Murine gamma/delta-expressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens

T cells with antidonor specificities have been isolated from human recipients experiencing graft rejection after allogeneic bone marrow transplantation (BMT). Partial T-cell depletion of unrelated BM grafts with an anti- T-cell receptor (TCR) monoclonal antibody (MoAb) directed against the TCR alpha/beta heterodimer have shown that the incidence of graft-versus-host disease is low and that the incidence of durable engraftment is high. These studies suggest either that the number of residual TCR alpha/beta+ cells was sufficient to permit alloengraftment or that the preservation of cells other than TCR alpha/beta+ cells was beneficial for engraftment. With respect to the latter, one such candidate cell is the TCR gamma/delta+ T cell. Because no studies have specifically examined whether TCR gamma/delta+ cells might be capable of eliminating BM-derived hematopoietic cells, we established a new graft rejection model system in which transgenic (Tg) H-2d mice (termed G8), known to express gamma/delta heterodimers on high proportion of peripheral T cells, were used as BMT recipients. These Tg TCR gamma/delta+ cells respond vigorously to target cells that express the nonclassical major histocompatibility complex (MHC) class lb region gene products encoded in H-2T region of H-2T(b)+ strains. G8 Tg mice were used as recipients for C57BL/6 (B6: H-2(b); H-2T(b)) T-cell-depleted (TCD) donor BM. We show that G8 Tg (H-2(d), H-2T(d)) mice are potent mediators of B6 BM graft rejection and that the rejection process was inhibited by anti-TCR gamma/delta MoAbs. In contrast, BM from a B6 congenic strain that expresses the H-2T(a) allele, B6.A-Tl(a)/BoyEg, was readily accepted, suggesting that H-2T antigens on repopulating donor BM cells are the targets of host graft rejecting T cells that express the TCR gamma/delta heterodimer. PB chimerism studies were performed at > or = 1.5 months post-BMT using TCD BM from severe combined immunodeficient allogeneic donors, which is highly susceptible to rejection by the host. The addition of donor G8 TCR gamma/delta+ cells to TCD donor BM was shown to significantly increase alloengraftment in B6 recipients. These results show that (1) host TCR gamma/delta+ cells can reject repopulating donor cells, presumably by responding to nonclassical MHC class lb gene products expressed on BM-derived hematopoietic progenitor cells; and (2) donor TCR gamma/delta+ cells can facilitate the alloengraftment of rigorously TCD donor BM.     

Allogeneic cell therapy for a murine mammary carcinoma

The effect of allogeneic cell therapy on tumor growth was studied in a murine model of mammary carcinoma (4T1) as an experimental model of solid tumors in humans. i.v. inoculation of 4T1 (H-2d) cells into syngeneic mice [BALB/c or (BALB/cXC57BL/6)F1] (F1) carrying the H-2d histocompatible antigens results in tumor colonies in the lungs that finally cause the death of all of the mice. Sublethally irradiated F1 mice were inoculated with 4T1 cells to simulate minimal residual disease and with immunocompetent splenocytes derived from naive donors of F1 (syngeneic), BALB/c (syngeneic to the tumor but semiallogeneic to the host), or C57BL/6 (allogeneic to the tumor and semiallogeneic to the host) mice. The survival of F1 tumor-bearing mice that were treated with allogeneic C57BL/6 splenocytes was significantly prolonged (P < 0.02) compared with hosts given F1 or BALB/c-derived splenocytes that are syngeneic to 4T1 tumor cells. Adoptive transfer of lung cells that were isolated from F1 primary mice inoculated with 4T1 cells and syngeneic BALB/c or F1 splenocytes led to local tumor growth and death in secondary recipients. In contrast, only 1 of 22 secondary recipients developed tumors when inoculated with lung cells derived from F1 mice given allogeneic C57BL/6 splenocytes. All of the 21 secondary hosts survived disease-free for a follow-up time of >200 days. These results indicate that immunocompetent cells allogeneic to the mammary carcinoma cells were able to inhibit tumor development in the primary hosts and to prevent tumor growth in the adoptive recipients, which suggests that allogeneic cell therapy may be an efficient antitumor tool to eradicate minimal residual disease in human solid tumors.     

