T-cell Deficient Mice Research Articles

CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury

ObjectiveThe present study analyzed the effect of CD4+ Forkhead box protein 3 negative (Foxp3−) T-cells and Foxp3+ CD4+ T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. Approach and resultsWe examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4+ T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4+ T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4+ T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4+ T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4+ T-cells in CD4 KO mice increased the infarct size only when including the Foxp3+ CD25+ subset. Depletion of CD4+ Foxp3+ T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. ConclusionsCD4+ Foxp3+ T-cells enhance myocardial ischemia-reperfusion injury. CD4+ T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition.

Abstract # 1775 Attenuation of opioid analgesia in T cell deficient mice

It is now known that neurons are not the only cell type involved in pain processing, which involves Schwann cells, satellite cells, and cells of the immune system, such as microglia, macrophages, and T cells. Many pain researchers have adopted the use of T-cell deficient mice in their experimental methods to elucidate the role of T-cells in neuropathic pain (Fitzgerald et al., 2009; Zuo et al., 2013), and T cells have been shown to release endogenous opioids (Dietrich et al., 2011). While it is well known that opioids have varying effects on the immune system, very little attention has been given to how the immune system may affect opioid regulation. We now have evidence that T-cell deficient mice (CD-1 nude and Rag1 null mutant) exhibit pronounced deficiencies in morphine analgesia, measured using the tail withdrawal or formalin test. Furthermore, T-cell deficient mice do not exhibit stress-induced analgesia after restraint. Adoptive transfer of splenocytes from CD-1 mice into T-cell deficient mice rescues opioid analgesia. These results suggest that T-cells play a role in opioid-mediated analgesia, not previously observed. Current experiments are investigating the mechanism behind this phenomenon.

Abstract # 1773 Cytotoxic T cells may induce pain hypersensitivity in female but not male mice after nerve injury in an interferon-g-receptor-dependent manner

In our previous study, we observed that female mice, unlike males, do not require microglia to produce pain hypersensitivity after neuropathic or inflammatory injury. Using T-cell deficient mice, we found that female mutant mice “switch” to the “male” microglial system. Important questions include how microglial or T cell systems are recruited, how and which T cells are infiltrating into the spinal cord, and what T cells are releasing that can sensitize pain-relevant neurons. Immune cells were extracted from lumbar spinal cord of WT mice and IFNgRKO mice post SNI and CFA injection, and analyzed using flow cytometry. Additionally, mechanical allodynia was assessed. Analyzing the spinal cord microenvironment after SNI revealed that female mice displayed: (1) a 2-fold increase in CD8+ cytotoxic T cells, (2) upregulation of the anti-inflammatory microglia population, and (3) downregulation of anti-inflammatory T regulatory cells and pro-inflammatory microglia. In contrast, males showed: (1) a 2-fold decrease in CD8+ cytotoxic T cells, and (2) an increase in pro-inflammatory microglia. IFNgRKO female mice showed the same pain behavioural response as WT males wherein minocycline reversed allodynia. Consistent with this, IFNgRKO female, like WT male, mice did not show upregulation of CD8+ cytotoxic T cells. Our current data further suggest that female mice, unlike males, are using cytotoxic CD8+ T cells to induce neuropathic pain, and that this mechanism appears to be dependent on IFNgRs.

