Interleukin-2 blocks the antitumour activity caused by depletion of CD25 cells in a murine renal adenocarcinoma model

OBJECTIVE: To test the effectiveness of antimouse CD25 monoclonal antibody (mAb) against murine renal adenocarcinoma (RENCA) cells, as immunoregulatory/suppressor cells are known to be involved in tumour development in vivo, but the functions of these cells are not yet clear, and eliminating naive CD25 (interleukin-2 receptor alpha)-positive T cells elicits potent immune responses to syngeneic tumours in vivo. MATERIALS AND METHODS: Aliquots of 1 x 10(4) or 1 x 10(5) RENCA cells were implanted into the subcapsule of the left kidney of syngeneic male Balb/c mice. Mice were injected with 125 micro g of antimouse CD25 mAb to deplete CD25(+) cells before RENCA implantation. Then 10(4) units of recombinant human interleukin-2 (rhIL-2) were subcutaneously injected twice daily for 7 days. Fourteen or 25 days later the tumour size was determined by laparotomy, and cells sorted using two-colour flow cytometry. RESULTS: Depletion of naive CD25(+) cells with anti-CD25 mAb and rhIL-2 administration effectively induced anti-RENCA tumour activity in Balb/c hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth. RENCA implantation reduced the proportion of CD4(+) cells among splenocytes, whereas anti-CD25 mAb treatment increased it. The proportion of CD25(+)CD8(+) cells among splenocytes and that of CD25(+) cells among CD8(+) cells were markedly reduced by co-administration of anti-CD25 mAb and rhIL-2 with RENCA implantation. Both CD4(+) and CD8(+) cells were stained around the remnant microscopic RENCA tumour after anti-CD25 mAb treatment. CONCLUSION: Either depletion of naive CD25(+) cells or rhIL-2 administration suppressed RENCA tumour growth in murine hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth.     

The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts

Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

T lymphocytes and silica-induced pulmonary inflammation and fibrosis in mice.

BACKGROUND: Silica-induced pulmonary inflammation and fibrosis in animals provides a good model for chronic pulmonary inflammation and fibrosis. Although lymphocytes are implicated in the pathogenesis of pulmonary fibrosis, experimental models using silica-treated athymic nude mice have not been successful in showing the fibrogenic mechanism regulated by T cells. The aim of this study was to re-evaluate the role of T lymphocytes in the development of silicosis by comparing the response to silica administration of nude athymic mutants with that of euthymic animals. METHODS: Suspensions of silica particles were transnasally administered to nude athymic mice (Balb/c nu/nu) as well as to their euthymic littermates (Balb/c nu/+). The degree of pulmonary inflammation and fibrosis was assessed on days 14, 28, and 56 based upon histological observation, analysis of collagen deposition in the lungs, and analysis of the cellular constituent, protein, and phospholipid content in the bronchoalveolar lavage fluid. RESULTS: Histologically, athymic mice developed less severe interstitial pneumonitis than euthymic mice. In euthymic mice the lung hydroxyproline content increased with time after silica administration from 6.48 (0.38) micrograms hydroxyproline/mg dry lung weight on day 0 to 8.87 (0.41) micrograms/mg on day 56. A gradual increase in lung hydroxyproline content was also observed in athymic mice but the increase was significantly smaller than in euthymic mice (6.63 (0.43) micrograms/mg on day 0, 7.90 (0.19) micrograms/mg on day 56). Administration of silica resulted in an increase in the number of macrophages and neutrophils and in the total protein and phospholipid content of the bronchoalveolar lavage (BAL) fluid in both mouse strains. No significant difference was detected between athymic and euthymic mice in the numbers of macrophages, but the increase in neutrophils in the BAL fluid of athymic mice was significantly smaller than in euthymic mice on days 14 and 56. The total protein and phospholipid content of the BAL fluid from athymic mice was lower than that from euthymic mice. CONCLUSIONS: T lymphocytes appear to be involved in the pathogenesis of silica-induced pneumonitis. Since pulmonary fibrosis develops even in nude athymic mice, T cells do not seem to play a primary part in fibrogenic response but they regulate, at least to some extent, the response of inflammatory cells and fibrogenesis of the lung.     

