Maria de la Luz Garcia-Hernandez

Tc17, a Unique Subset of CD8 T Cells That Can Protect against Lethal Influenza Challenge

We show here that IL-17-secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and that blocking Ab to IL-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naive CD8 T cells from OT-I TCR-transgenic mice. T cell numbers expand 20-fold and a majority secretes IL-17, but little IFN-γ. Many of the IL-17-secreting cells also secrete TNF and some secrete IL-2. Tc17 are negative for granzyme B, perforin message, and cytolytic activity, in contrast to Tc1 effectors. Tc17 populations express message for orphan nuclear receptor γt and FoxP3, but are negative for T-bet and GATA-3 transcription factors. The FoxP3-positive, IL-17-secreting and IFN-γ-secreting cells represent three separate populations. The IFN-γ-, granzyme B-, FoxP3-positive cells and cells positive for IL-22 come mainly from memory cells and decrease in number when generated from CD44low rather than unselected CD8 T cells. Cells of this unique subset of CD8 effector T cells expand greatly after transfer to naive recipients following challenge and can protect them against lethal influenza infection. Tc17 protection is accompanied by greater neutrophil influx into the lung than in Tc1-injected mice, and the protection afforded by Tc17 effectors is less perforin but more IFN-γ dependent, implying that different mechanisms are involved.

The development of inducible bronchus-associated lymphoid tissue depends on IL-17.

Ectopic or tertiary lymphoid tissues, such as inducible bronchus-associated lymphoid tissue (iBALT), form in nonlymphoid organs after local infection or inflammation. However, the initial events that promote this process remain unknown. Here we show that iBALT formed in mouse lungs as a consequence of pulmonary inflammation during the neonatal period. Although we found CD4+CD3− lymphoid tissue–inducer cells (LTi cells) in neonatal lungs, particularly after inflammation, iBALT was formed in mice that lacked LTi cells. Instead, we found that interleukin 17 (IL-17) produced by CD4+ T cells was essential for the formation of iBALT. IL-17 acted by promoting lymphotoxin-α-independent expression of the chemokine CXCL13, which was important for follicle formation. Our results suggest that IL-17-producing T cells are critical for the development of ectopic lymphoid tissues.

Prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of autoimmunity.

Prostate stem cell antigen (PSCA) is an attractive antigen to target using therapeutic vaccines because of its overexpression in prostate cancer, especially in metastatic tissues, and its limited expression in other organs. Our studies offer the first evidence that a PSCA-based vaccine can induce long-term protection against prostate cancer development in prostate cancer-prone transgenic adenocarcinoma mouse prostate (TRAMP) mice. Eight-week-old TRAMP mice displaying prostate intraepithelial neoplasia were vaccinated with a heterologous prime/boost strategy consisting of gene gun-delivered PSCA-cDNA followed by Venezuelan equine encephalitis virus replicons encoding PSCA. Our results show the induction of an immune response against a newly defined PSCA epitope that is mediated primarily by CD8 T cells. The prostates of PSCA-vaccinated mice were infiltrated by CD4-positive, CD8-positive, CD11b-positive, and CD11c-positive cells. Vaccination induced MHC class I expression and cytokine production [IFN-gamma, tumor necrosis factor-alpha, interleukin 2 (IL-2), IL-4, and IL-5] within prostate tumors. This tumor microenvironment correlated with low Gleason scores and weak PSCA staining on tumor cells present in hyperplastic zones and in areas that contained focal and well-differentiated adenocarcinomas. PSCA-vaccinated TRAMP mice had a 90% survival rate at 12 months of age. In contrast, all control mice had succumbed to prostate cancer or had heavy tumor loads. Crucially, this long-term protective immune response was not associated with any measurable induction of autoimmunity. The possibility of inducing long-term protection against prostate cancer by vaccination at the earliest signs of its development has the potential to cause a dramatic paradigm shift in the treatment of this disease.

Multifunctional CD4 Cells Expressing Gamma Interferon and Perforin Mediate Protection against Lethal Influenza Virus Infection

ABSTRACT CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ−/− CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.

