Mouse Mixed Lymphocyte Reaction Research Articles

CS12192: A novel selective and potent JAK3 inhibitor mitigates acute graft-versus-host disease in bone marrow transplantation

BackgroundDespite the significant role of JAK3 in various autoimmune diseases, including graft-versus-host disease (GVHD), there has been a lack of potent and selective JAK3 inhibitors specifically studied for GVHD. In our preclinical investigations, we evaluated a novel JAK3 inhibitor called CS12192, which is already undergoing clinical investigation in autoimmune diseases. MethodsWe evaluated the efficacy of CS12192 in GVHD through mixed lymphocyte reaction (MLR) in both mouse and human cells, as well as allogeneic bone marrow transplantation (BMT) in a murine model. ResultsCS12192, starting at a concentration of 0.5 μM, dose-dependently reduced the intracellular positivity for cytokines TNF-α and IFN-γ in CD4+ T cells (p < 0.05 to p < 0.0001) and CD8+ T cells (p < 0.01 to p < 0.0001) during mouse allogeneic MLR assays. This effect was observed for both single and double positivity of the cytokines. Moreover, In MLR assays with three different human donors, CS12192 also demonstrated a dose-dependent reduction in the proportion of IFN-γ positive CD4+ T cells (p < 0.0001) and CD8+ T cells (p < 0.01 to p < 0.0001). Additionally, it suppressed T cell proliferation in the mouse MLR (p < 0.05 to p < 0.0001), but this effect was observed in only one human donor (p < 0.001 to p < 0.0001). Furthermore, the administration of CS12192 at 40 and 80 mg/kg BID significantly improved the survival rate in the BMT model, resulting in cumulative 62-day survival rates of 88.89% (p < 0.01) and 100% (p < 0.001), respectively, compared with prednisolone (p < 0.05). ConclusionsCS12192 is a novel, potent and selective JAK3 inhibitor demonstrating great potential to mitigate acute GVHD.

Advances of the novel immunosuppressant brasilicardin A

Brasilicardin A (BraA) is a natural diterpene glycoside isolated from the pathogenic actinomycete Nocardia brasiliensis IFM 0406 with highly potent immunosuppressive activity (IC50=0.057 μg/mL). BraA potently inhibits the uptake of amino acids that are substrates for amino acid transport system L of T cells, which is different from the existing clinical immunosuppressants. BraA is more potent in a mouse mixed lymphocyte reaction and less toxic against various human cell lines compared with the known clinical immunosuppressants, such as cyclosporin A, ascomycin and tacrolimus. Therefore, BraA attracted more attention as a new promising immunosuppressant. However, the development of this promising immunosuppressant as drug for medical use is so far hindered because BraA has the unusual and synthetically challenging skeleton and shows the low-yield production in the natural pathogenic producer. This review introduces the molecular structure of BraA, its activity, mechanism of action, chemical synthesis of BraA analogs, heterologous expression of gene cluster, and an application of combining microbial and chemical synthesis for production of BraA, with the aim to facilitate the efficient production of BraA and its analogs.

Fullerene C60 derivatives suppress the acquired immune responses (THER5P.832)

Abstract Fullerene C60 derivatives are the most promising nanomaterials because of its unique ability to scavenge large numbers of free radicals. Therefore fullerene C60 derivatives with antioxidant effects have been expected to apply to the medicine for inflammatory disease such as inflammatory bowel disease. We have previously shown that fullerene C60 derivatives have the anti-inflammatory effects independent of an antioxidant effects in vitro. However, little is known about the effect of fullerene C60 derivatives on acquired immune system. Here, we examined the effects of fullerene C60 derivatives on the acquired immune responses. At first, we examined the effects of fullerene C60 derivatives on ovalbumin (OVA)-induced immune responses in vivo. Treatment of OVA plus fullerene C60 derivatives induced a lower level of OVA-specific IgE and IgG than did treatment of OVA alone, suggesting that fullerene C60 derivatives suppressed acquired immunity. Next, we investigated the influence of C60 fullerene derivatives on T cell proliferation in a mouse mixed lymphocyte reaction. The fullerene C60 derivatives suppressed the production of IL-2 from responder splenocytes. Furthermore, fullerene C60 derivatives also suppressed the IL-2 production from CD4+ T cell stimulated by anti-CD3 and anti-CD28 antibodies, although NAC (anti-oxidant medicines) didn’t show such effect. These results show that the fullerene C60 derivatives have the effect to suppress the acquired immune system.