The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation

BackgroundHeterotopic ossification (HO) is the pathologic process of extraskeletal bone formation. Although the exact etiology remains unknown, inflammation appears to catalyze disease progression. The goal of this study is to determine the impact of the adaptive immune system on HO. MethodsHO was induced in 8-wk-old control C57BL/6 and immunocompromised Rag1tm1Mom (Rag1 KO) male mice deficient in B- and T-lymphocytes via combined Achilles tenotomy and burn injury. Microcomputed tomography quantified the extent of HO formation at the tenotomy site. Adipose-derived mesenchymal stem cells were harvested to evaluate osteogenic differentiation potential. ResultsAreas of developing HO demonstrated substantial enrichment of CD45 + leukocytes at 3 wk after injury. HO from Rag1 KO mice was substantially less mature with foci of cartilage and disorganized trabecular bone present 12 wk after injury. Rag1 KO mice formed 60% less bone compared to immunocompetent controls (4.67 ± 1.5 mm versus 7.76 ± 0.65 mm; P = 0.001). Tartrate-resistant acid phosphatase staining and immunofluorescent analysis of osteoprotegerin and nuclear factor kappa-light-chain-enhancer of activated B cells demonstrated no appreciable difference in osteoclast number or activation. Alizarin red staining in vitro demonstrated a significant decrease in osteogenic potential in immunocompromised mice compared to controls (29.1 ± 0.54 mm versus 12.1 ± 0.14 mm; P < 0.001). ConclusionsWe demonstrate a prominent role for the adaptive immune system in the development of HO. In the absence of mature B- and T-lymphocytes, HO growth and development are attenuated. Furthermore, we demonstrate that mesenchymal populations from B- and T-cell deficient mice are inherently less osteogenic. This study identifies a potential therapeutic role for modulation of the adaptive immune system in the treatment of HO.

Abstract 424: Chemokine CXCL14 is a multistep tumor suppressor

Abstract PURPOSE: The multistep nature of tumor formation has been well established and each step depends on the mutation or abnormal regulation of various genes. In order to elucidate the in vivo function of CXCL14, we used CXCL14 transgenic (Tg) mice and investigated the effects of this chemokine at multiple stages during cancer development, in addition to the effects on the overall survival rate. Furthermore, we also sought to determine the role of CXCL14 in the action of natural killer (NK) and natural killer T (NKT) cells. EXPERIMENTAL PROCEDURES: Carcinogenic rate was determined in mice undergoing AOM/DSS-induced colorectal carcinogenesis. The growth rate of implanted tumor cells was determined by injecting Lewis lung carcinoma (LLC) cells or B16 melanoma cells subcutaneously into the dorsolateral region of Tg or isogenic wild type C57BL/6 (Wt) mice. Tumor cells were also injected intravenously via a tail vein into Wt, T-cell and B-cell deficient SCID mice or T-cell, B-cell and NK cell-deficient NOG mice to investigate colonization to the lungs. Tg or Wt mice were also pre-injected with NK cell function inhibitory anti-asialo GM1 antibodies to see the role of NK cells in CXCL14-dependent tumor suppression in vivo. Melanoma cells that had been engineered to express the CXCL14 gene under the control of doxycycline (B16-LMT3-Tet/OnBRAK) were also used. RESULTS: The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that in Wt mice. When tumor cells were injected into these mice, the size of the tumors that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumor cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity plays an important role during CXCL14-mediated suppression of tumor growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed on the B16-LMT3-Tet/OnBRAK melanoma cells. The injection of alpha-galactosylceramide, an activator of NKT cells appeared to decrease the degree of melanoma cell metastasis in both the Wt and Tg mice, but the effect was much stronger in the Tg mice than in the Wt mice, indicating that the presence of both CXCL14 and alpha-galactosylceramide resulted in a synergistic effect and even greater tumor suppression. Finally, the survival rates after tumor cell injection were significantly increased for the Tg mice. CONCLUSION: High expression of the CXCL14 gene either in host cells, or tumor cells resulted in suppression of growth and metastasis of tumor cells. As these Tg mice showed no obvious signs of abnormality, we propose CXCL14 to be a promising molecular target for cancer suppression/prevention. Citation Format: Ryu-Ichiro Hata, Kazuhito Izukuri, Yasumasa Kato, Soichiro Sasaki, Chihiro Miyamoto, Tetsu Akasaka, Xiaoyan Yang, Yojiro Maehata, Yoji Nagashima, Kazuyoshi Takeda, Tohru Kiyono, Naofumi Mukaida, Masaru Taniguchi. Chemokine CXCL14 is a multistep tumor suppressor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 424. doi:10.1158/1538-7445.AM2015-424

PolySia-Specific Retargeting of Oncolytic Viruses Triggers Tumor-Specific Immune Responses and Facilitates Therapy of Disseminated Lung Cancer.