Granulocyte colony-stimulating factor reduces the capacity of blood mononuclear cells to induce graft-versus-host disease: impact on blood progenitor cell transplantation

The feasibility of transplanting peripheral blood mononuclear cells (PBMC) from granulocyte colony-stimulating factor (G-CSF)-treated normal human donors to myeloablated allogeneic hosts has been demonstrated recently. The current work examined the ability of recombinant G-CSF to alter peripheral blood T-cell function and graft-versus-host disease (GVHD) in a murine model of allogeneic G-CSF-mobilized PBMC transplantation. Administration of recombinant G-CSF to C57BL/Ka mice markedly increased the capacity of PBMC to reconstitute lethally irradiated syngeneic hosts. T- and B-lineage lymphocytes were depleted about 10-fold in the bone marrow of the treated mice, and the T-cell yield in the blood was increased about fourfold. The ability of PBMC or purified CD4+ and CD8+ T cells to induce acute lethal GVHD in irradiated BALB/c mice was reduced after the administration of G-CSF. This was associated with decreased secretion of interferon gamma and interleukin-2 (IL-2) and an increased secretion of IL-4. The donor cell inoculum, which was most successful in the rescue of irradiated allogeneic hosts, was the low-density fraction of PBMC from G-CSF-treated mice. These low-density cells were enriched for CD4-CD8-NK1.1+ T cells and secreted about 10-fold more IL-4 than the unfractionated cells from the G-CSF-treated donors.     

Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium

BACKGROUND: Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs. METHODS: BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days). RESULTS: All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9. CONCLUSION: These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.     

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.     

Prevention of marrow graft rejection without induction of graft-versus-host disease by a cytotoxic T-cell clone that recognizes recipient alloantigens

In allogeneic marrow transplantation, donor T cells that recognize recipient alloantigens prevent rejection but also cause graft-versus-host disease (GVHD). To evaluate whether the ability to prevent marrow graft rejection could be dissociated from the ability to cause GVHD, we generated a panel of four different CD8 cytotoxic T-lymphocyte clones specific for H2(d) alloantigens. Three of the clones caused no overt toxicity when as many as 20 x 10(6) cells were infused intravenously into irradiated H2(d)-positive recipients, and one clone caused acute lethal toxicity within 1 to 3 days after transferring 10 x 10(6) cells into H2(d)-positive recipients. One clone that did not cause toxicity was able to prevent rejection of (C57BL/6J x C3H/HeJ)F1 marrow in 800 cGy-irradiated (BALB/cJ x C57BL/6J)F1 recipients without causing GVHD. Large numbers of cells and exogenously administered interleukin-2 were required to prevent rejection. These results with different CD8 clones suggest that GVHD and prevention of rejection could be separable effects mediated by distinct populations of donor T cells that recognize recipient alloantigens.     

A critical role for CD48 antigen in regulating alloengraftment and lymphohematopoietic recovery after bone marrow transplantation

The binding of CD2, present on T cells, to its counterreceptor CD48 facilitates adhesion, signaling, alloantigen-induced cytokine production, and cytotoxic T-lymphocyte responses. Because these T-cell functions have been implicated in graft-versus-host disease (GVHD) pathogenesis, we have analyzed the effects of the CD2:CD48 pathway on GVHD mediated by CD4(+) and CD8(+) T cells infused into sublethally irradiated recipients. CD4(+) T-cell-mediated, and to a lesser extent, CD8(+) T-cell-mediated GVHD was inhibited by CD2 + 48 monoclonal antibody (MoAb) infusion. To assess the effects of combined MoAb infusion on alloengraftment, two different alloengraftment bone marrow transplantation (BMT) models were used. In both, MoAb infusion markedly inhibited alloengraftment and hematopoietic recovery post-BMT. To determine if the adverse effects on lymphohematopoiesis in the allogeneic BMT recipients were caused by an immune or nonimmune mechanism, studies were performed in congenic BMT recipients to preclude an immune mechanism as the cause for delayed recovery post-BMT. MoAb infusion resulted in impaired lymphohematopoietic recovery in congenic BMT recipients and markedly reduced day 12 colony-forming unit-spleen formation in syngeneic BMT recipients, consistent with a nonimmune mediated mechanism. Because the spleen is a site of early hematopoietic recovery post-BMT, studies were performed using adult splenectomized syngeneic BMT recipients. MoAb infusion delayed recovery in both nonsplenectomized and splenectomized recipients post-BMT, indicating that the delayed hematopoietic recovery was not the consequence of an abnormal homing pattern of hematopoietic progenitors to the spleen early post-BMT. CD48 MoAb was necessary and sufficient for the inhibition of GVHD lethality and delayed lymphohematopoietic effects of the combined MoAb regimen. CD48 MoAb was found to induce a profound modulation of CD48 antigen expression on BM cells, suggesting that the CD48 antigen may have an important function in hematopoiesis in the BM compartment. Taken together, these data provide evidence that the CD48 antigen plays a critical role in regulating hematopoiesis in post-BMT.     

