Biopsy-derived oral keratinocytes - A model to potentially test for oral mucosa radiation sensitivity.

Purpose: To establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and Methods: Oral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation. Results: Stable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm2 up to a median of 119.2 mm2 (range: 54.4-290). Radiated cells spread to only 100.7 mm2 (2 Gy; range: 55.3-266.7); 73.2 mm2 (4 Gy; 15-240.4); 47 mm2 (6 Gy; 2-111.9), and 22.7 mm2 (8 Gy; 0-80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h). Conclusion: In vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations.

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance.

Pancreatic stellate cells in pancreatic cancer: In focus.

Pancreatic stellate cells are stromal cells that have multiple physiological functions such as the production of extracellular matrix, stimulation of amylase secretion, phagocytosis and immunity. In pancreatic cancer, stellate cells exhibit a different myofibroblastic-like morphology with the expression of alpha-smooth muscle actin, the activated form is engaged in several mechanisms that support tumorigenesis and cancer invasion and progression. In contrast to the aforementioned observations, eliminating the stromal cells that are positive for alpha-smooth muscle actin resulted in immune-evasion of the cancer cells and resulted in worse prognosis in animal models. Understanding the cancer-stromal signaling in pancreatic adenocarcinoma will provide novel strategies for therapy. Here we provide an updated review of studies that handle the topic "pancreatic stellate cells in cancer" and recent experimental approaches that can be the base for future directions in therapy.

IFNγ and perforin cooperate to control infection and prevent fatal pathology during persistent gammaherpesvirus infection in mice.

Infection with murine gammaherpesvirus 68 has become an accepted model for studying the virus/host interactions with regard to gammaherpesvirus infections. Previous studies using gene-deficient mice have revealed that neither IFNγ nor perforin is essential in controlling the outcome of infection or the virus load during chronic infection in C57BL/6 mice. However, pronounced multiorgan fibrosis and splenic atrophy are observed in mice lacking IFNγ or the IFNγ receptor. To study the interplay between perforin and IFNγ in controlling the virus-induced pathology and the viral load during chronic gammaherpesvirus infection, we infected IFNγ/perforin double-deficient C57BL/6 mice and followed the course of infection. While absence of perforin prevented the splenic atrophy in IFNγ-deficient mice, fibrosis did not disappear. Moreover, double-deficient mice developed extreme splenomegaly, were unable to control the viral load and displayed chronic immune activation. Thus, IFNγ and perforin act in concert to minimize pathology and control the viral load in mice chronically infected with MHV68. Furthermore, while certain aspect of the virus-induced pathology in IFNγ-deficient mice may be alleviated in double-deficient mice, other aspects are exaggerated, and the normal architecture of the spleen is completely destroyed. We believe that these findings add to the understanding of the virus/host interaction during chronic gammaherpes virus infection.

Founder effect in the Horn of Africa for an insulin receptor mutation that may impair receptor recycling.

AIMS/HYPOTHESIS: Genetic insulin receptoropathies are a rare cause of severe insulin resistance. We identified the Ile119Met missense mutation in the insulin receptor INSR gene, previously reported in a Yemeni kindred, in four unrelated patients with Somali ancestry. We aimed to investigate a possible genetic founder effect, and to study the mechanism of loss of function of the mutant receptor. METHODS: Biochemical profiling and DNA haplotype analysis of affected patients were performed. Insulin receptor expression in lymphoblastoid cells from a homozygous p.Ile119Met INSR patient, and in cells heterologously expressing the mutant receptor, was examined. Insulin binding, insulin-stimulated receptor autophosphorylation, and cooperativity and pH dependency of insulin dissociation were also assessed. RESULTS: All patients had biochemical profiles pathognomonic of insulin receptoropathy, while haplotype analysis revealed the putative shared region around the INSR mutant to be no larger than 28 kb. An increased insulin proreceptor to ß subunit ratio was seen in patient-derived cells. Steady state insulin binding and insulin-stimulated autophosphorylation of the mutant receptor was normal; however it exhibited decreased insulin dissociation rates with preserved cooperativity, a difference accentuated at low pH. CONCLUSIONS/INTERPRETATION: The p.Ile119Met INSR appears to have arisen around the Horn of Africa, and should be sought first in severely insulin resistant patients with ancestry from this region. Despite collectively compelling genetic, clinical and biochemical evidence for its pathogenicity, loss of function in conventional in vitro assays is subtle, suggesting mildly impaired receptor recycling only.

CCR5 and CXCR3 are dispensable for liver infiltration, but CCR5 protects against virus-induced T-cell-mediated hepatic steatosis.