Polysialic acid (polySia) is expressed on several malignant tumors of neuroendocrine origin, including small cell lung cancer. In this study, we investigated the therapeutic efficacy of tumor-directed T-cell responses, elicited by polySia-retargeted oncolytic adenovirus infection, in an orthotopic murine model of disseminated polySia-positive lung cancer. In several cell lines, we demonstrated highly polySia-selective retargeting of adenoviral infection using a bispecific adapter comprising the ectodomain of the coxsackievirus/adenovirus receptor and a polySia-recognizing single-chain antibody domain. PolySia-dependent systemic infection in vivo facilitated effective uptake of viruses in subcutaneous polySia-expressing human tumors, whereas hepatic viral load and hepatotoxicity were significantly reduced. The impact and nature of antitumoral immune responses triggered by systemic delivery of polySia-retargeted oncolytic adenoviruses were investigated in an orthotopic model of disseminated lung cancer. Interestingly, improved transduction by polySia-retargeted oncolytic adenoviruses led to CD45-positive cell infiltrates in close association with large lytic areas. Consistently, enhanced tumor regression and prolonged survival was only observed in immunocompetent mice, but not in T-cell-deficient mice. To investigate whether improved systemic infection by polySia retargeting would elicit a tumor-specific T-cell response, we screened the used lung cancer cells for mutated oncogenes by complete exon sequencing. In agreement with our other results, only retargeted oncolysis was able to induce a significant response specific for the tumor-associated neoepitope Gsta2-Y9H. In conclusion, we demonstrated that effective retargeting of oncolytic adenovirus against polySia-expressing tumors elicits an effective tumor-directed T-cell response after systemic virus delivery and facilitates therapy of disseminated lung cancer.

The Thymus-Specific Serine Protease TSSP/PRSS16 Is Crucial for the Antitumoral Role of CD4+ T Cells

In cancer, immune cells can play conflicting roles, either protective, by elimination of tumor cells during immune surveillance, or detrimental, by promoting carcinogenesis during inflammation. We report here that the thymus-specific serine protease (TSSP), which is involved in CD4(+) Tcell maturation in the thymus, exerts a tumor suppressor activity. Mice genetically deficient for TSSP are highly prone to spontaneous cancer development. The absence of TSSP also increases the rate of induced colitis-associated colorectal (CAC) tumor formation, through exacerbated colon inflammation. Adoptive transfer of Tcells in various combinations (CD4(+) and CD8(+) from wild-type and/or knockout mice) into Tcell-deficient mice showed that the TSSP-deficient CD4(+) Tcell compartment promotes tumor development, associated with high levels of the cytokine IL-17A. Inhibition of IL-17A during CAC tumor formation prevents the increased carcinogenesis and colic immune disequilibrium observed in TSSP-deficient mice. Therefore, our data demonstrate that antitumoral immune surveillance requires thymic TSSP-driven production of CD4(+) Tcells contributing to inflammatory balance.

Sexual dimorphism, weight gain and glucose intolerance in a B- and T-cell deficient mouse model