The protein efficiency ratios of 30:70 mixtures of animal:vegetable protein are similar or higher than those of the animal foods alone

C57BL/6 mice receiving pretransplant immunization with C3H.SW spleen cells via the portal vein, but not the vena cava, show Ag-specific delayed rejection of allogeneic C3H.SW skin grafts. This delayed rejection is not seen if preimmunization is performed in gamma delta TCR knockout (C57BL/6-Tcrdtm1Mom) mice. gamma delta TCR+ and alpha beta TCR+ hybridoma cells were prepared from Peyer's patch cells harvested from C57BL/6 mice 4 days following portal venous immunization with 100 x 10(6) irradiated C3H.SW spleen cells and skin grafting with C3H.SW tail skin. After recloning, these hybridoma cells were tested for cytokine production in vitro following restimulation with irradiated C3H.SW spleen cells and for their ability to delay rejection of C3H.SW skin grafts after adoptive transfer to C57BL/6 mice. Delayed graft rejection was a function of cells that showed preferential production of IL-10, not IFN-gamma, in vitro, independent of the source (vena cava or portal vein immunized mice) or the TCR phenotype of the hybridoma. Simultaneous infusion of anti-IL-10 mAb abolished this graft prolongation effect of transferred gamma delta TCR+ hybridomas. Hybridoma cells producing IL-10 on restimulation could polarize cytokine production from freshly stimulated mesenteric lymph node away from production of IL-2 and IFN-gamma, and toward IL-4, IL-10, and TGF-beta production. This immunoregulation by hybridoma cells in vivo and in vitro was observed even for third party Ag-stimulated mice/cells as long as the hybridoma cells themselves received stimulation with their specific Ag.     

Graft-versus-host-disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function

Allogeneic bone marrow transplant recipients often exhibit a graft-versus-host-disease (GVHD)-associated immune deficiency that can be prolonged and lead to life-threatening infections. We have examined the role of donor T cell-mediated cytotoxic function in the development of GVHD-associated immune deficiency. A major histocompatibility complex-matched model of allogeneic bone marrow transplantation was employed in which lethally irradiated C3H.SW mice received a nonlethal dose of T cells from either perforin-deficient (B6-perforin 0/0), Fas-ligand (FasL)-defective (B6-gld), or normal (B6) allogeneic donor mice. T cell-depleted marrow from B6-Ly-5.1 congenic donor mice was transplanted along with the donor T cell populations to determine the effects of donor T cell-mediated cytotoxicity on engraftment. Our results demonstrate that recipients of perforin-deficient or normal allogeneic T cells exhibit profound lymphoid hypoplasia and severely reduced splenic proliferative responses to lipopolysaccharide in vitro. In contrast, GVHD-associated lymphoid hypoplasia is dramatically reduced and in vitro B cell function is intact in recipients of FasL-defective allogeneic T cells. Engraftment of myeloid and erythroid lineage cells occurs irrespective of donor T cell cytotoxic function. Although recipients of perforin-deficient or normal allogeneic T cells exhibited hematopoietic engraftment exclusively of donor origin, recipients of FasL-defective donor T cells exhibited significant mixed chimerism (Ly-5.1/Ly-5.2). Because only marrow of donor origin was transplanted, this finding suggests that Fas-mediated antirecipient cytotoxicity is required for clearance of residual hematopoietic stem cells of host origin that persist following lethal irradiation.     

Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell-depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vbeta6(+) and Vbeta3(+)) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-gamma (IFN-gamma) to host antigens in vitro. These in vitro responses correlated with increased IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.     

