Thomas Kammertoens

CD4-Positive T Lymphocytes Provide a Neuroimmunological Link in the Control of Adult Hippocampal Neurogenesis

Adult hippocampal neurogenesis occurs in an exceptional permissive microenvironment. Neuroimmunological mechanisms might be prominently involved in the endogenous homeostatic principles that control baseline levels of adult neurogenesis. We show in this study that this homeostasis is partially dependent on CD4-positive T lymphocytes. Systemic depletion of CD4-positive T lymphocytes led to significantly reduced hippocampal neurogenesis, impaired reversal learning in the Morris water maze, and decreased brain-derived neurotrophic factor expression in the brain. No such effect of CD8 or B cells was observed. Repopulation of RAG2−/− mice with CD4, but not with CD8 cells again increased precursor cell proliferation. The T cells in our experiments were non-CNS specific and rarely detectable in the healthy brain. Thus, we can exclude cell-cell contacts between immune and brain cells or lymphocyte infiltration into the CNS as a prerequisite for an effect of CD4-T cells on neurogenesis. We propose that systemic CD4-T cell activity is required for maintaining cellular plasticity in the adult hippocampus and represents an evolutionary relevant communication route for the brain to respond to environmental changes.

Adaptive peripheral immune response increases proliferation of neural precursor cells in the adult hippocampus

To understand the link between peripheral immune activation and neuronal precursor biology, we investigated the effect of T‐cell activation on adult hippocampal neurogenesis in female C57Bl/6 mice. A peripheral adaptive immune response triggered by adjuvant‐induced rheumatoid arthritis (2 μg / μl methylated BSA) or staphylococcus enterotoxin B (EC50 of 0.25 μg/ml per 20 g body weight) was associated with a transient increase in hippocampal precursor cell proliferation and neurogenesis as assessed by immunohistochemistry and confocal microscopy. Both treatments were paralleled by an increase in corticosterone levels in the hippocampus 1‐ to 2‐fold over the physiological amount measured by quantitative radioimmunoassay. In contrast, intraperitoneal administration of the innate immune response activator lipopolysaccaride (EC50 of 0.5 μg/ml per 20 g body weight) led to a chronic 5‐fold increase of hippocampal glucocorticoid levels and a decrease of adult neurogenesis. In vitro exposure of murine neuronal progenitor cells to corticosterone triggered either cell death at high (1.5 nM) or proliferation at low (0.25 nM) concentrations. This effect could be blocked using a viral vector system expressing a transdomain of the glucocorticoid receptor. We suggest an evolutionary relevant communication route for the brain to respond to environmental stressors like inflammation mediated by glucocorticoid levels in the hippocampus.—Wolf, S. A., Steiner, B., Wengner, A., Lipp, M., Kammertoens, T., Kempermann, G. Adaptive peripheral immune response increases proliferation of neural precursor cells in the adult hippocampus. FASEBJ. 23, 3121–3128 (2009). www.fasebj.org

Efficient gene transfer into primary human CD8+ T lymphocytes by MuLV-10A1 retrovirus pseudotype.

Efficient and stable gene transfer into primary human T lymphocytes would greatly improve their use for adoptive transfer to treat acquired disorders, viral diseases, and cancer. We have constructed retroviral vector pseudotypes of amphotropic murine leukemia viruses (A-MuLV, MuLV-10A1), gibbon ape leukemia virus (GaLV), and feline endogenous virus (RD114) containing the enhanced green fluorescent protein (GFP) as a marker gene. Transduction of primary human CD8+ T lymphocytes by the different GFP-retrovirus pseudotypes revealed the superiority of MuLV-10A1 in comparison with A-MuLV, GaLV, and RD114, respectively. The superior transduction efficacy of CD8+ T cells by MuLV-10A1 correlates with a longer half-life of this pseudotype in comparison with A-MuLV and, as shown by interference analysis with the human T cell line HUT78, by the utilization of both the A-MuLV receptor (Pit2) and the GaLV receptor (Pit1) for cell entry.

Cross-Talk between T Cells and Innate Immune Cells Is Crucial for IFN-γ-Dependent Tumor Rejection

Though the importance of IFN-γ in tumor immunity has been well-demonstrated, little is known about its source and how it is induced. By using various bone marrow chimeric mice, we show here that IFN-γ essential for tumor immunity is solely produced by hemopoietic cells. Surprisingly, IFN-γ derived from T cells was not necessary for tumor immunity in this model. In the immunized mice, in which only innate immune cells have the IFN-γ-producing potential, tumors were efficiently rejected. The innate immune cells, such as NK1.1+ cells and CD11b+ cells, can provide sufficient amounts of IFN-γ which requires, however, the help of T cells. The close cooperation between T cells and innate immune cells during tumor regression is likely mediated by IL-2. Together, our results clearly illustrate how T cells cooperate with innate immune cells for IFN-γ-mediated tumor rejection and this may have important indications for clinical trials of tumor immunotherapy.

Tumour ischaemia by interferon-γ resembles physiological blood vessel regression

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ–GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

Combined chemotherapy of murine mammary tumors by local activation of the prodrugs ifosfamide and 5-fluorocytosine.

