| ObjectiveBone marrow transplantation (BMT) is a major method for treatment of leukemia. There are two kinds of BMT, autogeneic or syngeneic bone marrow transplantation and allogeneic bone marrow transplantation. The application of allogeneic bone marrow tranplantation is limited because of severe complication(graft-versus-host disease,GVHD),and absence of donor bone marrow.Now autogeneic bone marrow transplantation in our country is major type of BMT for it has no complication . But major problem following autogeneic BMT is the high relapsed rate.The data from clinical statistics demonstrated that patients sufferred from GVHD following allogeneic BMT have lower relapsed rate than similar patients without GVHD. It was confirmed in animal experiment that GVHD has graft-versus-leukemia (GVL) effects and can decrease relapsed rate of leukemia. These results suggested that one reason of leukemia recrudescence following auto-BMT is that aut-BMT has no GVL effect. If a GVL effect can be induced by artifial methods in patients following auto-BMT,it will prevent the recrudescence of leukemia.Glazier and Bryson reported that GVHD-like syndrome, syngeneic GVHD(SGVHD), developed following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a 21-day course of the immunosuppressive agent cyclosporin A (CsA) in rat or murine, respectively. More importantly, the syngeneic GVHD possessed GVL effect. Phase I trail of the cyclosporine-induced auologous graft-versus-leukemia was carried out for treatment of patients with leukemia. But it is difficult to control intensity of SGVHD becausethe mechanism of SGVHD is unclear. So the application of the treatment method was limited.Purpose of this article is to study the immunological mechanism in induction of SGVHD and to provide new experimental data and strategy for prevention of recrudescence of leukemia following auto-BMT.Materials and Methods1. Induction of murine SGVHDRecipient mice were lethally irradiated. The animals were reconstituted with syngeneic T cell-depleted BM. Beginning on the day of transplantation , the mice were treated daily for 21 days with CsA.2. Evaluation of murine SGVHDFollowing cessation of CsA, the animals were weighed three times per week and observed for clinical signs of the development of SGVHD (weight loss, diarrhea). The tissue sections of liver and colon were prepared and then stained with a standard H&E staining procedure. All tissue sections were analyzed by pathologist with double-blind methods and graded for inflammation caused by SGVHD using a previously published grading scale. To perform immunohistochemistry, tissue samples were placed in tissue-freezing medium and snap frozen in liquid nitrogen Tissues were cut into thin sections and then were stained with primary mAb against CD4 and CD8. The cells were visualized using HRP-conjugated anti-rat secondary Abs.3. Isolation and purification of spleen cells and colon IELThe spleen and colons were removed. Single spleen cell suspensions were prepared from individual spleen according to described methods. IEL were then isolated according to a modification of the method of Lefraneois and Lycke. Briefly, the colon from two to four mice was cut longitudinally, then cut latitudally into 0.5-lmm sections, washed with CMF, and placed into CMF containing 1 mM DTT and 1 mM EDTA. The tissue was shaken for 30 min at 37°C. The resuspended cells were then passed over a nylon wool column. Following concentration by centrifugation, the cells were resuspended in 44% Percoll and layered onto a 66%Percoll cushion. The tubes were centrifuged and then the purified IEL were removed from the 44/67% Pereoll interface and washed.4. Flow cytometry for analysis of spleen and colon IEL cell phenotypesIsolated lymphoid cells from the spleen or colon were placed in staining buffer. To reduce nonspecific staining, cells were incubated with Ab against FcII/FcIIlR. The cells were then stained with fluorochrome-conjugated mAb against various lymphoid surface markers. Conjugated mAb against CD4, CD8 , CD3, a(5 TCR , ySTCR, were used for two- and three-color analysis using a BD Biosciences FACSCalibur flow cytometer.5. RT-PCR for detection of cytokinesAdd 2X 105 IEL into 96-well plate immobilized or unimmobilized by anti-CD3 monoclonal antibody and incubated at 37°C for 4-6 hr. Total RNA was isolated from the IEL. RNA was reversely transcribed into cDNA using a reverse transcription system. PCR reactions were then performed using primers for P-actin, IFN-7, TNF-a and IL-12,11-4, IL-10. The PCR products were analyzed using the ImageMaster Gel Analysis Software. OD values for each individual fragment were normalized to P -actin expression from the same tissue fragment.6. T cell depletion in vivo by anti-CD4 and anti-CD8 mAb treatment(1) The normal mice were also treated with anti-CD4 mAb and anti-CD8 mAb daily for 1 week, and spleen cells and IEL of colon were analyzed by flow cytometry.