BackgroundEstrogen is thought to aid maintenance of insulin sensitivity potentially through modulation of a counter-regulatory mechanism that interferes with the contribution of adaptive and innate immune systems to visceral fat deposition. We evaluated the impact of estrogen on long-term high fat diet (HFD) intake in B- and T-cell deficient and immunocompetent animals comparatively. MethodsA total of 16 BALB and 16 SCID mice, 8 of each sex and strain, were randomized to receive low fat diet, 4.1% fat or HFD, 35% fat, such that there was a group of both each sex and each strain receiving each diet. Biweekly levels of adiponectin, leptin and insulin levels were assessed and a glucose tolerance test (GTT) was performed after 13weeks. ResultsUnlike their male counterparts, HFD-fed SCID females neither gained weight, nor became insulin resistant. Meanwhile, in the HFD-fed BALB groups both males and females gained weight similarly, but remarkable sexual dimorphism was nonetheless observed. The females had notable higher adiponectin levels as compared to males (10–60μg/mL vs. 6–10μg/mL respectively) causing the adiponectin-to-leptin (A/L) ratio to reach 80 one week after HFD initiation. The A/L dropped to 10, still higher than males, by week 13, but dropped to 2 by the end of the study in agreement with inverse insulin trends. None of the HFD-fed female groups developed insulin resistance (IR) by week 13, while all male counterparts had. Similar results were observed in the HFD-fed SCID groups whereby the females did not develop IR and had a higher A/L; however, adiponectin levels were comparable between groups (5–11μg/mL). ConclusionsThe present study provides lacking evidence indicating that estrogen may be sufficient to prevent weight gain and development of glucose intolerance in high-fat fed B- and T-cell deficient mice.

Retargeting of oncolytic adenoviruses by bispecific adapter molecules reduces viral liver load and improves therapy by triggering antigen-specific CD8 T-cell responses

Polysialic acid (PolySia) represents an excellent tumor-selective target for oncolytic adenovirus-based therapies since it is selectively expressed on several malignant tumors. In this study, we used a strategy to retarget oncolytic Adenoviruses (OAV) to PolySia-expressing tumors using the bispecific adapter protein CARsc-pSia consisting of the soluble CAR ectodomain and a PolySia-binding scFv domain. By infection experiments in cells of defined PolySia status using CARsc-pSia coated OAV particles, we could show that viral cell entry occurred in a highly PolySia-selective manner. Furthermore, only CARsc-pSia treatment allowed efficient adenoviral infection of normally refractory PolySia-expressing human tumor cells. After systemic administration we could show improved adenoviral infection of s.c. grown PolySia-expressing human tumor xenografts. Masking of the fiberknob by the molecular adapter led to significant reduction of the natural tropism since we could observe a significantly reduced adenoviral liver load and hepatotoxicity in mice. When we investigated the involvement of antitumoral immune responses, therapeutic investigations were therefore performed in an orthotopic animal model in both immunocompetent C67/Bl6 and immunodeficient nude mice. In these models, only CARsc-pSia-complexing of viruses allowed for significant transduction. Interestingly, improved transduction led to improved local tumor regression and prolonged survival in immunocompetent mice but not in T-cell deficient nude mice. Consistently, large infiltrations of CD45-pos. cells in virus-affected, lytic areas were only detectable in mice which received retargeted OAV. Most importantly we could show that retargeted OAV induced an epitope-specific immune response against Gsta-2, a mutated tumor-specific antigen that we could identify by exome sequencing, epitope prediction and in vivo validation. In conclusion, our results show that specific retargeting of OAV represents a promising hepatotoxocity-reduced viroimmuntherapeutic strategy of clinically relevant tumor entities.

Pretreatment with TCDD exacerbates liver injury from Concanavalin A: critical role for NK cells.

For many liver diseases, including viral and autoimmune hepatitis, immune cells play an important role in the development and progression of liver injury. Concanavalin A (Con A) administration to rodents has been used as a model of immune-mediated liver injury resembling human autoimmune hepatitis. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to alter the development of immune-mediated diseases. Mice pretreated with TCDD developed exacerbated liver injury in response to administration of a mild dose (6 mg/kg) of Con A. In the present study, we tested the hypothesis that TCDD pretreatment exacerbates Con A-induced liver injury by enhancing the activation and recruitment of accessory cell types including neutrophils, macrophages, and natural killer (NK) cells. Mice were treated with 0, 0.3, 3, or 30 μg/kg TCDD and 4 days later with Con A or saline. TCDD pretreatment with doses of 3 and 30 μg/kg significantly increased liver injury from Con A administration. The plasma concentrations of neutrophil chemokines were significantly increased in TCDD-pretreated mice after Con A administration. NKT cell-deficient (CD1d KO) mice were used to examine whether NKT cells were required for TCDD/Con A-induced liver injury. CD1d KO mice were completely protected from liver injury induced by treatment with Con A alone, whereas the injury from TCDD/Con A treatment was reduced but not eliminated. However, T-cell deficient (RAG1 KO) mice were protected from liver injury induced by Con A irrespective of pretreatment with TCDD. TCDD/Con A treatment increased the percentage of NK cells expressing the activation marker CD69. Depletion of NK cells prior to treatment resulted in significant reductions in plasma interferon-γ and liver injury from TCDD/Con A treatment. In summary, exposure to TCDD exacerbated the immune-mediated liver injury induced by Con A, and our findings suggest that NK cells play a critical role in this response.

