Shayna Street

Suppression of Lymphoma and Epithelial Malignancies Effected by Interferon γ

The immunosurveillance of transformed cells by the immune system remains one of the most controversial and poorly understood areas of immunity. Gene-targeted mice have greatly aided our understanding of the key effector molecules in tumor immunity. Herein, we describe spontaneous tumor development in gene-targeted mice lacking interferon (IFN)-γ and/or perforin (pfp), or the immunoregulatory cytokines, interleukin (IL)-12, IL-18, and tumor necrosis factor (TNF). Both IFN-γ and pfp were critical for suppression of lymphomagenesis, however the level of protection afforded by IFN-γ was strain specific. Lymphomas arising in IFN-γ-deficient mice were very nonimmunogenic compared with those derived from pfp-deficient mice, suggesting a comparatively weaker immunoselection pressure by IFN-γ. Single loss of IL-12, IL-18, or TNF was not sufficient for spontaneous tumor development. A significant incidence of late onset adenocarcinoma observed in both IFN-γ– and pfp-deficient mice indicated that some epithelial tissues were also subject to immunosurveillance.

The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent.

IL-12 has been demonstrated to have potent anti-tumor activities in a variety of mouse tumor models, but the relative roles of NK, NKT, and T cells and their effector mechanisms in these responses have not been fully addressed. Using a spectrum of gene-targeted or Ab-treated mice we have shown that for any particular tumor model the effector mechanisms downstream of IL-12 often mimic the natural immune response to that tumor. For example, metastasis of the MHC class I-deficient lymphoma, EL4-S3, was strictly controlled by NK cells using perforin either naturally or following therapy with high-dose IL-12. Intriguingly, in B16F10 and RM-1 tumor models both NK and NKT cells contribute to natural protection from tumor metastasis. In these models, a lower dose of IL-12 or delayed administration of IL-12 dictated a greater relative role of NKT cells in immune protection from tumor metastasis. Overall, both NK and NKT cells can contribute to natural and IL-12-induced immunity against tumors, and the relative role of each population is tumor and therapy dependent.

Innate Immune Surveillance of Spontaneous B Cell Lymphomas by Natural Killer Cells and γδ T Cells

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking β-2 microglobulin (β2m; and thus MHC class I, CD1d, and CD16) and/or perforin, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perforin gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked β2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/β2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perforin, but not IFN-γ, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1+ and γδTCR+ T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and γδTCR+ T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.

Enrichment of circulating interleukin‐17–secreting interleukin‐23 receptor–positive γ/δ T cells in patients with active ankylosing spondylitis

OBJECTIVE Ankylosing spondylitis (AS) is a common inflammatory arthritis affecting primarily the axial skeleton. IL23R is genetically associated with AS. This study was undertaken to investigate and characterize the role of interleukin-23 (IL-23) signaling in AS pathogenesis. METHODS The study population consisted of patients with active AS (n = 17), patients with psoriatic arthritis (n = 8), patients with rheumatoid arthritis, (n = 9), and healthy subjects (n = 20). IL-23 receptor (IL-23R) expression in T cells was determined in each subject group, and expression levels were compared. RESULTS The proportion of IL-23R-expressing T cells in the periphery was 2-fold higher in AS patients than in healthy controls, specifically driven by a 3-fold increase in IL-23R-positive γ/δ T cells in AS patients. The proportions of CD4+ and CD8+ cells that were positive for IL-17 were unchanged. This increased IL-23R expression on γ/δ T cells was also associated with enhanced IL-17 secretion, with no observable IL-17 production from IL-23R-negative γ/δ T cells in AS patients. Furthermore, γ/δ T cells from AS patients were heavily skewed toward IL-17 production in response to stimulation with IL-23 and/or anti-CD3/CD28. CONCLUSION Recently, mouse models have shown IL-17-secreting γ/δ T cells to be pathogenic in infection and autoimmunity. Our data provide the first description of a potentially pathogenic role of these cells in a human autoimmune disease. Since IL-23 is a maturation and growth factor for IL-17-producing cells, increased IL-23R expression may regulate the function of this putative pathogenic γ/δ T cell population.

Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients.

Citrullinated peptide-exposed DCs induced immune regulatory effects in HLA risk genotype–positive RA patients. Immunotherapy out of joint Autoantibodies to anti–citrullinated peptides (ACPA) are found in most patients with rheumatoid arthritis (RA), especially those with HLA-DRB1 risk alleles. Benham et al. report a first-in-human phase 1 trial of a single injection of autologous dendritic cells modified with an NF-κB inhibitor that have been exposed to four citrullinated peptide antigens. They find that HLA risk genotype–positive RA patients had reduced numbers of effector T cells and decreased production of proinflammatory cytokines compared with untreated RA patient controls. The therapy was safe and did not induce disease flares. These data support larger studies of antigen-specific immunotherapy for RA. In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritis in an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti–citrullinated peptide autoantibodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated with HLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCs modified with a nuclear factor κB (NF-κB) inhibitor exposed to four citrullinated peptide antigens, designated “Rheumavax,” in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered once intradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype–positive RA patients with citrullinated peptide–specific autoimmunity. Sixteen RA patients served as controls. Rheumavax was well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a reduction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15 (IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin447–455–Cit450 relative to controls. Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreased within 1 month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety and biological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides, and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen immunomodulatory therapy in RA.

