Characterization of Donor Variability for γδ T Cell ex vivo Expansion and Development of an Allogeneic γδ T Cell Immunotherapy.

Gamma delta (γδ) T cells recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against both hematological and solid tumors. Moreover, γδ T cells provide a platform for the development of allogeneic cell therapies, as they can recognize antigens independent of MHC recognition and without the requirement for a chimeric antigen receptor. However, γδ T cell adoptive cell therapy depends on ex vivo expansion to manufacture sufficient cell product numbers, which remains challenging and limited by inter-donor variability. In the current study, we characterize the differences in expansion of γδ T cells from various donors that expand (EX) and donors that fail to expand, i.e., non-expanders (NE). Further, we demonstrate that IL-21 can be used to increase the expansion potential of NE. In order to reduce the risk of graft vs. host disease (GVHD) induced by an allogeneic T cell product, αß T cell depletions must be considered due to the potential for HLA mismatch. Typically, αß T cell depletions are performed at the end of expansion, prior to infusion. We show that γδ T cell cultures can be successfully αß depleted on day 6 of expansion, providing a better environment for the γδ T cells to expand, and that the αß T cell population remains below clinically acceptable standards for T cell-depleted allogeneic stem cell products. Finally, we assess the potential for a mixed donor γδ T cell therapy and characterize the effects of cryopreservation on γδ T cells. Collectively, these studies support the development of an improved allogeneic γδ T cell product and suggest the possibility of using mixed donor γδ T cell immunotherapies.

Induction of an IL7-R(+)c-Kit(hi) myelolymphoid progenitor critically dependent on IFN-gamma signaling during acute malaria.

Although the relationship between hematopoietic stem cells and progenitor populations has been investigated extensively under steady-state conditions, the dynamic response of the hematopoietic compartment during acute infection is largely unknown. Here we show that after infection of mice with Plasmodium chabaudi, a c-Kit(hi) progenitor subset positive for interleukin 7 receptor-alpha (IL-7Ralpha) emerged that had both lymphoid and myeloid potential in vitro. After being transferred into uninfected alymphoid or malaria-infected hosts, IL-7Ralpha(+)c-Kit(hi) progenitors generated mainly myeloid cells that contributed to the clearance of infected erythrocytes in infected hosts. The generation of these infection-induced progenitors was critically dependent on interferon-gamma (IFN-gamma) signaling in hematopoietic progenitors. Thus, IFN-gamma is a key modulator of hematopoiesis and innate and adaptive immunity during acute malaria infection.

Discovery of a vaccine antigen that protects mice from Chlamydia pneumoniae infection.

Chlamydiae are atypical intracellular bacteria that infect via mucosal surfaces causing, for example, trachoma, pneumonia, cervicitis, urethritis and infertility. Existing antibiotics are only partially effective and no vaccines are available. Using surface expressed or secreted proteins previously identified by genomics and proteomics we tested five as vaccines against intranasal challenge with Chlamydia pneumoniae. One antigen, LcrE, induced CD4+ and CD8+ T cell activation, type 1 cytokine secretion and neutralising antibodies and was completely effective in eliminating infection. Such antigens are highly conserved and essential to all Chlamydial species. The discovery of an effective vaccine for Chlamydiae pneumoniae has potential wide benefits for human health.

In the absence of reactive oxygen species, T cells default to a Th1 phenotype and mediate protection against pulmonary Cryptococcus neoformans infection.

In recent years, the prevalence of invasive fungal infections has increased, attributed mostly to the rising population of immunocompromised individuals. Cryptococcus neoformans has been one of the most devastating, with an estimated 6-8% of AIDS-infected patients succumbing to Cryptococcus-associated meningitis. Reactive oxygen species (ROS) are potent antimicrobial agents but also play a significant role in regulating immune cell phenotype, but cause immunopathology when produced in excess. We now show that mice lacking phagocyte NADPH oxidase have heightened macrophage and Th1 responses and improved pathogen containment within pulmonary granulomatous lesions. Consequently, dissemination of this fungus to the brain is diminished, an effect that is independent of IL-12. Similar results are described using the metalloporphyrin antioxidant manganese(III) tetrakis(N-ethyl pyridinium-2-yl)porphyrin, which also promoted a protective Th1 response and reduced dissemination to the brain. These findings are in sharp contrast to the protective potential of ROS against other fungal pathogens, and highlight the pivotal role that ROS can fulfill in shaping the profile of the host's immune response.