B7h (ICOS-L) Maintains Tolerance at the Fetomaternal Interface

In a successful pregnancy, the semiallogeneic fetus is not rejected by the maternal immune system, which implies tolerance mechanisms protecting fetal tissues from maternal immune attack. Here we report that the ICOS-B7h costimulatory pathway plays a critical role in maintaining the equilibrium at the fetomaternal interface. Blockade of this pathway increased fetal resorption and decreased fetal survival in an allogeneic pregnancy model (CBA female × B6 male). Locally in the placenta, levels of regulatory markers such as IDO and TGF-β1 were reduced after anti-B7h monoclonal antibody treatment, whereas levels of effector cytokines (eg, IFN-γ) were significantly increased. In secondary lymphoid organs, enhanced IFN-γ and granzyme B production (predominantly by CD8(+) T cells) was observed in the anti-B7h-treated group. The deleterious effect of B7h blockade in pregnancy was maintained only in CD4 knockout mice, not in CD8 knockout mice, which suggests a role for CD8(+) Tcells in immune regulation by the ICOS-B7h pathway. In accord, regulatory CD8(+) T cells (in particular, CD8(+)CD103(+) cells) were significantly decreased after anti-B7h monoclonal antibody treatment, and adoptive transfer of this subset abrogated the deleterious effect of B7h blockade in fetomaternal tolerance. Taken together, these data support the hypothesis that B7h blockade abrogates tolerance at the fetomaternal interface by enhancing CD8(+) effector response and reducing local immunomodulation mediated by CD8(+) regulatory T cells.

Gamma delta T‐cells regulate myeloid cell activity after injury

Gamma delta (γδ) T‐cells have been shown to be important in post‐burn inflammation and wound healing, but their role in the regulation of myeloid cells is unknown. To study this wild‐type (WT) and γδ T‐cell deficient (δTCR−/−) mice were subjected to a major burn or sham treatment. Three days later skin was collected and subjected to dispase/trypsin digestion or frozen. The number of CD11b+ myeloid cells increased by ~70% in the wound skin of WT mice. This was due to increased numbers of myeloid‐derived suppressor cells (MDSC; CD11b+ GR1+), as macrophage (Mϕ; CD11b+ F4/80+) numbers decreased by ~80% after burn. In δTCR−/− mice, burn increased myeloid cell numbers 7‐fold due to both a MDSC (20‐fold) and a Mϕ (3‐fold) influx. Burn also increased skin cytokine levels for IL‐1β, IL‐6, TNF‐α and MIP‐1β. TNF‐α, MIP‐1β levels were further elevated in the injured skin of δTCR−/− mice. In conclusion these data show that γδ T‐cells regulate myeloid cell infiltration of the wound site and act to quell inflammation. NIH grant GM079122

Combined B- and T-cell deficiency does not protect against obesity-induced glucose intolerance and inflammation