(2) On the day of BMT, mice were individed into four groups: control group (no treatment) , CsA-treated group ( CsA treatment for 21 days), and mAb-treated control group(mAb treatment daily for 7 days and then every other day for an additional 14 days) and mAb-CsA- treated group(CsA treatment and mAb treatment).Then spleen cells and IEL were isolated; and the level of T cell depletion was determined by flow cytometry.(3) On the day of BMT, mice were individed into four groups: control group (no treatment) , CsA-treated group ( CsA treatment for 21 days), and mAb-treated control group(mAb treatment daily for 7 days and then every other day for an additional 14days) and mAb-CsA- treated group(CsA treatment and mAb treatment)_Following cessation of CsA and mAb therapy, the animals were monitored for the development of clinical symptoms of SGVHD. In some experiments, mice were euthanatized. The sections of liver and colon were detected pathologically.7. Statistical analysisStatistical differences between control and SGVHD samples were determined using Mann-Whitney test, Student's t test, Fisher's exact test. Differences p<0.05 were considered statistically different.Results1. The change of T cell subsets in spleen of SGVHD miceThe total number of T cells and its subsets in SVGD mice did not increase but decrease, compared to normal or control mice (p<0.05).2. Alterations of T cell phenotype in IEL of SGVHD miceThe results by immunohistochemistry demontrated that CD4+ T cells in colonic mucosa increased in SGVHD mice related to control mice. The analysis for phenotype of colonic IEL by FCM showed that number of CD4+ T cells significantly increased and CD3+CD4'CD8" double negative (DN) T cells distinctly decreased in SGVHD mice. But the number of CD8+ T cells and total number of T cells were similar in SGVHD, in normal and in control mice. The augmentation of CD4+ T cells was a result of increased CD4+ctP T cells. The number of CD4+apT cells in EELs was 12% in normal or control. But it increased to 28% of EEL in SGVHD, double times than normal or control mice (p<0.001). Reduction of DN T cells was a result of decreased DN apT cells .The number of DN ap T cells decreased from 10% in normal or control mice to 2.8% in SGVHD mice. But the numbers of y5T cells were similar in normal, control or GVHD mice. In addition, we found that proportion of CD4+apT cells and DN ap T cells increased from 1.0-1.5:1 to 13-25:1. These results show that unbalance of CD4+ apT cells and DN apT cells is important to induction of SGVHD.3. Expression of cytokines of colonic IEL in SGVHD miceTotal mRNA was extracted from murine IEL and transcribed reversely intocDNA.The expression level of cytokines(IL-4, IL-10, IL-12, IFN-Y , TNF-a) are detected by half-quantitative RT-PCR. These results showed that expresssion level of IL-2 and IFN- Y increased in GVHD mice. IEL stimulated by anti-CD3 antibody expressed an enhanced IFN-y and TNF-a than that of unstimulated IEL. But that of IL-12 was invariable in anti-CD3mAb-stimulated and anti-CD3mAb-unstimulated group. Expression of IL-4 and IL-10 had not been detected in normal or control or SGVHD mice.4. Expression of IEL phenotypes and cytokines during inducing course of SGVHDThe above findings demonstrated that enhanced CD4+ T cells were present in the colon during active disease, but it was not clear as to when the T cells began to accumulate within the colon of CsA- treated animals. Following BMT and CsA treatment, spleen cells and IEL were isolated from one-week, two-week and three-week mice and phenotype of spleen cells and IEL was analyzed by Flow cytometry and the expression levels of cytokines were detectd by RT-PCR. The results showed that subsets of colon IEL and expression of cytokines are similar in CsA-induced mice and in control mice after therapy of CsA for one week. At two weeks post-CsA, number of CD4+ aPT cells started to increase and DN aPT cells slightly decrease. But the expression of cytokines had not been detected in control mice and in CsA-induced mice. At the third week post-CsA, number of CD4+ccPT cells in colon IEL increased and DN apT cells decreased significantly. Expression of IFN- Y enhanced but that of IL-12 and TNF-a had not been detetected. These results demonstrated that at the third week after therapy of CsA, the proportion of CD4+aP T and DN apT cells had disregulated and CD4+aP T has been activated to secrete IFN-Y.5. Effect of T cell depletion on the development of SGVHDThe above findings demonstrated that activated CD4+ cells were important for the occurrence of SGVHD. But Flanagan reported that anti-CD4 and anti-CD8 antibody therapy could not inhibit GVHD. For the reason, we used anti-CD4 andanti-CD8 antibody to treat mice for seven days according to Flanagan's design. Then we analyzed CD4+ and CD8+ T cells population of splenic cells and IEL. Though CD4+ and CD8+ T cells in spleen are almost depleted by antibody treatment, CD4+ and CD8+ T cells in colonic IEL are similar in normal mice and antibody-treated mice. So another in vivo T cell depletion studies were designed. The animals were given 500 u g of mAb