A critical role for ICOS co-stimulation in immune containment of pulmonary influenza virus infection.

Lung pathology observed during influenza infection is due to direct damage resulting from viral replication and bystander damage caused by overly exuberant antiviral immune mechanisms. In the absence of universally effective vaccines and antiviral therapies, knowledge of the cellular components required for immune containment of influenza is essential. ICOS is a late co-stimulatory molecule expressed by T cells 12-24 h after activation. We show for the first time that inhibition of ICOS with a monoclonal antibody reduces pulmonary T cell inflammation and associated cytokine expression. Surprisingly however, this reduction in T cells was not accompanied by an alleviation of weight loss and illness. Furthermore, lung viral titres were elevated following anti-ICOS treatment, suggesting that the beneficial outcome of reducing T cell pathology was masked by enhanced virus-induced damage and innate inflammation. This study demonstrates the delicate balance that exists between pathogen burden and pulmonary T cell inflammation during influenza infection and highlights the critical role of ICOS in this response.

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.

An absence of reactive oxygen species improves the resolution of lung influenza infection.

Three influenza virus pandemics occurred in the last century, in 1918 killing 40-50 million people. In the absence of strain-specific vaccines, with potential resistance to antivirals and the threat of an imminent pandemic, strategies that alleviate symptoms are a priority. Reactive oxygen species are potent antimicrobial agents but cause immunopathology when produced in excess. Mice lacking a functional phagocyte NADPH oxidase (Cybb tm1 mice) or treated with the metalloporphyrin antioxidant manganese (III) tetrakis (N-ethyl pyridinium-2-yl) porpyhrin (MnTE-2-PyP) show heightened inflammatory infiltrates in their airways in response to pulmonary influenza infection, with augmented macrophage populations and a Th1-skewed T cell infiltrate. Underlying this exuberant macrophage response was a significant reduction in apoptosis and down-regulation of the myeloid inhibitory molecule CD200. Both, Cybb tm1 and MnTE-2-PyP-treated mice exhibited a reduced influenza titer in the lung parenchyma. Inflammatory infiltrate into the lung parenchyma was markedly reduced and lung function significantly improved. Manipulation of the homeostatic control of myeloid cells by inflammatory mediators therefore represents a novel therapeutic strategy in the treatment of influenza virus infection.

Mycobacterial mutants with defective control of phagosomal acidification.

The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.

TGF-beta prevents eosinophilic lung disease but impairs pathogen clearance.

Respiratory infections are the third leading cause of death worldwide. Complications arise directly as a consequence of pathogen replication or indirectly due to aberrant or excessive immune responses. In the following report, we evaluate the efficacy, in a murine model, of nasally delivered DNA encoding TGF-beta1 to suppress immunopathology in response to a variety of infectious agents. A single nasal administration suppressed lymphocyte responses to Cryptococcus neoformans, influenza virus and respiratory syncytial virus. The suppression did not depend on the phenotype of the responding T cell, since both Th1 and Th2 responses were affected. During Th2-inducing infection, pulmonary eosinophilic responses were significantly suppressed. In all cases, however, suppressed immunity correlated with increased susceptibility to infection. We conclude that nasal TGF-beta treatment could be used to prevent pulmonary, pathogen-driven eosinophilic disease, although anti-pathogen strategies will need to be administered concordantly.

Stimulation via Toll-like receptor 9 reduces Cryptococcus neoformans-induced pulmonary inflammation in an IL-12-dependent manner.

Cytosine-phosphate-guanosine-containing oligodeoxynucleotides (CpG ODN) are important vaccine adjuvants that promote Th1-type immune responses. Cryptococcus neoformans is a serious human pathogen that replicates in the lung but may disseminate systemically leading to meningitis, particularly in immunocompromised individuals. Immunization of susceptible C57BL/6 mice with CpG ODN deviates the immune response from a Th2- toward a Th1-type response following infection with C. neoformans. CpG also induces IL-12, TNF, MCP-1 and macrophage nitric oxide production. CD4(+) and CD8(+) T cells producing IFN-gamma increase in frequency, while those producing IL-5 decrease. More importantly, pulmonary eosinophilia is significantly reduced, an effect that depends on IL-12 and CD8(+) T cells but not NK cells. CpG treatment also reduces the burden of C. neoformans in the lung, an effect that is IL-12-, NK cell- and T cell-independent and probably reflects a direct effect of CpG on pathogen opsonization or an enhancement of macrophage antimicrobial activity. An equivalent beneficial effect is also observed when CpG ODN treatment is delivered during established cryptococcal disease. This is the first study documenting that promotion of lung TLR9 signaling using synthetic agonists enhances host defense. Activation of innate immunity has clear therapeutic potential and may even be beneficial in patients with acquired immune deficiency.