Interleukin‐10 promotes B16‐melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

The aim of this study was to investigate the mechanisms by which interleukin‐10 (IL‐10) induces tumour growth in a mouse‐melanoma model. A B16‐melanoma cell line (B16‐0) was transfected with IL‐10 cDNA and three clones that secreted high (B16‐10), medium and low amounts of IL‐10 were selected. Cell proliferation and IL‐10 production were compared in vitro, and tumour growth, percentages of necrotic areas, tumour cells positive for proliferating cell nuclear antigen (PCNA), IL‐10 receptor (IL‐10R) and major histocompatibility complex type I (MHC‐I) and II (MHC‐II), as well as infiltration of macrophages, CD4+ and CD8+ lymphocytes and blood vessels were compared in vivo among IL‐10‐transfected and non‐transfected tumours. Proliferation and tumour growth were greater for IL‐10‐transfected than for non‐transfected cells (P < 0·001), and correlated with IL‐10 concentration (r ≥ 0·79, P < 0·006). Percentages of tumour cells positive for PCNA and IL‐10R were 4·4‐ and 16·7‐fold higher, respectively, in B16‐10 than in B16‐0 tumours (P < 0·001). Macrophage distribution changed from a diffuse pattern in non‐transfected (6·4 ± 1·7%) to a peripheral pattern in IL‐10‐transfected (3·8 ± 1·7%) tumours. The percentage of CD4+ lymphocytes was 7·6 times higher in B16‐10 than in B16‐0 tumours (P = 0·002). The expression of MHC‐I molecules was present in all B16‐0 tumour cells and completely negative in B16–10 tumour cells. In B16‐0 tumours, 89 ± 4% of the whole tumour area was necrotic, whereas tumours produced by B16‐10 cells showed only 4·3 ± 6% of necrotic areas. IL‐10‐transfected tumours had 17‐fold more blood vessels than non‐transfected tumours (61·8 ± 8% versus 3·5 ± 1·7% blood vessels/tumour; P < 0·001). All the effects induced by IL‐10 were prevented in mice treated with a neutralizing anti‐IL‐10 monoclonal antibody. These data indicate that IL‐10 could induce tumour growth in this B16‐melanoma model by stimulation of tumour‐cell proliferation, angiogenesis and immunosuppression.

Adoptive Transfer of Tumor-Specific Tc17 Effector T Cells Controls the Growth of B16 Melanoma in Mice

In vitro generated OVA-specific IL-17–producing CD8 T effector cells (Tc17) from OT-1 mice, adoptively transferred into B16-OVA tumor-bearing mice, controlled tumor growth in early and late stage melanoma. IL-17, TNF, and IFN-γ from the Tc17 effectors all played a role in an enhanced recruitment of T cells, neutrophils, and macrophages to the tumor. In addition, Tc17 cells and recently recruited, activated neutrophils produced further chemokines, including CCL3, CCL4, CCL5, CXCL9, and CXCL10, responsible for the attraction of type 1 lymphocytes (Th1 and Tc1) and additional neutrophils. Neutrophils were rapidly attracted to the tumor site by an IL-17 dependent mechanism, but at later stages the induction of the chemokine CXCL2 by Tc17-derived TNF and IFN-γ contributed to sustain neutrophil recruitment. Approximately 10–50 times as many Tc17 effectors were required compared with Tc1 effectors to exert the same level of control over tumor growth. The recruitment of neutrophils was more prominent when Tc17 rather than Tc1 were used to control tumor and depletion of neutrophils resulted in a diminished capacity to control tumor growth.

In vivo Effects of Vaccination with Six-Transmembrane Epithelial Antigen of the Prostate: A Candidate Antigen for Treating Prostate Cancer

