cells both in naive CD4+ T cell and terminally differentiated effector CD4+ T cell compartments with concomitant reduction of CD181+ cells in effector memory CD4+ T cell subset. The level of T cell activation was assessed judging from the surface expression of CD25 (IL‐2 receptor &agr;‐chain). We demonstrate that IL‐8 treatment (0.01–10.0 ng/ml concentration range) reduced the activation status of both CD4− and CD4+ effector memory T cells, as well as terminally differentiated effector T cells, without significantly affecting the activation of naive T cells or central memory T cells. In addition, IL‐8 up‐regulated IL‐2 and down‐regulated IL‐10 production by activated T cells, with no effect on interferon‐gamma (IFN‐&ggr;) and IL‐4 production. Data obtained suggests the importance of IL‐8 in the direct regulation of adaptive T cell reactivity. HighlightsActivation of T‐lymphocytes leads to an increase in T‐cell production of IL‐8.IL‐8 is able to modulate IL‐8 receptor expression on activated T cells.IL‐8 is capable of affecting directly TCR‐mediated activation of both CD4+ and CD4− T subsets.IL‐8 is a modulator of cytokine production by activated T cells.

Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3+ T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA+/CD197+ naive T cells, CD45RA-/CD197+ central memory T cells, CD45RA-/CD197- effector memory T cells and CD45RA+/CD197- terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114+ T cells in central memory CD4+ T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38+ T cells in CD4+ naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4- central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells.

A Possible Role for Idiotype/Anti-idiotype B–T Cell Interactions in Maintaining Immune Memory

Variable regions of both B-cell receptors (BCRs) and T-cell receptors (TCRs) are completely formed in the postnatal period, and, consequently, no innate immune tolerance against these structures exists in adulthood. Indeed, antibodies (Abs) specific to TCRs have been found in both animals and humans. These facts clearly indicate the existence of B cells able to directly interact with T cells through binding of BCRs to TCRs without implicating major histocompatibility complex molecules. A novel paradigm is proposed in that the immune memory is based on idiotype/anti-idiotype interactions occurring between BCRs and TCRs following clearance of the antigen that elicited immune responses. It is envisaged that direct contact between memory T and B cells could provide co-stimulatory signals needed to sustain viability, growth, and differentiation of the interacting immune cells. In contrast, plasma cells originating from memory B-cells could produce anti-TCR Abs that inhibit direct BCR-to-TCR interactions, thereby downregulating the B- to T-cell contact-based immune memory via a negative feedback mechanism.

Xenogeneic cell-based vaccine therapy for stage III melanoma: safety, immune-mediated responses and survival benefits.

BackgroundNew therapies for melanoma have yielded promising results, but their application is limited because of serious side-effects and only moderate impact on patient survival. Vaccine therapies may offer some hope by targeting tumor-specific responses, considering the immunogenic nature of melanomas.ObjectivesTo investigate the safety profile and efficiency of a xenogeneic cell-based vaccine therapy in stage III melanoma patients and evaluate the survival rate in treated patients.Materials and MethodsTwenty-seven stage III melanoma patients were immunized with a lyophilized xenogeneic polyantigenic vaccine (XPV) prepared from murine melanoma B16 and carcinoma LLC cells.ResultsNeither grade III/IV toxicities, nor clinically significant changes in blood and biochemical parameters were noted after an induction course of 10 XPV subcutaneous immunizations. No laboratory or clinical signs of systemic autoimmunity were documented. Following 10 vaccinations, a relative increase in the numbers of circulating memory CD4+CD45RO+ T cells (but not CD8+ CD45RO+ T cells)was observed. Peripheral blood mononuclear cells obtained from XPV-treated patients demonstrated increased proliferative responses to human BRO melanoma-associated antigens and marked increases in serum levels of IFN and IL-8. Serum levels of TNF-, IL-4 and IL-6 were not affected. The overall five-year survival rate in the treated patients was significantly higher than that in 27 control patients with matched clinical and prognostic characteristics (55% vs 18%).ConclusionXPV-based immunotherapy could be maximally effective when started as early as possible before or after surgical excision of the primary tumor and local metastases, i.e. when tumor-mediated suppressive effects on immunity are minimal.

Induction of mixed allogeneic chimerism for leukemia

Hybrid (C56BL/6 x DBA) (BDF1; H-2b/H-2d) mice bearing the P815 leukemia (H-2d) were grafted with a (CBA x C57BL/6)F1 (CBF1; H-2k/H-2b) cell suspension, comprising bone marrow cells (BMC; 25 x 10(6)/mouse) and spleen cells (SC; 55 x 10(6)/mouse) on day-4, then treated with cyclophosphamide (200 mg/kg) on day-2 and finally grafted once more with CBF1 cells (25 x 10(6) BMC + 7 x 10(6) SC) on day 0. Allogeneic cell graftings performed in this way induced durable mixed hematopoietic chimerism and significantly prolonged the survival of recipients, compared with that of leukemia-bearers grafted with syngeneic cells. The results obtained raise the possibility of using allogeneic hematopoietic tissue transplantation in combination with non-lethal cytoreductive therapy to induce a long-lasting graft-vs-leukemia effect.