Innate imprinting by the modified heat-labile toxin of Escherichia coli (LTK63) provides generic protection against lung infectious disease.

In a healthy individual, the lung contains few lymphoid cells. However, amplified immune responses, as exemplified during lung infection, can cause extensive tissue damage. We have previously demonstrated that one lung infection modulates the immunopathological outcome to a subsequent unrelated pathogen. Mimicking heterologous immunity may provide a means of enhancing both innate and acquired immunity. We now show that prior lung administration of a modified heat-labile toxin from Escherichia coli (LTK63) enhances immunity to respiratory syncytial virus, influenza virus, and the fungus Cryptococcus neoformans. Treatment with LTK63 decreased lung inflammation and tissue damage and improved the ability to resolve the infection. APCs expressing the activation markers MHC class II, CD80, and CD40 increased in number in the lung. LTK63 treatment increased the pathogen-specific IgA response in the nasal mucosa and simultaneously decreased inflammatory cytokine production (IFN-gamma and TNF-alpha) after infection. The number of activated CD8(+)CD44(+) T cells and the respiratory syncytial virus- or influenza-specific CD8-proliferative responses increased, although the total inflammatory infiltrate was reduced. LTK63 treatment matured lung APCs (LTK63 prevented efficient presentation of whole OVA to DO11.10 cells, whereas OVA peptide presentation was unaffected), enhanced immunity in both a Th1 and Th2 environment, was long lasting, and was not pathogen or host strain specific; the protective effects were partially independent of T and B cells. Innate imprinting by toxin-based immunotherapeutics may provide generic protection against infectious disease in the lung, without the need for coadministered pathogen-specific Ag.

Mucosal vaccination against serogroup B meningococci: induction of bactericidal antibodies and cellular immunity following intranasal immunization with NadA of Neisseria meningitidis and mutants of Escherichia coli heat-labile enterotoxin.

Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

A critical role for OX40 in T cell-mediated immunopathology during lung viral infection.

Respiratory infections are the third leading cause of death worldwide. Illness is caused by pathogen replication and disruption of airway homeostasis by excessive expansion of cell numbers. One strategy to prevent lung immune-mediated damage involves reducing the cellular burden. To date, antiinflammatory strategies have affected both antigen-specific and naive immune repertoires. Here we report a novel form of immune intervention that specifically targets recently activated T cells alone. OX40 (CD134) is absent on naive T cells but up-regulated 1-2 d after antigen activation. OX40-immunoglobulin fusion proteins block the interaction of OX40 with its ligand on antigen-presenting cells and eliminate weight loss and cachexia without preventing virus clearance. Reduced proliferation and enhanced apoptosis of lung cells accompanied the improved clinical phenotype. Manipulation of this late costimulatory pathway has clear therapeutic potential for the treatment of dysregulated lung immune responses.

OX40 ligation on activated T cells enhances the control of Cryptococcus neoformans and reduces pulmonary eosinophilia.

Pulmonary eosinophilia induced in C57BL/6 mice after Cryptococcus neoformans infection is driven by CD4(+) Th2 cells. The immunological mechanisms that protect against eosinophilia are not fully understood. Interaction of OX40 (CD134) and its ligand, OX40L, has been implicated in T cell activation and cell migration. Unlike CD28, OX40 is only expressed on T cells 1-2 days after Ag activation. Manipulation of this pathway would therefore target recently activated T cells, leaving the naive repertoire unaffected. In this study, we show that engagement of OX40 by an OX40L:Ig fusion protein drives IFN-gamma production by CD4(+) T cells and reduces eosinophilia and C. neoformans burden in the lung. Using gene-depleted mice, we show that reduction of eosinophilia and pathogen burden requires IL-12 and/or IFN-gamma. C. neoformans infection itself only partially induces OX40L expression by APCs. Provision of exogenous OX40L reveals a critical role of this pathway in the prevention of C. neoformans-induced eosinophilia.