daily for 1 week, then every other day through the end of CsA treatment. The results from analysis of FCM showed that both CD4+ and CD8+ T cells were depleted efficiently from the spleen and IEL of control and CsA-treated mAb-treated animals. Further experiment using this treatment design demonstrated that treatment with anti-CD4, but not anti-CD8, reduced induction rate of SGVHD and eliminated clinical GVHD symptoms and colon pathology. Interestingly, neither anti-CD4 nor anti-CD8 therapy affected the development of liver pathology associated with SGVHD. These findings demonstrated that the pathological mechanisms in liver and colon of SGVHD were different each other. CD4+ T cells initiate development of the intestinal inflammation associated with murine SGVHD.But both CD4+ T and CD8+ T did not associated with inflamation of liver. Maybe other factor took part in inflamation of liver.such as macrophage and NK cells. Conclusion and innovation1. The number of CD4+apT cells increases and the number of DN aPT cells decreases in colonic IEL of SGVHD mice. Their proportion is disregulated.2. Expression of Thl-type cytokines (IL-12,EFN-y,TNF-a) from colonic BEL of SGVHD mice increases significantly.3. Therapy of CsA disturbs balance of CD4+aP T and DN aPT cells in normal colonic IEL and induces expression of cytokines.4. CD4+T cells not CD8+ T cells have an important effect on induction of SGVHD and immunopathology of colon.5. A new hypothesis about mechanism of SGVHD was suggested: Normally, the positive regulatory CD4apT cells keep balance with negative regulatory DN a.pT cells. This balance maintains immunological homeostasis, especially in local part ofintestines. During syngeneic BMT, DN T cells are depleted through pretreatment of transplantation. Additionally, therapy of CsA disturbs negative selection in thymus and self-reactive T cells are not depleted. The self-reactive T cells are transported from thymus to peripheral, such as colon. It makes proportion of CD4+aBT cells and DN apT cells maladjust. Because restraint of DN T cells is lowed, self-reactive CD4+aP T cells activate, proliferate and release cytokines (IFN-y, TNF-a) to induce immunopathological lession of tissue. The hypothesisespecially emphasizes negative regulation of DN T cells which are easily ignored and balance of CD4+aP and DN apT cells are important for homeostasis of immune system. It belongs to original innovation.ObjectiveCD137 and CD137L is another pair of important costimulatory molecules besides CD28-B7 for T cell activation. As an inducible T cell surface receptor, CD137 is a member of tumor necrosis.factor receptor superfamily and mainly expresses on activated T cells. CD 137 ligand (CD137L) mainly expresses on activated antigen presenting cells including dendritic cells, monocyte-macrophages and B cells. CD137-CD137L costimulatory signal can activate T cells by depending or independing on CD28-B7 pathway to play an important role in immune reponses, immune regulations, tumor immunity and so on, which has broad prospects for clinical application.Now the expressions and functions of murine CD 137 have been researched in detail, but there were limited data about human CD 137. The data from some research demonstrated that human CD 137 was different from murine CD 137 on some aspects. It is more useful to research human CD 137 pathway further. Since 1996 we have studied the expression feature of CD137 on T cells and T cell subsets, the relation of CD 137 expression with RA and SLE. The objective of this paper is first to study the expression feature of CD 137 on human DCs and the impact of CD 137 on the direct inhibiton of tumor mediated by DCs, second to study the expression feature and the function of CD137-CD137L on tumor line cells from different sources, human solid tumor tissues and leukemia cells, so as to study further the function of CD137-CD137L and its applied value on the diagnosis and the therapy of tumor. Materials and Methods1. The expression and function of CD137 and CD137L on human peripheral blood DCs(1) The induction of human peripheral blood DCs: The monocytes derived from PBMC were induced into immature DCs by culturing with hrGM-CSF and hrIL-4 for 5-7 days. The morphology and the number of nonadherent cells were observed and calculated by microscope.(2) The expression of CD137 and CD137L on human peripheral blood DCs: The related phenotypes of the DC cells were detected by Flow Cytometry.(3) The effect of agonistic anti-CD137 monoclonal antibody on the DCs-mediated direct inhibition of tumor cells by MTT method: The immature DCs was stimulated by LPS to induce maturation. The direct inhibition of tumor cells (Hela, HT29, Lovo, HepG2, HepG2.2.15) mediated by human DCs was measured by method of MTT. Agonistic anti-CD137 antibody was added into the above system and then direct inhibition of tumor cells was measured by MTT method.2. The expression and function of CD137 and CD137L on human tumor(1) The expression of CD137L on human tumor cell lines: The expression of CD137L on human tumor cell lines were detected by RT-PCR on RNA level and by FCM on protein level..