Efficacy of anti-intercellular adhesion molecule-1 immunotherapy on immune responses to allogeneic hepatocytes in mice

Adhesion molecules appear to play important roles in vascularized organ allograft rejection, because antibodies directed against them are effective in prolonging survival of vascularized organ allografts in rodents. However, the efficacy of these agents for cellular allografts is unknown. The current studies were undertaken to determine the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on host immune responses to purified hepatocytes. Host mice (C3H, H-2(k)) grafted with hepatocytes in sponge matrix allografts (HC-SMA) received IgG isotype control, anti-ICAM-1, or anti-VCAM-1 monoclonal antibody (mAb) on days 0 through 9 after grafting. Twelve to 14 days later, host cells infiltrating the HC-SMA were assessed for the development of allospecific cytolytic T cells (allo-CTLs). Treatment with anti-ICAM-1 or anti-VCAM-1 mAb resulted in significantly decreased recruitment of host cells into HC-SMA (P < .035). However, only anti-ICAM-1 mAb resulted in abrogation of development of allo-CTLs in HC-SMA (P = .001). C3H (H-2(k)) hosts grafted with allogeneic hepatocytes from control C57BL/6 (H-2(b)) or ICAM-1 knockout [H-2(b)] mice elicited the development of allo-CTLs in HC-SMA (P = not significant). Furthermore, there was no difference in the development of allo-CTLs in HC-SMA of control hosts [C57BL/6, H-2(b)] compared with ICAM-1 knockout hosts (H-2(b)) (P = not significant). Treatment with anti-ICAM-1 mAb had no effect on the development of allo-CTLs in ICAM-1 knockout (H-2(b)) hosts bearing HC-SMA. The immunosuppressive effect of host treatment with anti-ICAM-1 mAb does not appear to be a consequence of simple blockage of donor hepatocyte or host immune cell expression of ICAM-1, but suggests a potential inhibitory effect on host immune cell activation or function, as well as an effect on recruitment of host cells to the allograft.     

Analysis of the role of CD4+ T-cells during murine cytomegalovirus infection in different strains of mice

We examined the role of CD4+ T-cells in peritoneal exudate cells (PECs) during the course of acute murine cytomegalovirus (MCMV) infection in two strains of mice. Cell counts of PECs and cytofluorometric analysis showed that C57BL/6, a resistant strain, had more CD4+ T-cells than BALB/c, a susceptible strain, after intraperitoneal infection of 3 x 10(3) PFU of the Smith strain of MCMV, though both strains had an equivalent number of CD8+ T-cells. CD4+ T-cells of both strains expressed mRNA of IFN-gamma, IL-2, and IL-4 on days 5 and 7 after infection, with much higher expression of these cytokines in C57BL/6 than in BALB/c. At the same time point after infection, macrophages were shown to express mRNA of IL-1 alpha and TNF-alpha with higher expression of IL-1 alpha in C57BL/6 than in BALB/c. Production of nitric oxide, recently shown to be one of the antiviral effector mechanisms of macrophages, by macrophages of both strains was examined showing more production of nitric oxide on day 7 after infection in C57BL/6 than in BALB/c. From these findings, we suggest the possibility that CD4+ T-cells contribute to the protection against MCMV infection via the secretion of cytokines and the resultant activation of macrophages to produce nitric oxide.     

Effects of interleukin-6 on hematopoiesis in allogeneic and syngeneic bone marrow chimeras

Effects of interleukin-6 (IL-6) on hematopoietic progenitor cells were analyzed in murine bone marrow chimeras. When IL-6 was injected into syngeneic [C3H/He-->C3H/He] bone marrow chimeras from day 1 to day 12, the numbers of highly proliferative potential colony-forming units (CFU-HPP) or colony-forming units mix (CFU-Mix) in spleen cells and bone marrow cells increased on day 14 although there was a marked increase in spleen cells but not in bone marrow cells on day 21. The numbers of CFU-HPP increased in spleen cells from allogeneic [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 on days 14 and 21. In syngeneic bone marrow chimeras, the numbers of colony-forming units granulocyte/macrophage (CFU-GM) and burst colony-forming units (BFU-E) increased similarly to those of CFU-HPP and CFU-Mix on day 14. On day 21, these were mainly increased in spleen cells. In allogeneic bone marrow chimeras, IL-6 decreased the numbers of CFU-GM and BFU-Mix dose-dependently on day 14. Only 10 micrograms of IL-6 increased the numbers of CFU-GM and BFU-E on day 21. In our previous work, we showed that platelet counts increased on day 14 in syngeneic bone marrow chimeras injected with IL-6, whereas platelet and leukocyte counts increased on days 14 and 24 in allogeneic bone marrow chimeras injected with IL-6, correlating inversely with the numbers of hematopoietic progenitor cells. Overall, primitive hematopoietic progenitors (i.e., CFU-HPP and CFU-Mix) existed primarily in spleen cells of allogeneic bone marrow chimeras on day 14, whereas those in spleen cells of syngeneic bone marrow chimeras were found on day 21. These findings indicate that the effect of IL-6 on hematopoiesis in allogeneic bone marrow chimeras is completely different from that in syngeneic bone marrow chimeras, probably via graft-versus-host reaction (GVHR) but not GVH disease (GVHD).     