SAR studies of brasilicardin A for immunosuppressive and cytotoxic activities

Eleven derivatives ( 5– 13, 15, and 16) of an immunosuppressive and cytotoxic tricyclic terpenoid, brasilicardin A ( 1), were prepared and assayed for inhibitory effects to the mouse mixed lymphocyte reaction (MLR) and seven human tumor cell lines. The 17 N-methyl form ( 8) of 1 showed the most potent immunosuppressive activity in mouse MLR, while induction of more bulky group for N-17 resulted in significant decrease of the activity. Compound 8 also showed potent cytotoxic activity against DLD-1, Lu-65, A549, K562, and MOLT-4 cells, while the benzyl ester ( 13) of 1 exhibited potent cytotoxicity against K562, MOLT-4, and jarkat leukemia cell lines. The 17 N-acetyl derivative ( 11) of 1 selectively inhibited the cell growth of DLD-1 cells. The methyl ester ( 5) of 1 showed potent cytotoxic activity against K562, MOLT-4, and Ball-1 cell lines, the last of which was resistant to 1, 8, and 13.

Dipeptide boronic acid, a novel proteasome inhibitor, prevents islet-allograft rejection.

We have demonstrated previously in vitro that proteasome inhibitors suppress the proliferation, and induce the apoptosis, of activated T cells. This implies that they could be used as a novel category of immunosuppressants to block allograft rejection. Therefore, in this study, dipeptide boronic acid (DPBA) was tested for its effect on mouse islet transplantation. First, DPBA was investigated in vitro for its effect on mouse mixed lymphocyte reaction (MLR) and cytotoxic T-lymphocyte (CTL) activity. DPBA was then used in vivo to treat mouse islet-allograft rejection. Both MLR and CTL were dose dependently suppressed by the proteasome inhibitor. A 17-day DPBA regimen resulted in islet-allograft survival in 50% of the recipients for a duration of up to 60 days, whereas the control group without immunosuppressants rejected the islet graft in 7 days. DPBA showed moderate side effects according to blood biochemistry; the function of endogenous islets after treatment appeared normal on glucose challenge. The proteasome inhibitor could inhibit islet-allograft rejection in mice without serious side effects at therapeutic dose levels. This has opened a new dimension in the development of better immunosuppression regimens for islet transplantation.

SNF4435C and D, Novel Immunosuppressants Produced by a Strain of Streptomyces spectabilis. III. Immunosuppressive Efficacy.

Novel immunosuppressants, SNF4435C and D produced by a strain of Streptomyces spectabilis, were examined for their pharmacodynamical profiles. SNF4435C and D suppressed the responses of both murine splenocytes and human peripheral blood lymphocytes in the mixed lymphocyte reaction (MLR) with IC50 values of 0.5 microM and 0.2 microM, respectively. In the mouse MLR experiments, SNF4435C and D did not block the production of interleukin-2 (IL-2) and the compounds-induced suppression was not restored by the addition of exogeneous IL-2. In addition, the significant inhibitory action was still retained even when SNF4435C or D was added after 48 hours from the start of the culture. These results were distinct from the behaviors observed with FK-506. SNF4435C, the major component, suppressed mouse delayed type hypersensitivity (DTH) and prolonged rat skin allograft survival.

Brasilicardin A, a new terpenoid antibiotic from pathogenic Nocardia brasiliensis: fermentation, isolation and biological activity.

A novel tricyclic diterpenoid antibiotic, brasilicardin A, was isolated from the culture broth of Nocardia brasiliensis IFM 0406. The antibiotic exhibited immunosuppressive activity in a mouse mixed lymphocyte reaction (MLR) assay system and its IC50 value was 0.057 microg/ml. Although the inhibitory activity of cyclosporin A (CyA) against IL-2 production was confirmed in the MLR assay system, brasilicardin A did not have the activity. The results of in vitro toxicity testing of brasilicardin A against various human cell lines were compared with those of CyA.