Immunotherapy may provide an alternative treatment for cancer patients, especially when tumors overexpress antigens that can be recognized by immune cells. The identification of markers and therapeutic targets that are up-regulated in prostate cancer has been important to design new potential treatments for prostate cancer. Among them, the recently identified six-transmembrane epithelial antigen of the prostate (STEAP) is considered attractive due to its overexpression in human prostate cancer tissues. Our study constitutes the first assessment of the in vivo effectiveness of STEAP-based vaccination in prophylactic and therapeutic mouse models. Two delivery systems, cDNA delivered by gene gun and Venezuelan equine encephalitis virus-like replicon particles (VRP), both encoding mouse STEAP (mSTEAP) and three vaccination strategies were used. Our results show that mSTEAP-based vaccination was able to induce a specific CD8 T-cell response against a newly defined mSTEAP epitope that prolonged the overall survival rate in tumor-challenged mice very significantly. This was achieved without any development of autoimmunity. Surprisingly, CD4 T cells that produced IFNgamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2) played the main role in tumor rejection in our model as shown by using CD4- and CD8-deficient mice. In addition, the presence of high IL-12 levels in the tumor environment was associated with a favorable antitumor response. Finally, the therapeutic effect of STEAP vaccination was also assessed and induced a modest but significant delay in growth of established, 31 day old tumors. Taken together, our data suggest that vaccination against mSTEAP is a viable option to delay tumor growth.

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs

Significance Ectopic clusters of immune cells that mimic the structure and function of secondary lymphoid organs are defined as tertiary lymphoid organs (TLOs). They have been observed at sites of chronic inflammation for decades, but their formation and function have remained enigmatic. TLOs are thought to contribute to disease pathogenesis by promoting autoreactive lymphocyte survival and autoantibody production. In this study we identify a novel role for the cytokine IL-22 in TLO development and biology. We provide evidence that IL-22 expression within TLOs is instrumental for the production of the lymphoid chemokines, chemokine (C-X-C motif) ligand 13 and chemokine (C-X-C motif) ligand 12, which in turn orchestrate B-cell clustering, lymphoid aggregation, and autoantibody production. Our data provide a strong rationale for targeting IL-22 in TLO-associated autoimmune diseases. The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22–blocking agents in B-cell–mediated autoimmune conditions.

Multiple Redundant Effector Mechanisms of CD8+ T Cells Protect against Influenza Infection

We have previously shown that mice challenged with a lethal dose of A/Puerto Rico/8/34-OVAI are protected by injection of 4–8 × 106 in vitro–generated Tc1 or Tc17 CD8+ effectors. Viral load, lung damage, and loss of lung function are all reduced after transfer. Weight loss is reduced and survival increased. We sought in this study to define the mechanism of this protection. CD8+ effectors exhibit multiple effector activities, perforin-, Fas ligand–, and TRAIL-mediated cytotoxicity, and secretion of multiple cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, IL-17, IL-21, IL-22, IFN-γ, and TNF) and chemokines (CCL3, CCL4, CCL5, CXCL9, and CXCL10). Transfer of CD8+ effectors into recipients, before infection, elicits enhanced recruitment of host neutrophils, NK cells, macrophages, and B cells. All of these events have the potential to protect against viral infections. Removal of any one, however, of these potential mechanisms was without effect on protection. Even the simultaneous removal of host T cells, host B cells, and host neutrophils combined with the elimination of perforin-mediated lytic mechanisms in the donor cells failed to reduce their ability to protect. We conclude that CD8+ effector T cells can protect against the lethal effects of viral infection by means of a large number of redundant mechanisms.

Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion

In humans, vWF levels predict the risk of myocardial infarction and thrombosis; however, the factors that influence vWF levels are not completely understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels, though the functional relationship between STXBP5 and vWF is unknown. We hypothesized that STXBP5 inhibits endothelial cell exocytosis. We found that STXBP5 is expressed in human endothelial cells and colocalizes with and interacts with syntaxin 4. In human endothelial cells reduction of STXBP5 increased exocytosis of vWF and P-selectin. Mice lacking Stxbp5 had higher levels of vWF in the plasma, increased P-selectin translocation, and more platelet-endothelial interactions, which suggests that STXBP5 inhibits endothelial exocytosis. However, Stxbp5 KO mice also displayed hemostasis defects, including prolonged tail bleeding times and impaired mesenteric arteriole and carotid artery thrombosis. Furthermore, platelets from Stxbp5 KO mice had defects in platelet secretion and activation; thus, STXBP5 inhibits endothelial exocytosis but promotes platelet secretion. Our study reveals a vascular function for STXBP5, validates the functional relevance of a candidate gene identified by GWAS, and suggests that variation within STXBP5 is a genetic risk for venous thromboembolic disease.