(2) The expression of CD137 and CD137L in human solid tumor tissue: The expression of CD137 and CD137L on normal tissue, benign and malignant tumor tissue was detected by Immunohistochemistry method.(3) The expression of CD137 and CD137L on peripheral blood white cells from patients with leukemia: The expressions of CD137 and CD137L on white blood cells of peripheral blood from normal people and different leukemia people were detected using FCM.(4) The function study of CD137L on human tumor cells (ELISA): T cells were purified using RosetteSep? ANTIBODY COCKTAIL. T cells co-cultured with tumor cells L78> HepG2.2.15 expressing CD137L for 48h, supernant was collected and the expression of IFN- y was detedcted by ELISA. The tumor cells HLEn A2^ HepG2.2.15 on which CD137L expressed cocultured with CHO cells on which CD 137 expressed for 48 h and supernatant was collected . The expression level ofIL-8 was detected by ELISA. Results1. he expression and function of CD137 on human peripheral blood DCs(1) The induction of human peripheral blood DCs: After 5 to 7 days' culture, the non-adherent cells from PBMC had the typical morphology of dendritic cells. The results from FCM showed these cells expressed 64.02% of HLA-DR and 2.34% of CD 14 (marker of monocytes).(2) The expression of CD137 on human DCs: The result from FCM showed that human immature DCs expressed 9.77% of CD137 and 41.03% of CD137L. On human mature DCs stimulated by LPS, the expression of CD137 increased to 36.06%.(3) The enhanced effect of agonistic anti-CD137 mAb on the direct inhibition of tumor cells mediated by human DCs: At E to T ratio of 5:1 (optimal E to T ratio) , human DCs could inhibit directly the growth of about 20.16% of HT29, 25.44% of Hela and 75.41% of HepG2.2.15 (PO.05) . But human DCs couldn't inhibit the growth of Lovo and HepG2 (P > 0.05) . LPS and DCs supernatant didn't show obvious growth-inhibition effect on tumor cells (P > 0.05 ) . Agonistic anti-CD 137 monoclonal antibody could enhance the direct inhibition of tumor cells mediated by human DCs (PO.05) , the average inhibition rate increased about 20%. For tumor cells HT29, Hela, HepG2.2.15 and HepG2, the inhibition rate increased from 20.16%, 25.44%, 75.41%, 0.15% to 40.31%, 57.07%, 97.49%, 17.81%, respectively.2. The expression and function of CD137 and CD137L in human tumor(1) The expression of CD137L on human tumor cell lines: The result from RT-PCR and FCM showed that 9 human tumor cell lines from different tissues expressed CD137L on different levels. BEL 7402, HL-60, A2 expressed high-level CD137L; HepG2.2.15, L78 expressed medium-level CD137L; HT-29, U937, H6, HLE expressed low-level CD137L; the embryonic venous endotheliocytes (ECV 304) expressed medium-level CD137L; the PBMC from normal human didn't express CD137L.(2) The expression of CD137 and CD137L on human solid tumor tissue: Theresult from immunohistochemistry showed that normal tissues (12 cases) didn't express CD137 or CD137L; some of benign tumor (15 cases) and malignant (38 cases) tumors expressed both CD137 and CD137L. The positive rate of CD137 in malignant tumor group was 36.84%, which was no significantly higher than of benign tumor group (36.84% vs 13.33%, p>0.05). The positive rate of CD137L in malignant tumor group was 60.53%, which was significantly higher than of benign tumor group (60.53% vs 20.00% , p<0.05). CD 137 only expressed in wall of blood vessel in tumor tissue, CD137L expressed only in tumor cells not other parts.(3) The expression of CD137 and CD137L on peripheral blood white cells from patients with leukemia: No CD137 or CD137L expression was detected on white blood cells (lymphocytes and granular cells) from normal person, CD 137 did not express on white blood cells from patients with leukemia too. But the expression of CD137L was detected on white blood cells from all patients with leukemia studied(ALL and AML).(4) The function of CD137L on human tumor cells: CD137L on tumor cell L78 could enhance levels of IFN- Y secreted by T cells with anti-CD3. The enhanced levels of IFN- Y could be block by anti-CD137L. However, HepG2.2.15 expressing similar level of CD137L to L78 couldn't stimulate T cells to secrete IFN- y . CD137L on tumor cells had different influence on EL-8 secretion of tumor cells: it could enhance levels of IL-8 secreted by HepG2.2.15 about two times. But it couldn't elevate IL-8 secretion of tumor cells HLE and A2 significantly.Conclusion and innovation1. It was found that human immature DCs derived from peripheral blood monocytes not only express CD137L, but also express constitutionally CD137. The expression rate of CD 137 increases on mature DCs.2. The triggering of CD137 molecule on human DCs by agonistic anti-CD137 monoclonal antibody could enhance the direct growth-inhibition of tumor cells mediated by human DCs significantly, which demonstrated CD 137 on human DCs...
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