Strain difference in the induction of T-cell activation-associated, interferon gamma-dependent hepatic injury in mice

A single intravenous injection of concanavalin A (Con A) induces T-cell activation-associated inflammatory injury selectively in the liver. This study investigated the strain difference in the development of Con A-induced hepatic injury. Normal C57BL/6 and BALB/c spleen cells produced comparable levels of T-cell-derived lymphokines (interferon gamma [IFN-gamma], tumor necrosis factor alpha [TNF-alpha], and interleukin-2 [IL-2]) following in vitro stimulation with Con A. A single intravenous injection of Con A to C57BL/6 mice induced the plasma levels of TNF-alpha and IL-2 comparable with or slightly higher than those observed in BALB/c mice, whereas the same treatment resulted in an apparently lower level of IFN-gamma production in C57BL/6 mice. RNA from livers of Con A-treated C57BL/6 mice exhibited lower levels of IFN-gamma mRNA than RNA of BALB/c livers. Unexpectedly, a dramatic difference in the severity of hepatic injury was observed between C57BL/6 and BALB/c. Namely, the peak alanine transaminase (ALT) level was more than 15,000 U/L and inducible as early as 8 hours after injection of 0.2 mg Con A per mouse in the C57BL/6 strain, whereas the peak was approximately 3,000 U/L and induced as late as 24 hours after Con A injection in the BALB/c strain. The increase in plasma ALT levels was limited to less than 10% by injection of anti-IFN-gamma monoclonal antibody (mAb) in both strains. The C57BL/6 strain inducing lower levels of IFN-gamma exhibited higher IFN-gamma responsiveness as exemplified by the intrahepatic expression of an IFN-gamma-inducible gene, an inducible type of nitric oxide (NO) synthase (iNOS). These results indicate that, while IFN-gamma produced in vivo by activated T cells induces hepatic injury, there exists a striking strain difference in the induction of IFN-gamma-dependent hepatic injury.     

IGIF does not drive Th1 development but synergizes with IL-12 for interferon-gamma production and activates IRAK and NFkappaB

In these studies, IFN gamma-inducing factor (IGIF), unlike IL-12, did not drive Th1 development in BALB/c or C57BL/6 mice, but like IL-1alpha, potentiated IL-12-driven Th1 development in BALB/c mice. IGIF and IL-12 synergized for IFN gamma production from Th1 cells. Unlike IL-1alpha, IGIF had no effect on Th2 cells. IGIF signaled through IRAK, IL-1 receptor-associated kinase, to induce nuclear translocation of p65/p50 NFkappaB in Th1 cells. IL-1alpha had no effect on proliferation, cytokine production, or NFkappaB activation in Th1 cells but activated NFkappaB and proliferation in Th2 cells. Thus, Th1 and Th2 cells may differ in responsiveness and receptor expression for IL-1 family molecules. IGIF and IL-1alpha may differentially amplify Th1 and Th2 effector responses, respectively.     

Surgical treatment of locally advanced rectal cancer. Options and strategies

To evaluate the contribution of the subsets of T helper lymphocyte (Th) to the development of pulmonary lesion in mycoplasma pneumonia, we compared the pathological findings between Th1 dominant mice (C57BL/6) and Th2 dominant mice (BALB/c) in experimental Mycoplasma pulmonis (M. pulmonis) pneumonia. Mice (ICR, C57BL/6, and BALB/c) were intranasally inoculated with 0.03 ml of a solution containing M. pulmonis (1 x 10(8)) colony forming units per ml. Another M. pulmonis inoculated ICR mice were treated with interleukin-2 (IL2; 4.8 micrograms/day), days 3-9, intracutaneously). All mice were sacrificed at day 14, and the lung specimens were examined. Peribronchial lymphocyte cuffing was more prominent in C57BL/6 mice than that of ICR mice, and intra-alveolar inflammatory-cell infiltration in BALB/c mice was more prominent than in ICR mice. Pathological patterns of the lung in IL-2 treated ICR mice were mimicking those of C57BL/6 mice. These results suggested that pathological patterns of mycoplasma pneumonia in mice might be altered by the imbalance of host T helper lymphocyte subset.     

IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6)

CBA/J (highly resistant), BALB/c (moderately resistant), and C57BL/6 (susceptible) mice displayed three resistance patterns following intratracheal inoculation of Cryptococcus neoformans 52. The inability to clear the infection correlated with the duration of the eosinophil infiltrate in the lungs. The role of IL-5 in promoting the pulmonary eosinophilia and subsequent inflammatory damage in susceptible C57BL/6 mice was investigated. C57BL/6 mice developed a chronic alveolar, peribronchiolar, and perivascular eosinophilia following C. neoformans infection. This resulted in the accumulation of intracellular Charcot-Leyden-like crystals in alveolar macrophages by wk 4 and the extracellular deposition of these crystals in the bronchioles with associated destruction of airway epithelium by wk 6. IL-5 mRNA was expressed in the lungs, and injections of anti-IL-5 mAb prevented eosinophil recruitment and crystal deposition but did not alter cryptococcal clearance. Depletion of CD4+ T cells (but not CD8+) ablated IL-5 production by lung leukocytes in vitro and eosinophil recruitment in vivo. Neutralization of IL-5 also inhibited the recruitment of macrophages, CD8+ T lymphocytes, and B lymphocytes by 47 to 57%. Anti-IL-5 mAb inhibited CD4+ T lymphocyte recruitment by 30% but did not affect neutrophil recruitment. Thus, the development of a chronic eosinophil infiltrate in the lungs of C. neoformans-infected C57BL/6 mice is a nonprotective immune response that causes significant lung pathology. Furthermore, IL-5 promotes the recruitment and activation of eosinophils, resulting in the recruitment of additional macrophages and lymphocytes into the lungs.     

Glucose-modified low density lipoprotein enhances human monocyte chemotaxis

In response to stimulation with immobilized anti-CD3 antibody, splenocytes from C57BL/6 and BALB/c mice principally produced INF-gamma and IL-4, respectively. However, both splenocytes equally proliferated in response to ConA. We compared the changes after inoculation with BCG (1 mg/mouse) in their capacity to produce IL-4 or IFN-gamma in response to anti-CD3 antibody and to proliferate in response to ConA. Splenocytes from C57BL/6 and BALB/c mice, that had been inoculated with BCG 4 weeks before, produced IFN-gamma with diminished IL-4 production in response to anti-CD3 antibody. Furthermore these splenocytes became anergic to ConA stimulation and died due to cell apoptosis in stead of proliferation. However, we observed the strain difference at 12 weeks after BCG-infection. BCG-primed C57BL/6 splenocytes, that continuously produced IFN-gamma in response to anti-CD3 antibody, failed to proliferate in response to ConA. In contrast, BCG-primed BALB/c splenocytes, that increased IL-4 production but decreased IFN-gamma production when stimulated with anti-CD3 antibody, could proliferate well in response to ConA. Since the splenocytes of BALB/c mice became ConA responsive along with their shifting from Th1 dominant immune response at 4 weeks to Th2 dominant immune response at 12 weeks after BCG-inoculation, IL-4 was assumed to play a crucial role in activation of anergic T cells. Therefore, we stimulated splenocytes from both strains of mice infected with BCG 4 weeks before with ConA in the presence or absence of IL-4. Splenocytes from BCG-infected BALB/c mice showed marked proliferation, while those from BCG-infected C57BL/6 mice failed. We found that IL-4 protected against ConA-induced cell apoptosis in BALB/c splenocytes but not C57BL/6 splenocytes.     

Ambiguous role of interleukin-12 in Yersinia enterocolitica infection in susceptible and resistant mouse strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-gamma) production in NK and CD4+ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-gamma-receptor-deficient (IFN-gamma R-/-) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55-/-) mice. IFN-gamma R-/- mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-gamma R+/+ mice. Administration of IL-12 was protective in IFN-gamma R+/+ mice but not in IFN-gamma R-/- mice, suggesting that IFN-gamma R-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor beta (TGF-beta). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-beta. While administration of IL-12 alone increased TNF-alpha levels, administration of TGF-beta or TGF-beta plus IL-12 decreased both TNF-alpha and IFN-gamma levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55-/- mice, suggesting that TNF-alpha accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.