TNF-receptor-1 adaptor protein FAN mediates TNF-induced B16 melanoma motility and invasion.

BACKGROUND: Locomotion of cancer cells can be induced by TNF and other motogenic factors secreted by cells of the tumour microenvironment such as macrophages. Based on our recent findings that the TNF receptor adaptor protein FAN mediates TNF-induced actin reorganisation and regulates the directed migration of immune cells responding to chemotactic cues, we addressed the role of FAN in cancer cell motility and the formation of invadopodia, a crucial feature in tumour invasion. METHODS: In B16 mouse melanoma cells, FAN was downregulated and the impact on FAN on cell motility and invasion was determined using in vitro assays and in vivo animal models. RESULTS: Like FAN(-/-) murine embryonic fibroblasts, FAN-deficient B16 melanoma cells showed defective motility responses to TNF in vitro. In vivo FAN-deficient B16 melanoma cells produced significantly less disseminated tumours after i.v. injection into mice. Danio rerio used as a second in vivo model also revealed impaired spreading of FAN-deficient B16 melanoma cells. Furthermore, FAN mediated TNF-induced paxillin phosphorylation, metalloproteinase activation and increased extracellular matrix degradation, the hallmarks of functionally active invadopodia. CONCLUSION: The results of our study suggest that FAN through promoting melanoma cellular motility and tumour invasiveness is critical for the tumour-promoting action of TNF.

Differential effects of CXCR4-CXCL12- and CXCR7-CXCL12-mediated immune reactions on murine P0106-125 -induced experimental autoimmune neuritis.

AIM: The role of chemokines and their receptors, which regulate trafficking and homing of leucocytes to inflamed organs in human or murine autoimmune neuritis, has not yet been elucidated in detail, Therefore, the role of the chemokine receptors CXCR4 and CXCR7 and their ligand CXCL12 was studied in autoimmune-mediated inflammation of the peripheral nervous system. METHODS: CXCL12/CXCR4 and/or CXCL12/CXCR7 interactions were specifically inhibited by the compounds AMD3100 or CCX771, respectively, in experimental autoimmune neuritis (EAN) of C57BL/6J mice immunized with P0106-125 peptide. RESULTS: Disease activity was significantly suppressed by blocking CXCR7 while antagonization of CXCR4 enhanced disease activity. Enhanced disease activity was accompanied by significantly increased transcription of IFN-γ, IL-12 and TNF-α mRNA in regional lymph nodes and spleen as well as by increased serum levels of IFN-γ. Furthermore, by blocking CXCR4, expression of the cell adhesion molecules ICAM-1 and VCAM-1 was upregulated on vascular endothelial cells of the sciatic nerve, which coincided with significantly increased infiltration of the sciatic nerve by CD4+ T cells and macrophages. Remarkably, combined antagonization of both CXCR4 and CXCR7 significantly suppressed disease activity. This was accompanied by increased frequencies of activated and highly IFN-γ-expressing, P0106-125 -specific T cells in regional lymph nodes and spleen; however, these cells were unable to infiltrate the sciatic nerve. CONCLUSION: These data suggest differential and hierarchically ordered roles for CXCR4/CXCL12- vs. CXCR7/CXCL12-dependent effects during EAN: CXCR7/CXCL12 interaction is a gatekeeper for pathogenic cells, regardless of their CXCR4/CXCL12-dependent state of activation.

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells.

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.

Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.

Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

Relationship among immunodominance of single CD8+ T cell epitopes, virus load, and kinetics of primary antiviral CTL response.

The primary CTL response of BALB/c mice infected with the lymphocytic choriomeningitis (LCM) virus strain WE is directed exclusively against one major epitope, n118, whereas a viral variant, ESC, that does not express n118 induces CTL against minor epitopes. We identified one minor epitope, g283, that induces primary lytic activity in ESC-infected mice. Infections of mice with WE and ESC were used to study the hierarchical control of a T cell response. Presentation of minor epitopes is not reduced in WE-infected cells. Generation of CTL against n118 does not suppress the generation of minor epitope-specific CTL systemically, as mice coinfected with WE and ESC developed CTL against n118 and g283. However, elimination of ESC and development of minor epitope-specific CTL in ESC infection were slower than elimination of WE and development of CTL against n118. CD8+ T cells against the minor epitope were activated in ESC and WE infection, but did not expand in the latter to show lytic activity in a primary response. We explain the absence of minor epitope-specific lytic activity in WE infection by the fast reduction of virus load due to the early developing n118-specific CTL. Immunodominance of CTL epitopes in primary virus infections thus can be explained as a kinetic phenomenon composed of 1) expansion of CD8+ T cells specific for individual epitopes, 2) stimulatory effect of virus load, and 3) negative feedback control on virus load by the fastest CTL population.

Protective immunity in BALB/c mice against the simian virus 40-induced mKSA tumor resulting from injection of recombinant large T antigen. Requirement of CD8+ T lymphocytes.