Synthetic analogues of TNP-470 and ovalicin reveal a common molecular basis for inhibition of angiogenesis and immunosuppression

TNP-470 ( 1), a synthetic derivative of the natural product fumagillin ( 2), potently inhibits angiogenesis in vivo and the growth of endothelial cell cultures in vitro. The structurally related natural product ovalicin ( 3) also inhibits angiogenesis but possesses potent immunosuppressive activity. The recent finding that all three drugs bind and inhibit the same target, methionine aminopeptidase 2 (MetAP2), raised the question of whether TNP-470 is also immunosuppressive and whether inhibition of MetAP2 underlies both activities of ovalicin. To address these questions, we synthesized a series of analogues of TNP-470 and ovalicin and tested them for their abilities to inhibit the proliferation of either endothelial cell or mixed lymphocyte cultures. TNP-470 and its analogues were found to possess both immunosuppressive and anti-angiogenic activities. A strong correlation was observed between the ability of compounds to inhibit bovine and human endothelial cell growth and their ability to inhibit the mouse mixed lymphocyte reaction (MLR), implying that the two activities share a common molecular basis, i.e., inhibition of MetAP2. Interestingly, ovalicin and several other compounds behaved differently in the human MLR than in either the mouse MLR or human endothelial cell proliferation assays, pointing to possible species-specific and cell type-specific differences in the metabolism or uptake of these compounds.

FR-900520 and FR-900523, novel immunosuppressants isolated from a Streptomyces. II. Fermentation, isolation and physico-chemical and biological characteristics.

FR-900520 and FR-900523, novel neutral macrolide immunosuppressants, were isolated from the cultured broth of Streptomyces hygroscopicus subsp. yakushimaensis No. 7238. Their molecular formulae were determined as C43H69NO12 and C42H67NO12, respectively. The compounds suppressed immune response in vitro. IC50 values of FR-900520 and FR-900523 for mouse mixed lymphocyte reaction were 0.55 nM and 1.6 nM, respectively. FR-900520, the major component, clearly prolonged skin allograft survival in rats.

SUPPRESSION OF CELLULAR IMMUNE REACTIONS BY RAT OVARIAN CELLS

The influence on the immunologic response by rat ovarian cells of follicle and stromal tissue origin was investigated using the mixed lymphocyte reaction (MLR), the phytohemagglutinin (PHA) assay, the cytotoxic T lymphocyte (CTL) assay, and a cytotoxic T-cell-mediated microcytotoxicity test. The MLR between BN/Mai Pfd (RT-In) stimulator splenocytes (mitomycin-C treated) and R/A Pfd (RT-1u) responder lymph node cells was markedly suppressed by ovarian cells of follicle and/or stromal tissue origin. A similar in vitro inhibition was observed with cell-free supernatants from ovarian cells, not only in the rat MLR but also in the mouse MLR between Balb/c stimulator splenocytes and C57B1 responder lymph node cells. Moreover, the reactivity of rat lymphocytes was suppressed when they were cocultured with ovarian cells in the PHA and CTL assays. Preincubation of the supernatants with serial diluted antiprogesterone serum (APS) and antiestradiol serum (AES) revealed that the suppressive effect of these supernatants could be completely abolished with APS--and, to a lesser extent, by AES. As shown by the cell-mediated microcytoxicity assay both the ovarian cells and the steroids secreted by these cells were, however, ineffective in suppressing the effector phase. It is suggested that the steroid secretion by the ovary may play an important role in the prolonged survival of ovarian grafts.

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera.

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due to H-21-region differences. In both cases of MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. - Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in re-stimulation during MLR-II. - These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.