CCR5 and CXCR3 are important molecules in regulating the migration of activated lymphocytes. Thus, the majority of tissue-infiltrating T cells found in the context of autoimmune conditions and viral infections express CCR5 and CXCR3, and the principal chemokine ligands are expressed within inflamed tissues. Accordingly, intervention studies have pointed to nonredundant roles of these receptors in models of allograft rejection, viral infection, and autoimmunity. In spite of this, considerable controversy exists, with many studies failing to support a role for CCR5 or CXCR3 in disease pathogenesis. One possible explanation is that different chemokine receptors may take over in the absence of any individual receptor, thus rendering individual receptors redundant. We have attempted to address this issue by analyzing CCR5(-/-), CXCR3(-/-), and CCR5/CXCR3(-/-) mice with regard to virus-induced liver inflammation, generation and recruitment of effector cells, virus control, and immunopathology. Our results indicate that CCR5 and CXCR3 are largely dispensable for tissue infiltration and virus control. In contrast, the T-cell response is accelerated in CCR5(-/-) and CCR5/CXCR3(-/-) mice and the absence of CCR5 is associated with the induction of CD8(+) T-cell-mediated immunopathology consisting of marked hepatic microvesicular steatosis.

Role of very late antigen-1 in T-cell-mediated immunity to systemic viral infection.

The T-cell response to lymphocytic choriomeningitis virus was studied in mice lacking very late antigen-1 (VLA-1). The generation of virus-specific effector T cells was unimpaired in VLA-1(-/-) mice. In the memory phase, VLA-1 deficiency did not influence the number of memory CD8(+) T cells or their distribution between lymphoid and nonlymphoid organs. Regarding a functional role of VLA-1, we found that intracerebral infection of both VLA-1(-/-) and wild-type (wt) mice resulted in lethal T-cell-mediated meningitis, and quantitative and qualitative analyses of the cellular exudate did not reveal any significant differences between the two strains. Expression of VLA-1 was also found to be redundant regarding the ability of effector T cells to eliminate virus from internal organs of i.v. infected mice. Using delayed-type hypersensitivity (DTH) assays to evaluate subdermal CD8(+) T-cell-mediated inflammation, no significant influence of VLA-1 was found either in the primary response or in the memory phase. However, alpha-VLA-4 antibody reduced the DTH-like reaction in VLA-1(-/-) mice to a higher degree than in wt mice, suggesting a synergistic effect of blocking both integrins. Taken together, the current findings indicate that the expression of VLA-1 is not pivotal for T-cell-mediated antiviral immunity to a systemic infection.

Stimulation of human neutrophils and monocytes by staphylococcal phenol-soluble modulin.

Modulins represent microbial products that stimulate cytokine production in host cells. The modulins responsible for gram-positive sepsis remain poorly understood. Staphylococci release a factor (or factors) that activates nuclear factor-kappa B and stimulates cytokine production in cells of macrophage lineage. This factor, termed phenol-soluble modulin (PSM), has been recently isolated from culture supernatant of Staphylococcus epidermidis. We examined the effects of PSM on proinflammatory properties of human neutrophils and monocytes in vitro. PSM activated the respiratory (oxidative) burst in neutrophils and primed neutrophils for enhanced respiratory burst activity in response to formyl-methionyl-leucyl-phenylalanine. PSM also stimulated neutrophil degranulation as reflected by increased surface expression of CD11b and CD18, which was accompanied by rapid shedding of L-selectin. Spontaneous apoptosis of both neutrophils and monocytes was inhibited by PSM. Furthermore, PSM also functioned as a chemoattractant factor for both neutrophils and monocytes. Thus, the proinflammatory properties of PSM resemble those of both lipopolysaccharide and bacterial chemotactic peptides. These findings suggest that PSM may play a role in the pathogenesis and systemic manifestations of sepsis caused by staphylococci.

CD11b expression as a marker to distinguish between recently activated effector CD8(+) T cells and memory cells.

CD8(+) T cells in different activation states have been difficult to identify phenotypically. In this study we have investigated whether Mac-1 (CD11b) expression can be used as a criterion to distinguish between recently activated effector cells and memory cells belonging to the CD8(+) T cell subset. Polyclonal virus-specific effector and memory CD8(+) T cells from lymphocytic choriomeningitis- and vesicular stomatitis virus-infected mice were visualized through staining for intracellular IFN-gamma or binding of MHC-peptide tetramers, and Mac-1 expression was evaluated. Naive T cells and most virus-specific memory CD8(+) T cells express little or no Mac-1 independent of the virus model employed. In contrast, the majority of CD8(+) T cells present during acute infection express a significant level of Mac-1 and, similarly, Mac-1 expression is found on secondary effectors generated in response to viral re-exposure. We therefore suggest that high Mac-1 expression defines a subset of circulating effector cells and that the presence of this marker on antigen-specific CD8(+) T cells signifies recent activation.

Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus.