Presence of Functional Mouse Regulatory CD4+CD25+T Cells in Xenogeneic Neonatal Porcine Thymus-Grafted Athymic Mice

Xenotransplantation with porcine thymus is emerging as a possible means to reconstitute host cellular immunity and to induce immune tolerance in rodents and large animals. However, the presence of regulatory T cells (Treg cells) in this model needs to be determined. We herein demonstrated that efficient repopulation of mouse CD4+CD25+Treg cells was achieved in Balb/c nude mice by grafting neonatal porcine thymic tissue (NP THY). Mouse CD4+CD25+T cells expressed normal levels of Foxp3 in NP THY-grafted nude mice. Furthermore, these CD4+CD25+Treg cells showed significant inhibitory effects on the cell proliferation or interleukin-2 products of syngeneic T cells to alloantigens, Con A or a peptide antigen, although the potent immunosuppressive function might be lower than CD4+CD25+Treg cells in Balb/c mice. CD4+CD25+T cells in NP THY-grafted nude mice showed significantly stronger inhibition on the response to donor porcine antigens of CD4+CD25(-)T cells than CD4+CD25+Treg cells in Balb/c mice. Both CD4+CD25+Treg cells in NP THY-grafted nude and Balb/c mice prevented the development of autoimmune disease mediated by syngeneic CD4+CD25(-)T cells in a similar efficient way in the secondary recipients. These findings provide evidence for the potential involvement of CD4+CD25+Treg cells in keeping self-tolerance and transplant tolerance in this xeno-thymus transplantation model.     

Selectively expanding subsets of T cells in mice by injection of interleukin-2/antibody complexes: implications for transplantation tolerance

The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti-IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti-IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.     

Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance

Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to na?ve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.     

体内注射白细胞介素10和转化生长因子质粒延长小鼠移植皮肤存活时间

目的 探讨体内注射白细胞介素10(IL-10)和转化生长因子β1(TGF-β1)质粒对小鼠移植皮肤存活时间的影响.方法 构建含IL-10和TGF-β1基因的质粒,以Balb/c小鼠为受者、Balb/c小鼠与C57BL/6小鼠杂交的F1代小鼠为供者,行皮片移植.移植当天,经尾静脉分别给受者快速注射不含基因的空白质粒(空白组)、含IL-10基因质粒(IL-10组)、含TGF-β1基因质粒(TGF-β1组)以及含IL-10和TGF-β1双基因的质粒(联合组),以后每2天注射1次,20 μg/次,共注射6次,观察移植皮肤存活时间.另取Balb/c小鼠,在输注C57BL/6小鼠脾细胞后,按前述分组及方法接受质粒快速注射,注射5次后,分离其脾细胞,以流式细胞仪检测脾细胞中CD4+ CD25+ T淋巴细胞含量.结果 移植皮片存活时间,空白组为(13.50±1.04)d,IL-10组为(13.83±1.16)d,TGF-β1组为(15.33±1.50)d,联合组为(21.33±3.20)d,联合组移植皮片存活时间明显长于其他3组(P<0.01).脾细胞中CD4+ CD25+ T淋巴细胞的含量,空白组为(6.58±1.86)%,IL-10组为(10.52±1.13)%,TGF-β1组为(14.44±0.42)%,联合组为(14.25±1.24)%,TGF-β1组和联合组的CD4+ CD25+ T淋巴细胞含量明显高于空白组和IL-10组(P<0.01).结论 体内注射IL-10和TGF-β1质粒可延长小鼠移植皮肤存活时间,并能提高CD4+ CD25+ T淋巴细胞含量.     

Despite efficient intrathymic negative selection of host-reactive T cells,autoimmune disease may develop in porcine thymus-grafted athymic mice: evidence for failure of regulatory mechanisms suppressing autoimmunity

BACKGROUND: CD4 T-cell reconstitution and xenogeneic tolerance is achieved in T cell-depleted, thymectomized C57BL/6 (B6) mice and nude mice by grafting of fetal pig thymus (FP THY). Sixty percent of grafted nude mice and 10% of grafted thymectomized B6 mice develop a clinical illness resembling chronic graft-versus-host disease. METHODS: Negative selection of mouse T cells in FP THY grafts was studied in "AND" TCR transgenic mice with a negative selecting MHC. Pathologic and immunohistochemical examinations and adoptive transfer assays were performed to determine the role of mouse CD4+ cells in the occurrence of autoimmune disease in this model. RESULTS: Marked clonal deletion of mouse thymocytes bearing a transgenic TCR ("AND"), which recognizes H2s expressed by host hematopoietic cells, was observed in FP THY grafts. Pathologic and immunohistochemical examinations of the liver, skin, lungs, and kidneys of mice with wasting syndrome showed marked mouse CD4+ T-cell infiltration without detectable pig cells. After adoptive transfer of splenocytes, but not of CD4+ cell-depleted splenocytes, from sick mice along with B6 bone marrow cells to lethally irradiated syngeneic B6 mice, the secondary recipients developed a similar autoimmune syndrome as the donors. Cotransfer of na?ve syngeneic splenocytes prevented the occurrence of autoimmune disease in secondary recipients of splenocytes from healthy FP THY-grafted BALB/c nude mice. CONCLUSION: These results demonstrate a key role for mouse CD4+ T cells in causing autoimmune disease in this model and suggest the importance of regulatory mechanisms in addition to intrathymic clonal deletion for the maintenance of tolerance to recipient antigens.     

Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. METHODS: CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. RESULTS: Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). CONCLUSION: IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.     

CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

BACKGROUND: Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. METHODS: Neonatal BALB/c mice were injected with 108 live CAF, splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using 51Cr release assays of A/J or third-party targets. RESULTS: We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 to CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4+/CD25+ cells, but not CD4+/CD25- cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2. Moreover, when added back into cultures, purified CD4+/CD25+ cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. CONCLUSION: These data indicate that CD8 anergy is associated with the state of tolerance, and that CD4+CD25+ cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.     

移植抗原特异性转基因CD8+T细胞对白细胞介素2的依赖性

目的研究白细胞介素2（IL-2）在调节移植抗原特异性转基因CD8^＋T细胞介导的免疫排斥反应中的作用。方法将经荧光染科CFSE标记后的C57BL／6小鼠和2CTg小鼠（CD4敲除鼠）淋巴细胞分别植入经致死剂量γ射线照射过的两组DBA／2J小鼠体内，检测CD4^＋与CD8^＋T细胞在体内分裂增殖的时相，并用胞浆内IL-2标志染色方法测定活化后T细胞表达IL-2的能力。以Balb／c小鼠为供者，糖尿病2CTg小鼠和2C Tg-IL-2KO小鼠（IL-2敲除鼠）为受者，进行胰岛细胞移植。观察CD8^＋ T细胞在介导移植排斥中的作用。结果DBA／2J小鼠输注了C57BL／6小鼠的淋巴细胞后，CD4^＋与CD8^＋T细胞分裂增殖均非常明显，前者表达大量IL-2，后者则不表达。DBA／2J小鼠输注了2CTg小鼠的淋巴细胞后。在完全没有CD4^＋T细胞存在的情况下，CD8^＋T细胞仍明显分裂增殖并大量表达IL-2。2CTg和2CTg—IL-2KO小鼠移植胰岛细胞后，前者迅速发生排斥反应，胰岛移植物的平均存活时间仅为8d，而后者胰岛移植物的平均存活时间〉50d。结论CD8^＋T细胞在产生和利用IL-2时有很大的可塑性。CD4^＋T细胞存在时，CD8^＋T细胞能有效利用CD4^＋T细来源的IL-2进行分裂增殖，在缺乏CD4^＋T细胞时，则利用自身来源的IL-2进行分裂增殖；移植抗原特异性CD8^＋T细胞的效应功能完全依赖于IL-2，排斥反应由CD8^＋T细胞介导时，阻断IL-2／IL-2受体通路可诱导移植物长期存活。     

Mediation of skin allograft rejection in scid mice by CD4+ and CD8+ T cells.