BALB/c mice are often considered "low responders" or even "nonresponders" with regard to cytolytic CD8+ T lymphocytes and SV40 large T Ag (TAg). Large TAg and fragments thereof were produced by recombinant technology and injected into BALB/c mice that were subsequently challenged by i.p. injection of syngeneic TAg-expressing mKSA tumor cells. Two portions of the TAg were found to induce protective immunity, one stretching from amino acid residues 1-272 and the other from amino acid residues 683-708. In mice thus protected, the spleens were virtually free of cytotoxic T cells but CD8+ T lymphocytes obtained from the peritoneal cavity during rejection of the mKSA cells were directly lytic for TAg-expressing target cells. Depleting immune mice of CD4+ or CD8+ T lymphocytes by treatment with mAb abolished their ability to resist tumor development. We conclude that immunity against SV40 TAg-expressing tumor cells in BALB/c mice is dependent on both CD4+ and CD8+ T lymphocytes.

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.

Emergence of virus escape mutants after immunization with epitope vaccine.

BALB/c and C57BL/6J mice were immunized with recombinant vaccines consisting of lymphocytic choriomeningitis virus CD8+ T-lymphocyte epitopes and a carrier protein. During challenge infection with WE strain lymphocytic choriomeningitis virus, mutants with alterations in distinct amino acid residues of the epitopic nonapeptides appeared and multiplied. Splenocytes from WE-infected BALB/c mice lysed cells coated with the WE-type epitope; lysis was considerably less effective when the epitopic nonapeptide with which the syngeneic cells had been sensitized was the mutated form. Neither target was lysed by splenocytes from BALB/c mice infected with the variant virus. Mutants were not detected in F1 hybrid mice immunized with two viral epitopes that were restricted by class I molecules of both parents.

Murine hepatitis caused by lymphocytic choriomeningitis virus. II. Cells involved in pathogenesis.

BACKGROUND: Human virus hepatitides are often assumed to result from pathogenic immune responses rather than from direct viral cytopathic effects, but the details are largely unknown. Hepatitis of the mouse undergoing infection with lymphocytic choriomeningitis (LCM) virus is an immunopathologic phenomenon, and its analysis may help us to understand some of the events leading to the human illnesses. EXPERIMENTAL DESIGN: Mice were infected with LCM virus and were depleted of T cells or their subsets by inoculation of monoclonal antibodies; other infected mice lacked all T lymphocytes or the CD8+ subset because of genetic defects. Also, mice were infected and transfused with unsorted or CD(4+)-enriched LCM-immune spleen cells. Subsequently, the infectious titers were determined, the cytolytic activities of mononuclear cells isolated from the livers were measured, and the disease process was studied. RESULTS: In LCM virus-infected mice devoid of all T lymphocytes, pathologic alterations remained undetectable. In contrast, immunocompetent animals responded with a severe hepatitis, at the height of which the liver contained large numbers of cytolytic mononuclear leukocytes. Experiments with mice depleted of subset T lymphocytes revealed a predominantly CD8+ T lymphocyte-mediated phase, which was characterized by panlobular inflammation, whereas later there was a periportal inflammatory reaction, in which mainly CD4+ T lymphocytes were involved. Infusion of syngeneic immune spleen cells from immunocompetent donor mice into infected thymus-less mice resulted in virus elimination and damage to liver cells. With a similar protocol and the use of congenic mice, CD8+ T lymphocytes were observed to rapidly enter the recipients' livers, where they were present at the time liver cell injury was apparent. In mice genetically deficient in CD8+ T lymphocytes due to disruption of the gene for beta 2-microglobulin, a somewhat different type of LCM hepatitis developed that was largely dependent on CD4+ T lymphocytes. Liver cells were also damaged in infected nude mice that had been infused with positively selected CD4+ spleen cells from infected +/+ mice. CONCLUSIONS: Our findings published previously (Löhler J, Gossmann J, Kratzberg T, Lehmann-Grube F. Lab Invest 1994; 70:263-78) and related here suggest that the hepatitis in mice undergoing infection with LCM virus consists of three consecutive phases, which are mediated predominantly by NK cells, CD8+ T lymphocytes, and CD4+ T lymphocytes, respectively. Presumably, other elements of the immune system, such as mononuclear phagocytes and B lymphocytes, contribute to the pathogenesis.

T lymphocyte-mediated antiviral immune responses in mice are diminished by treatment with monoclonal antibody directed against the interleukin-2 receptor.