IFN-gamma-deficient (IFN-gamma(-/-)) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4(+) Th cells are required to establish and maintain this new equilibrium. The absence of IFN-gamma does not impair the generation of IL-2-producing CD4(+) cells, and depletion of these cells precipitates severe CD8(+) T cell-mediated immunopathology in IFN-gamma(-/-) mice, indicating an important role of CD4(+) T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-gamma(-/-) mice following CD4(+) cell depletion. Initially the antiviral CTL response did not require CD4(+) cells, but with time an impaired reactivity toward especially the glycoprotein 33--41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33--41 and nucleoprotein 396--404) CD8(+) T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4(+)-depleted mice, especially in mice also deficient in IFN-gamma. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-gamma(-/-) mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-gamma(-/-) hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8(+) effector T cells is one important factor influencing the requirement for T cell help during viral infection.

Persistent virus infection despite chronic cytotoxic T-lymphocyte activation in gamma interferon-deficient mice infected with lymphocytic choriomeningitis virus.

The role of gamma interferon (IFN-gamma) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-gamma-deficient (IFN-gamma -/-) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-gamma -/- mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-gamma -/- mice. The prolonged effector phase in infected IFN-gamma -/- mice was associated with elevated numbers of CD8(+) T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8(+) T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-gamma -/- mice than in wild-type mice. These findings indicate that in the absence of IFN-gamma, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-gamma-mediated antiviral activity on the dynamics between virus replication and CTL activity.

CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene.

An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its growth in a T cell-competent host adapts to the T cell system through a selective elimination of highly immunogenic tumour cells that are susceptible to CD8+ T cell-mediated lysis.

CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection.

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell-independent encephalitis, and infection resulted in a dramatic early up-regulation of chemokine gene expression. Similar marked up-regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV-infected mice, only LCMV-induced T cell-dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus-activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+-dependent CNS inflammation.

The status of half-cystine residues and locations of N-glycosylated asparagine residues in human eosinophil peroxidase.

The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.

A new theory of cytotoxic T-lymphocyte memory: implications for HIV treatment.

We use simple mathematical models to examine the dynamics of primary and secondary cytotoxic T-lymphocyte (CTL) responses to viral infections. In particular, we are interested in conditions required to resolve the infection and to protect the host upon secondary challenge. While protection against reinfection is only effective in a restricted set of circumstances, we find that resolution of the primary infection requires persistence of CTL precursors (GTLp), as well as a fast rate of activation of the CTLp. Since these are commonly the defining characteristics of CTL memory, we propose that CTL memory may have evolved in order to clear the virus during primary challenge. We show experimental data from lymphocytic choriomeningitis virus infection in mice, supporting our theory on CTL memory. We adapt our models to HIV and find that immune impairment during the primary phase of the infection may result in the failure to establish CTL memory which in turn leads to viral persistence. Based on our models we suggest conceptual treatment regimes which ensure establishment of CTL memory. This would allow the immune response to control HIV in the long term in the absence of continued therapy.

Cytokine vaccination: neutralising IL-1alpha autoantibodies induced by immunisation with homologous IL-1alpha.

High-affinity IgG autoantibodies (aAb) to IL-1alpha are among the most frequently found aAb to cytokines in humans. To establish an animal model with aAb to IL-1alpha, we immunised mice with recombinant murine IL-1alpha. Unprimed and Bacille Calmette-Guérin (BCG)-primed BALB/cA mice were vaccinated with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding of IL-1alpha to the murine T-cell line NOB-1 by simple competition and neutralised IL-1alpha, but not IL-1beta-induced IL-6 in vivo. The aAb did not induce visible discomfort in the animals. In conclusion, long-lasting and high levels of neutralising and specific IgG aAb to IL-1alpha can be induced in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients with immunoinflammatory diseases and cytokine-dependent tumours.

Role of CD40 ligand and CD28 in induction and maintenance of antiviral CD8+ effector T cell responses.

The primary aim of this report was to evaluate the immune responses of CD40 ligand-deficient (CD40L-/-) mice infected with two viruses known to differ markedly in their capacity to replicate in the host. Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen that replicates widely and extensively, whereas vesicular stomatitis virus (VSV) spreads poorly. We found that the primary response of CD40L-/- mice toward VSV is significantly impaired; proliferation of both CD4+ and CD8+ cells is reduced 2- to 3-fold, few CD8+ cells acquire an activated phenotype, and little functional activity is induced. Very similar results were obtained in VSV-infected, CD28-deficient mice. In contrast, neither CD40L nor CD28 was required for induction of a primary CD8+ response toward LCMV. Surprisingly, lack of CD4+ T cells had no impact on the primary immune response toward any of the viruses, even though the CD40 ligand dependence demonstrated for VSV would be expected to be associated with CD4 dependence. Upon coinfection of VSV-infected mice with LCMV, the requirement for CD40 ligand (but not CD28) could be partially bypassed, as evidenced by a 3-fold increase in the frequency of VSV-specific CD8+ T cells on day 6 postinfection. Finally, despite the fact that the primary LCMV-specific CD8+ response is virtually unimpaired in CD40L-/- mice, their capacity to maintain CD8+ effector activity and to permanently control the infection is significantly reduced. Thus, our results demonstrate that the importance of CD40/CD40L interaction for activation of CD8+ T cells varies between viruses and over time.