Obesity-induced inflammation is associated with insulin resistance and morphologically characterized by macrophage influx into the adipose tissue. Recently, various other immune cells, including B- and T-cells, have been shown to participate in modulating adipose tissue inflammation during the development of obesity. We show that HFD-feeding modulates the influx of B and T-cells into adipose tissue of obese animals, suggestive of a role of the adaptive immune system in the development of adipose tissue inflammation. Despite a lower bodyweight after HFD-feeding, gene expression levels of CD68, F4/80 and MCP-1 in white adipose tissue were enhanced in SCID animals that lack B- and T-cells. Moreover, conditioned medium from adipose tissue explants of HFD-fed SCID mice revealed increased release of IL-6 and CXCL1 compared to WT animals. Compared to WT mice, glucose tolerance was impaired in B- and T-cell deficient mice after HFD-feeding. Thus, complete B- and T-cell deficiency does not protect against HFD-induced adipose tissue inflammation and glucose intolerance. In contrast, SCID mice showed an increased pro-inflammatory status at the level of the adipose tissue in some cytokines. Our findings suggest that a delicate balance between various B- and T-cell populations controls adipose tissue inflammation.

IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival

Glioblastoma multiforme (GBM) is an aggressive adult brain tumor with a poor prognosis. One hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4(+)FoxP3(+)GITR(+) regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain tumors and the impact on Treg recruitment. To determine the clinical relevance of IDO expression in brain tumors, we first correlated patient survival to the level of IDO expression from resected glioma specimens. We also used novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-derived and peripheral IDO expression on Treg recruitment, GITR expression, and long-term survival was determined. Downregulated IDO expression in glioma predicted a significantly better prognosis in patients. Coincidently, both IDO-competent and deficient mice showed a survival advantage bearing IDO-deficient brain tumors, when compared with IDO-competent brain tumors. Moreover, IDO deficiency was associated with a significant decrease in brain-resident Tregs, both in orthotopic and transgenic mouse glioma models. IDO deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term survival advantage conferred by IDO deficiency was lost in T-cell-deficient mice. These clinical and preclinical data confirm that IDO expression increases the recruitment of immunosuppressive Tregs that lead to tumor outgrowth. In contrast, IDO deficiency decreases Treg recruitment and enhances T-cell-mediated tumor rejection. Thus, the data suggest a critical role for IDO-mediated immunosuppression in glioma and support the continued investigation of IDO-Treg interactions in the context of brain tumors.

Abstract 3451: Lack of CD47 in the tumor microenvironment enhances anti-tumor adaptive immune responses when combined with ionizing radiation

Abstract Over half of the new cancer patients diagnosed this year in the United States will require ionizing radiation (IR) therapy as part of their course of treatment. Achieving a curative response, however, is limited by detrimental side effects and off-target toxicities of IR to normal cells. We previously demonstrated that CD47 null mice or blockade of CD47 confers radioprotection to normal tissues including radiosensitive hematopoietic progenitors. On the other hand, blockade of CD47 enhanced the radiation-induced tumor growth delay in two syngenic mouse models. One possible explanation for this observation is that, by protecting tumor-associated immune cells, CD47 suppression increases tumor immune surveillance. To examine the role of T cell immunity, we used the F15-12 RM mouse fibrosarcoma model in athymic mice. Blockade of CD47 in combination with IR did not alter tumor growth in these T-cell deficient mice versus IR alone. However, adoptive transfer of CD8+ T-cells in combination with CD47 blockade and IR significantly enhanced the reduction in tumor growth when compared to tumors of mice irradiated in combination with the T cell transfer alone (186 ± 50 vs. 370 ± 123 mm3 respectively, N=8). In vitro experiments showed that blockade of CD47 enhances antigen dependent CD8+ mediated cytotoxicity of tumor cells. Furthermore, in a syngeneic tumor model of B16 melanoma we observed over a 50% reduction in volume of irradiated tumors in a CD47-/- host versus a WT host. Immunohistochemical staining indicated an increase of intratumoral CD8+ cytotoxic T-cells. Further analysis indicated that lack or blockade of CD47 in combination with IR resulted in a significant increase in granzyme B expression, further indicating that the increased target cell death may be mediated by this pathway. CD47 suppression may also reduce tumor growth by altering the immunosuppressive factor VEGF. In both tumor models, either blockade of or lack of CD47 in combination with IR was associated with a reduction in VEGF. VEGF is a potent pro-angiogenic factor and a potent immunosuppressive factor that inhibits activation of cytotoxic T-cells. Our findings indicate that agents targeting CD47 enhance anti-tumor T cell immunity when combined with radiation. Combining CD47 suppression with IR and adoptive T cell transfer may increase the percentage of curative responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3451. doi:1538-7445.AM2012-3451

Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model

We have previously shown that IsdB, a conserved protein expressed by Staphylococcus aureus, induces a robust antibody response which correlates with protection in a murine challenge model. Here we investigate the role of cellular immunity in IsdB mediated protection using lymphocyte deficient SCID mice. As opposed to WT CB-17 mice the CB-17 SCID mice were not protected against a lethal challenge of S. aureus after active and passive immunizations with IsdB. Adoptive transfer of in vitro isolated lymphocyte subsets revealed that reconstituting mice with IsdB specific CD3+ or CD4+ T-cells conferred antigen specific protection while CD8+ T-cells, CD19+ B-cells and plasma cells (CD138highB220intCD19lo) alone were not protective. A combination of CD3+ T-cells plus CD19+ B-cells conferred protection in CB-17 SCID mice, whereas bovine serum albumin (BSA) immune lymphocytes did not confer protection. Active immunization experiments indicated that IsdB immunized Jh mice (B-cell deficient) were protected against lethal challenge, while nude (T-cell deficient) mice were not. In vitro assays indicated that isolated IsdB specific splenocytes from immunized mice produced abundant IL-17A, much less IFN-γ and no detectable IL-4. IL-23 deficient mice were not protected from a lethal challenge by IsdB vaccination, pointing to a critical role for CD4+ Th17 in IsdB-mediated vaccination. Neutralizing IL-17A, but not IL-22 in vivo significantly increased mortality in IsdB immunized mice; whereas, neutralizing IFN-γ did not alter IsdB-mediated protection. These findings suggest that IL-17A producing Th17 cells play an essential role in IsdB vaccine-mediated defense against invasive S. aureus infection in mice.

Activation of CD4 + T Lymphocytes Improves Wound Healing and Survival After Experimental Myocardial Infarction in Mice

The role of adaptive immunity, especially CD4(+) T-helper cells, has not yet been systematically investigated in wound healing and remodeling after myocardial infarction (MI). Therefore, we studied whether CD4(+) T cells become activated and influence wound healing after experimental MI in mice. When we compared sham versus MI in wild-type (WT) mice, T-cell receptor-dependent activation of both conventional Foxp3(-) and regulatory Foxp3(+) CD4(+) T cells could be demonstrated in heart-draining lymph nodes within the first week after MI. Concomitantly, we found infiltration of CD4(+) T cells in infarcted myocardium. To study the role of CD4(+) T cells in wound healing and remodeling, CD4(+) T-cell-deficient mice (CD4 knockout [KO], MHCII(Δ/Δ)) and T-cell receptor-transgenic OT-II mice recognizing an irrelevant ovalbumin-derived peptide were studied. Serial echocardiography up to day 56 after MI revealed increased left ventricular dilation in CD4 KO compared with WT mice. Within the infarcted myocardium, CD4 KO mice displayed higher total numbers of leukocytes and proinflammatory monocytes (18.3±3.0 10(4)/mg WT versus 75.7±17.0 10(4)/mg CD4 KO, P<0.05). MHCII(Δ/Δ) and OT-II mice displayed significantly greater mortality (21% WT versus 48% OT-II, P<0.05, and WT 22% versus 52% MHCII(Δ/Δ), P<0.05) and myocardial rupture rates than WT mice. Collagen matrix formation in the infarct zone was severely disturbed in CD4 KO and MHCII(Δ/Δ) mice, as well as in OT-II mice. The present study provides the first evidence that CD4(+) T cells become activated after MI, presumably driven by recognition of cardiac autoantigens, and facilitate wound healing of the myocardium.