Blocking the interleukin-2 receptor's alpha-chain in lymphocytic choriomeningitis virus-infected mice by treatment with monoclonal antibodies diminished the increase of numbers of CD8+ T lymphocytes in spleens and prevented CD8+ T lymphocyte-mediated virus clearance from organs as well as generation of virus-specific cytotoxic T lymphocytes. Also, the CD8+ T cell-mediated early phase of the delayed-type hypersensitivity footpad swelling reaction was decreased. The same treatment had no effect on the number of CD4+ spleen T lymphocytes, which, however, did not enlarge during infection, but these cells' heightened DNA synthesis and cytokine production were reduced by antibody treatment; yet the generation of antiviral antibodies remained unaffected, and the CD4+ T lymphocyte-mediated second part of the footpad reaction was somewhat augmented. We conclude that blocking of the interleukin-2 receptor by antibody in lymphocytic choriomeningitis virus-infected mice diminishes both CD8+ and CD4+ T cell-mediated antiviral immune responses, the former more than the latter.

CD8+ T lymphocyte-mediated antiviral immunity in mice as a result of injection of recombinant viral proteins.

A major portion of the nucleoprotein (amino acids 67 through 300) and the glycoprotein-2 of lymphocytic choriomeningitis (LCM) virus were synthesized by using recombinant technology and were injected together with SDS twice in portions of 5 micrograms into BALB/c mice. As evidenced by diminished replication of LCM challenge virus, both proteins induced antiviral immunity, which was comparable in extent with the immunity caused by infection with LCM vaccinia recombinant viruses. Primed LCM-viral CTLs could not be demonstrated in these mice by culturing splenocytes in the presence of LCM virus, and Abs appeared slowly and in low quantities; but, after injection of large infectious doses, CTLs appeared faster and in higher numbers than in mice not previously treated with viral proteins. Depletion of CD8+ cells, but not of CD4+ cells, by treatment of mice with mAb abolished the antiviral immunity, demonstrating that protection was mediated by CD8+ T lymphocytes. Absence of CD4+ T lymphocytes before and during the period of immunization did not measurably affect the animals' antiviral immune status, indicating that activation of the CD8+ T lymphocytes was not dependent on help by CD4+ cells.

MHC class I molecule-restricted presentation of viral antigen in beta 2-microglobulin-deficient mice.

Normally, Ag is presented to CD8+ T lymphocytes as a tripartite complex consisting of peptide epitope, MHC-encoded class I heavy (alpha) chain, and beta 2-microglobulin (beta 2-m) light chain. Although there is agreement about the function of both peptide and alpha-chain, the role of beta 2-m has remained uncertain. In particular, can Ag be presented without its participation? We have sought to obtain an answer by using mice in which the gene for beta 2-m had been disrupted by homologous recombination. As a consequence, no light chains are synthesized and, furthermore, few if any CD8+ T lymphocytes are formed. Elimination of lymphocytic choriomeningitis (LCM) virus from the tissues of acutely infected mice is mediated solely by CD8+ T lymphocytes; hence, in the beta 2-m-lacking mutants the infection cannot be terminated. Here it is shown that infusion of immune spleen cells from syngeneic beta 2-m+/+ mice and from mice compatible in K or D of the MHC resulted in virus clearance. Approximately five times more cells were required to achieve antiviral effects in beta 2-m-deficient than in wild-type mice but attempts to improve elimination by treatment of the former with IFN-gamma or beta 2-m have failed. Depleting the immune splenocytes of CD8+ T lymphocytes but not of CD4+ T lymphocytes abolished the antiviral potential. We conclude that LCM virus-infected murine cells can present viral Ag to CD8+ effector T lymphocytes together with class I MHC molecules K and D, despite the total absence of beta 2-m.

Antiviral immune responses of lymphocytic choriomeningitis virus-infected mice lacking CD8+ T lymphocytes because of disruption of the beta 2-microglobulin gene.

Mice infected intracerebrally with lymphocytic choriomeningitis virus (LCM virus) develop a characteristic central nervous system disease and usually die. If the intravenous or intraperitoneal route is used, the infection leads to less severe clinical signs and the virus is eliminated. Illness and virus clearance are immunological phenomena, which are assumed to be caused exclusively by CD8+ T lymphocytes. In contrast, of the two phases of a delayed-type hypersensitivity reaction caused by inoculation of the virus into the mouse's foot, only the first is mediated by CD8+ cells, whereas the second is mediated by CD4+ cells. We have examined LCM virus-specific immune responses in mice devoid of CD8+ T lymphocytes as a result of disruption of the beta 2-microglobulin gene. As expected, the virus persisted but footpad swelling did not occur, although intracerebral infection resulted in CD4+ T-lymphocyte-mediated illness and antiviral antibodies were produced. Different results had been obtained by Fung-Leung et al. (W.-P. Fung-Leung, T. M. Kündig, R. M. Zinkernagel, and T. W. Mak, J. Exp. Med. 174:1425-1429, 1991), who, is essentially identical experiments but with mice lacking CD8+ T lymphocytes as a result of disruption of the Lyt-2-encoding gene, recorded control of the infection and development of a local delayed-type hypersensitivity reaction. We consider these differences important, because they provide us with clues that may help to understand the mode of action of the CD8+ T cells in cell-mediated antiviral immunity.