Migration of activated CD8(+) T lymphocytes to sites of viral infection does not require endothelial selectins.

Using mice deficient of E-selectin and E/P-selectin, we have studied the requirement for endothelial selectins in extravasation of leukocytes at sites of viral infection, with major emphasis on the recruitment of virus-specific T(C)1 cells. Lymphocytic choriomeningitis virus (LCMV)-induced meningitis was used as our primary experimental model. Additionally, localized subdermal inflammation and virus clearance in internal organs were analyzed during LCMV infection. The generation of CD8(+) effector T cells in infected mutants was unimpaired. Quantitative and qualitative analysis of the inflammatory exudate cells in intracerebrally infected mice gave identical results in all strains of mice. Expression of endothelial selectin was also found to be redundant regarding the ability of effector cells to eliminate virus in nonlymphoid organs. Concerning LCMV-induced footpad swelling, absent or marginal reduction was found in E/P-sel -/- mice, compared with wild-type mice after local challenge with virus or immunodominant viral MHC class I restricted peptide, respectively. Similar results were obtained after adoptive transfer of wild-type effector cells into E/P-sel -/- recipients, whereas footpad swelling was markedly decreased in P-sel/ICAM-1 -/- and ICAM-1 -/- recipients. LCMV-induced footpad swelling was completely inhibited in ICAM-deficient mice transfused with donor cell preincubated with soluble VCAM-1-Ig chimeric protein. Taken together, the current findings strongly indicate that the migration of T(C)1 effector cells to sites of viral infection can proceed in the absence of endothelial selectins, whereas ligands of the Ig superfamily are critically involved in this process. (Blood. 2000;95:1362-1369)

Host factors influencing viral persistence.

With the aim of characterizing the antiviral immune response to a non-cytocidal virus, we studied the outcome of lymphocytic choriomeningitis virus infection in a number of gene knockout mouse strains. Two virus strains differing markedly in their capacity to spread and replicate inside the murine host were used. Our results reveal that very different outcomes may be observed depending on virus strain and immunocompetence of the host. Thus while CD4+ cells are not critical during the initial phase of virus control, infectious virus reappear in mice lacking CD4+ cells, B cells or CD40 ligand. Reappearance of virus is associated with impaired long-term CD8+ T-cell mediated immune surveillance, and the time to virus resurgence is inversely correlated to the replication rate of the virus. Our studies also reveal that interferon-gamma is a central cytokine, and depending on the rate of virus replication, mice lacking the ability to produce interferon-gamma may develop either a severe, mostly fatal, T-cell mediated wasting syndrome or a chronic infection characterized by long-term coexistence of antiviral cytotoxic T lymphocytes and infectious virus. Mathematical modelling indicates that these different outcomes may be explained in relatively simple mathematical terms. This suggests that modelling may be used as a means to predict critical host and virus parameters. Therefore, combining mathematical modelling with precise, quantitative, in vivo analyses looks to be a promising approach in addressing central quantitative issues in immunobiology.

CD4(+) T cell-mediated protection against a lethal outcome of systemic infection with vesicular stomatitis virus requires CD40 ligand expression, but not IFN-gamma or IL-4.

To investigate the mechanism(s) whereby T cells protect against a lethal outcome of systemic infection with vesicular stomatitis virus, mice with targeted defects in genes central to T cell function were tested for resistance to i.v. infection with this virus. Our results show that mice lacking the capacity to secrete both IFN-gamma and perforin completely resisted disease. Similar results were obtained using IL-4 knockout mice, indicating that neither cell-mediated nor T(h)2-dependent effector systems were required. In contrast, mice deficient in expression of CD40 ligand were more susceptible than wild-type mice, and residual resistance in these mice was almost completely abrogated by depletion of CD8(+) T cells. In keeping with this, mice lacking both MHC class I and class II expression succumbed to the infection, whereas most class II-deficient mice normally survive. Adoptive transfer experiments using B cell- and T cell-deficient recipients revealed that no protection could be obtained in the absence of B cells, whereas treatment with virus-specific immune (IgG) serum controlled viral spreading to the central nervous system (CNS), but did not necessarily accomplish virus elimination. Taken together, these results underscore that B cells are essential in preventing early infection of the CNS, but T cells are required for long-term survival. CD4(+) T cells are most efficient in this context and the key function is to provide cognate help to B cells. However, if CD4(+) cell function is compromised, CD8(+) T cells become critical and may suffice for survival.