The success of chemotherapeutic intervention is limited because the necessary high local drug doses cannot be achieved without systemic toxicity. Application of suicide genes (SGs) and direct conversion of prodrugs (PDs) to toxic metabolites in situ by SGs may enhance the efficacy of chemotherapy. To evaluate this strategy in two murine breast cancer models, TS/A and GR, we injected cellulose sulfate capsules harboring cat kidney cells expressing the SGs cytosine deaminase and cytochrome P450 2B1 (CYP2B1) intratumorally. The PDs 5-fluorocytosine and ifosfamide were administered in 3-day intervals. The effect of in situ chemotherapy with each PD alone and the combination was analyzed over a period of 100 days. The results reveal that for TS/A tumors, the antitumoral effect mediated by CYP2B1 is more efficient than that of cytosine deaminase, whereas for GR tumors, both systems worked equally well. Furthermore, we find additive toxicity using both SG/PD systems for both TS/A and GR tumors.

Fas expression by tumor stroma is required for cancer eradication

The contribution of molecules such as perforin, IFN-γ (IFNγ), and particularly Fas ligand (FasL) by transferred CD8+ effector T (TE) cells to rejection of large, established tumors is incompletely understood. Efficient attack against large tumors carrying a surrogate tumor antigen (mimicking a “passenger” mutation) by TE cells requires action of IFNγ on tumor stroma cells to avoid selection of antigen-loss variants. Because “cancer-driving” antigens (CDAs) are rarely counterselected, IFNγ may be expected to be dispensable in elimination of cancers by targeting a CDA. Here, initial regression of large, established tumors required neither IFNγ, FasL, nor perforin by transferred CD8+ TE cells targeting Simian Virus (SV) 40 large T as CDA. However, cytotoxic TE cells lacking IFNγ or FasL could not prevent relapse despite retention of the rejection antigen by the cancer cells. Complete tumor rejection required IFNγ-regulated Fas by the tumor stroma. Therefore, TE cells lacking IFNγ or FasL cannot prevent progression of antigenic cancer because the tumor stroma escapes destruction if its Fas expression is down-regulated.

Tumor rejection by local interferon gamma induction in established tumors is associated with blood vessel destruction and necrosis

It has been shown that injecting a suspension of IFN‐γ‐secreting tumor cells results in their rejection. This effect has been attributed to IFN‐γ preventing tumor stroma formation but not to a direct effect on the cancer cells. However, it is not known, which influence IFN‐γ has on tumors with an established stroma. To address this question, the plasmacytoma cell line J558L was transduced with a vector allowing doxycycline‐inducible IFN‐γ gene expression. After the injection of the tumor cells into mice, IFN‐γ was induced at different time points. Tumors did not grow when inducing IFN‐γ immediately after tumor cell inoculation, while approximately half of the tumors were rejected when IFN‐γ was induced in early established tumors within 2 weeks. Induction of IFN‐γ 2–3 weeks after tumor cell inoculation was less efficient (0–17% rejection). IFN‐γ induction in established tumors led to a reduction of CD146+ endothelial cells and massive necrosis. Together, we show that vascularized tumors can be rejected by local IFN‐γ expression, but that rejection of established tumors was less efficient over time. This suggests that transplanted tumors became less susceptible to local IFN‐γ treatment the better they are established.

Composition of Intestinal Microbiota in Immune- Deficient Mice Kept in Three Different Housing Conditions

Background Abundance of commensals constituting the intestinal microbiota (IM) affects the immune system and predisposes to a variety of diseases, including intestinal infections, cancer, inflammatory and metabolic disorders. Housing conditions determine the IM and can hence influence the immune system. We analyzed how both variables affect the IM of four immune-compromized mouse lines kept under different housing conditions. Methodology/Principal Findings We investigated the IM composition in mice by quantitative 16S rRNA RT-PCR analysis of the main fecal bacterial groups (Enterobacteriaceae, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella (BP) spp., Clostridium leptum and coccoides groups). Mice were homozygous (HO) or heterozygous (HE) for a targeted inactivating mutation of either the IFN-γ Receptor (R), IFN-γ, Rag1 or IL-4 genes. Overall, differences in IM composition were subtle. However, in the SPF-barrier, total eubacterial loads were higher in Rag1 HE versus Rag1 HO mice as well as in IFN-γR HE versus IFN-γR HO and WT animals. Although absent in WT mice, bifidobacterial loads were higher in HO and HE IFN-γ and Rag1 as well as IL-4 HO mice. Furthermore, BP was slightly lower in HO and HE IFN-γR and IFN-γ mice as well as in IL-4 HO mice as compared to WT controls. Interestingly, IM compositions were comparable in WT mice when kept in individual ventilated cages (IVC) or open cages (OC). IFN-γ HO and HE mice, however, had higher enterobacteria and BP loads, but lacked bifidobacteria when kept in OC versus IVC, as was the case in HO and HE Rag1 mice. In addition, Rag1 HO mice harbored higher clostridial loads when housed in OC as compared to IVC. Unexpectedly, lactobacilli levels were higher in IFN-γR mice when kept in OC versus IVC. Conclusion/Significance Housing-dependent and immune-deficiency mediated changes in intestinal microbiota composition were rather subtle but may nevertheless impact immunopathology in experimental models.

Targeting human melanoma neoantigens by T cell receptor gene therapy

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.