Altered intestinal epithelium-associated lymphocyte repertoires and function in ApcMin/+ mice

ApcMin/+ mice spontaneously develop multiple intestinal adenomas along the length of the small intestine and colon. Currently little is known about the role of the immune system in regulating intestinal tumorigenesis in these animals. This study characterised small intestinal intraepithelial lympho-- cyte (IEL) populations in C56BL/6J ApcMin/+ mice and wild-type (Apc+/+) mice. We also determined the effect that T cells expressing either γδ or αβ encoded T cell receptors (TcR) exert on intestinal tumorigenesis. ApcMin/+ mice had significantly lower numbers of CD3+ IELs compared with Apc+/+ littermates and displayed reduced cytotoxicity against tumour target cells. Further analysis of IEL cytotoxicity revealed differences in the cytotoxic pathways utilised by IELs in ApcMin/+ and Apc+/+ mice with ApcMin/+ IELs displaying an absence of perforin/granzyme-mediated killing and increased levels of Fas-FasL-mediated cytotoxicity compared with wild-type IELs. Analysis of ApcMin/+ mice crossed with αβ T-cell deficient (TcRβ-/-) or γδ T-cell deficient (TcRδ-/-) mice on the same genetic background revealed decreased tumour multiplicity in the absence of both αβ and γδ T-cells. This study demonstrates that altered T-cell subsets play important roles in promoting tumorigenesis in ApcMin/+ mice and forms the basis for future mechanistic studies.

Enhancement of T-cell–Mediated Antitumor Response: Angiostatic Adjuvant to Immunotherapy against Cancer

Tumor-released proangiogenic factors suppress endothelial adhesion molecule (EAM) expression and prevent leukocyte extravasation into the tumor. This is one reason why immunotherapy has met with limited success in the clinic. We hypothesized that overcoming EAM suppression with angiogenesis inhibitors would increase leukocyte extravasation and subsequently enhance the effectiveness of cellular immunotherapy. Intravital microscopy, multiple color flow cytometry, immunohistochemistry, and various tumor mouse (normal and T-cell deficient) models were used to investigate the temporal dynamics of cellular and molecular events that occur in the tumor microenvironment during tumor progression and angiostatic intervention. We report that while EAM levels and T-cell infiltration are highly attenuated early on in tumor growth, angiostatic therapy modulates these effects. In tumor models with normal and T-cell-deficient mice, we show the active involvement of the adaptive immune system in cancer and differentiate antiangiogenic effects from antiangiogenic mediated enhancement of immunoextravasation. Our results indicate that a compromised immune response in tumors can be obviated by the use of antiangiogenic agents. Finally, with adoptive transfer studies in mice, we show that a phased combination of angiostatic therapy and T-cell transfer significantly (P < 0.0013) improves tumor growth inhibition. This research contributes to understand the cellular mechanism of action of angiostatic agents and the immune response within the tumor microenvironment, in particular as a consequence of the temporal dynamics of EAM levels. Moreover, our results suggest that adjuvant therapy with angiogenesis inhibitors holds promise for cellular immunotherapy in the clinic.

C‐Rel promotes type 1 and type 17 immune responses during Leishmania major infection

Recent studies demonstrated the crucial role of c-Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell-mediated autoimmune inflammation. We thus wondered whether these opposite functions of c-Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T-cell subsets on disease outcome. Here we show that c-Rel-deficient (rel(-/-) ) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN-γ and IL-17 production in response to L. major antigens was severely impaired in rel(-/-) mice. Reconstitution of Rag1(-/-) T-cell deficient mice with rel(-/-) CD4(+) T cells followed by L. major infection demonstrated that c-Rel-deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4(+) cells in vitro was normal. Notably, a selective defect in IL-12 and IL-23 production was observed in rel(-/-) DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c-Rel in myeloid cells is essential for clearance of L. major and that this c-Rel-mediated effect is dominant over the lack of Tregs.