We analyzed the role of CD4+ and CD8+ T cells in H-2-disparate skin allograft rejection in the mutant mouse strain C.B-17/Icr scid with severe combined immunodeficiency. On the day of skin allografting, scid mice were adoptively transferred with negatively selected CD4+ or CD8+ splenocytes from normal unsensitized C.B-17/Icr mice. These populations were obtained using a double-mAb--plus--complement elimination protocol using anti-CD4 or anti-CD8 mAb that resulted in no detectable CD4+ or CD8+ cells by FACS and negligible numbers of cytolytic T lymphocytes by limiting dilution analysis in anti-CD8 treated populations. Spleen cells were removed from grafted mice at the time of rejection and were tested in vitro for antidonor reactivity in several assays: mixed lymphocyte culture, cell-mediated lympholysis, and LDA for CTL and for IL-2-producing HTL. The presence of Thy 1.2+, CD4+, or CD8+ cells was determined by FACS. All control C.B-17 mice and scid mice adoptively transferred with nondepleted CD4+, and CD8+ cells rejected skin allografts with similar mean survival times (15.6 +/- 1.5, 18.8 +/- 3.4, 18.0 +/- 5.4, respectively), whereas control scid mice retain skin allografts indefinitely (all greater than 100 days). C.B-17 syngeneic grafts survived indefinitely in all groups. At the time of rejection, splenocytes from scid mice receiving CD4+ cells had negligible donor-specific cytotoxicity in CML and negligible numbers of CTL by LDA, but demonstrated a good proliferative response in MLC and IL-2-producing cells by LDA (frequency = 1/1764). There were no detectable CD8+ cells present by FACS analysis. Conversely, splenocytes from scid mice adoptively transferred with CD8+ cells had strong donor-specific cytotoxicity in CML (58.8% +/- 16.1%) and CTL by LDA (frequency = 1/3448), but no significant proliferation was detected in MLC. There were no detectable CD4+ cells by FACS, but there were small numbers of IL-2-producing cells by LDA (frequency = 1/10,204). These data demonstrate that CD4+ cells adoptively transferred into scid mice are capable of mediating skin allograft rejection in the absence of any detectable CD8+ cells or significant functional cytolytic activity. The adoptive transfer of CD8+ cells also results in skin allograft rejection in the absence of detectable CD4+ cells. The detection of small numbers of IL-2 secreting cells in these mice may indicate that CD(8+)-mediated allograft rejection in this model is dependent on IL-2-secreting CD8+ cells.     

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice

Abstract: Mouse CD4⁺ T cells efficiently develop in fetal pig thymus (FPTHY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8⁺ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of Vβ5.1/5.2⁺ and Vβ11⁺ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of Vβ6, Vβ7, and Vβ8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthyrnic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8⁺ cells can undergo normal functional and phenotypic maturation in FPTHY grafts. Thus, other explanations must be sought for the failure of CD8'cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THYLIV-grafted mice.     

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND: Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS: Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS: Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION: Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.     

Anti-tumour activity of heat-shock protein 60-recognizing CD4+ T cells against syngeneic murine renal cell carcinoma

OBJECTIVES: To determine whether heat-shock protein (HSP) 60-recognizing CD4(+) T cells show antitumour activity against renal carcinoma (RENCA) cells, as HSP is highly expressed by tumour cells and induced in cells by various stresses, including transformation. MATERIALS AND METHODS: RENCA, a renal cortical adenocarcinoma cell line of BALB/c origin, was used. Expression of major histocompatibility complex (MHC) class I, class II and HSP-60 on RENCA tumour cells was analysed by flow cytometry. BASL1.1, an autoreactive T-helper type 1 type CD4(+) T cell clone established by us, and that recognises HSP-60, was also used for a tumour-neutralising assay. RESULTS: The RENCA cells were negative for MHC class II, but expressed intracellular HSP-60. In the tumour-neutralising assay, BASL1.1 cells significantly suppressed the in vivo growth of RENCA cells. Three of five mice rejected RENCA cells when co-inoculated with BASL1.1 cells. CONCLUSIONS: These results indicate that HSP-60-recognizing CD4(+) T cells have the potential to eliminate renal cell carcinoma in vivo and that the eliciting of an anti-self T cell response at the tumour site can lead to regression of renal cancer.     

The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice

BACKGROUND: Xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult. METHODS: Neonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts. RESULTS: Repopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice. CONCLUSIONS: NP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.     

外源性IL-2对小鼠CD4^＋CD25^＋调节性T细胞影响的实验研究

目的：探讨IL-2对CD4＋CD25＋调节性T细胞（Tregs）的增殖及功能的影响。方法：提取B6小鼠脾脏细胞,流式细胞仪分离CD4＋CD25＋Tregs,将新鲜分离的CD4＋CD25＋Tregs与抗CD3单克隆抗体、同种同系抗原递呈细胞（APCs）及外源性IL-2共同培养,测定其增殖活性;并检测体外扩增后的CD4＋CD25＋Tregs的免疫抑制活性及其Foxp3的表达。结果：与外源性IL-2共同培养的CD4＋CD25＋Tregs增殖程度强烈,与对照组比较,差异有统计学意义（P〈0.05）;体外扩增的CD4＋CD25＋Tregs抑制CD4＋CD25-T细胞增殖活性的能力与新鲜分离的CD4＋CD25＋Tregs相似（P〉0.05）。体外扩增的CD4＋CD25＋Tregs的Foxp3表达与新鲜分离的CD4＋CD25＋Tregs亦相似（P〉0.05）。结论：外源性IL-2能够消除CD4＋CD25＋Tregs的无反应状态,且体外扩增的CD4＋CD25＋Tregs保持了其抑制活性。