Lymphokine Production in Primary Mixed Lymphocyte Reactions

The characteristics of the responder and stimulating cells involved in migration inhibition factor (MIF) production in primary 'one-way' mouse mixed lymphocyte reactions (MLR) were analyzed by using an indirect agarose droplet assay. T-lymphocytes are mainly responsible for MIF release, as shown by pretreatment with anti-Thy 1.2 serum plus complement or purification over a nylon wool column. On the other hand, macrophages and B-lymphocytes appear to be optimal stimulating cells. T-lymphocytes as stimulating cells induce MIF release, but to a much lesser degree than macrophages and B-lymphocytes. The kinetics of MIF production in MLR is related to the kind of stimulating cells employed. Lastly, the ability to release MIF is already present in the spleen of 1- to 2-week-old mice, lasts until 20 weeks of age and declines to undetectable levels at 50 weeks of age.

Lymphokine production in mouse mixed lymphocyte reaction (MLR)

We have investigated alloantigen differences which stimulate lymphokine release and3H-TdR uptake in primary ‘one-way’ MLC among allogeneic mice. When mice differing at the wholeH-2 region were tested, MIF and immune IF release was observed, along with a marked3H-TdR uptake. Differences atK, D, orI-S-G regions stimulate both lymphokine release and3H-TdR uptake, though stronger immune IF and3H-TdR responses were observed with differences atI-S-G regions. On the other hand, when mice differing in their minor histocompatibility antigens, and notably at theMls locus, were tested, lymphokine release took place even in the absence of proliferation. Lastly, in MLC between mice differing at multiple minor loci, butH-2 andMls matched, MIF release only, and not immune IF and3H-TdR responses were observed in a few combinations. These findings show that T lymphocytes can recognize alloantigens by releasing lymphokines even without going through proliferation. Moreover, different levels of T-lymphocyte activation exist, depending on the kind of stimulating alloantigens present.

Secondary mouse mixed lymphocyte reaction (MLR) in vitro: Correlation between primed cells reactivity and serological typing for Ia specificities

B10.M (H-2f) spleen lymphocytes were cultured for 14 days with X-irradiated B10.P (H-2p) lymphocytes. These primed cells were then tested for their capacity to respond to a secondary stimulation induced by a panel of mouse cells carrying differentH-2 haplotypes. The third-party cells induced various degrees of proliferation which could be expressed as a percentage of the proliferation induced by the primary stimulating strain (relative response = RR) and could then be classified according to the RR. Anti-Ia antibodies present in a B10.M anti-B10.P serum were studied by the dye exclusion lymphocytotoxicity technique (LCT) against the same panel, after absorption of H-2K, D antibodies on B10.P platelets. The strain panel could then be classified according to the LCT titers of the absorbed immune serum. A significant correlation (r=0.96,P<0.01) was found between both classifications. According to the Ia chart, the public specificities involved were Ia.6, 7, and 13, but these did not fully explain either the primed cell reactivity or the LCT results. An unexpected crossreactivity was observed between B10.P and B10. Absorption-elution experiments with A.TH anti-A.TL serum demonstrated that B10 and B10.P share the Ia.3 antigen. These results indicate that the structure(s) recognized by the primed lymphocytes is the Ia antigen(s).

Suppressor cell activity after isoprinosine treatment of lymphocytes from normal mice

Isoprinosine added in the range of 0.1–100 μ/ml to mouse lymphocyte cultures induced suppressor cell activity inhibiting mouse mixed lymphocyte reactions to approximately the same extent as Con A-activated suppressor cells.

Micromethods for induction and assay of mouse mixed lymphocyte reactions and cytotoxicity.

A new method in which induction of mixed lymphocyte responses and cytotoxicity can be performed under identical conditions is described. This enables the investigation of the relationship between MLR and cytotoxicity. A micro method for the 51Cr release assay for cytotoxicity is described using both mitogen-induced blasts and tumors as target cells. Under the conditions used data obtained is suitable for linear regression analysis, enabling quantitative comparisons to be made.

Dissociation of lymphocyte activating determinants from recognition sites in mouse MLR.

PREVIOUSLY, we were able to block mixed lymphocyte reactions (MLR) in man using anti-light chain antibody1 but we have been unable to confirm this observation in subsequent studies using other anti-light chain sera. We have now found that MLR in the mouse is unaffected by a variety of anti-immunoglobulin sera but using alloantisera it has become evident that the stimulator cells, but not the responder lymphocytes, can be blocked.