Cutting edge: granzymes A and B are not essential for perforin-mediated tumor rejection.

Controversy still exists regarding the biological function of granzyme serine proteases released with perforin from the cytotoxic granules of NK cells and CTLs. In particular, it is not clear whether the major granzymes, A and B, play an essential role in tumor rejection mediated by the perforin pathway. We have now examined the relative importance of perforin and granzyme A and B clusters in five different tumor models that stringently distinguish their importance. We conclude that granzyme A and B clusters are not essential for CTL- and NK cell-mediated rejection of spontaneous and experimental tumors, raising the likelihood that either perforin alone or in combination with an additional granzyme or granule component(s) mediates cytotoxicity of tumor cells in vivo.

T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles

IntroductionAnti-citrullinated peptide antibodies are found in rheumatoid arthritis (RA) patients with HLA-DRβ chains encoding the shared epitope (SE) sequence. Citrullination increases self-antigen immunogenicity, through increased binding affinity to SE-containing HLA-DR molecules. To characterise T-cell autoreactivity towards citrullinated self-epitopes, we profiled responses of SE+ healthy controls and RA patients to citrullinated and unmodified epitopes of four autoantigens.MethodsWe compared T-cell proliferative and cytokine responses to citrullinated and native type II collagen 1,237 to 1,249, vimentin 66 to 78, aggrecan 84 to 103 and fibrinogen 79 to 91 in six SE+ healthy controls and in 21 RA patients with varying disease duration. Cytokine-producing cells were stained after incubation with peptide in the presence of Brefeldin-A.ResultsAlthough proliferative responses were low, IL-6, IL-17 and TNF were secreted by CD4+ T cells of SE+ RA patients and healthy controls, as well as IFNγ and IL-10 secreted by RA patients, in response to citrullinated peptides. Of the epitopes tested, citrullinated aggrecan was most immunogenic. Patients with early RA were more likely to produce IL-6 in response to no epitope or to citrullinated aggrecan, while patients with longstanding RA were more likely to produce IL-6 to more than one epitope. Cytokine-producing CD4+ T cells included the CD45RO+ and CD45RO- and the CD28+ and CD28- subsets in RA patients.ConclusionProinflammatory cytokines were produced by CD4+ T cells in SE+ individuals in response to citrullinated self-epitopes, of which citrullinated aggrecan was most immunogenic. Our data suggest that the T-cell response to citrullinated self-epitopes matures and diversifies with development of RA.

Antigen-Specific Suppression of Inflammatory Arthritis Using Liposomes

Existing therapies for rheumatoid arthritis and other autoimmune diseases are not Ag specific, which increases the likelihood of systemic toxicity. We show that egg phosphatidylcholine liposomes loaded with Ag (OVA or methylated BSA) and a lipophilic NF-κB inhibitor (curcumin, quercetin, or Bay11-7082) suppress preexisting immune responses in an Ag-specific manner. We injected loaded liposomes into mice primed with Ag or into mice suffering from Ag-induced inflammatory arthritis. The liposomes targeted APCs in situ, suppressing the cells’ responsiveness to NF-κB and inducing Ag-specific FoxP3+ regulatory T cells. This regulatory mechanism suppressed effector T cell responses and the clinical signs of full-blown Ag-induced arthritis. Thus, liposomes encapsulate Ags and NF-κB inhibitors stably and efficiently and could be readily adapted to deliver Ags and inhibitors for Ag-specific suppression of other autoimmune and allergic diseases.

A Physiological Function of Inflammation-Associated SerpinB2 Is Regulation of Adaptive Immunity

SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB2−/− mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB2−/− mice. After immunization with OVA in CFA, SerpinB2−/− mice made ≈6-fold more IgG2c and generated ≈2.5-fold more OVA-specific IFN-γ–secreting T cells than SerpinB2+/+ littermate controls. In SerpinB2+/+ mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB2−/− macrophages promoted greater IFN-γ secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN-γ or cultured with wild-type T cells in vitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB2−/− myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 polymorphisms or dysregulated SerpinB2 expression.

Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer

The major limiting factor in the successful application of adjuvant therapy for metastatic disease is the lack of adjuvant specificity that leads to severe side effects. Reasoning that T cells of the immune system are highly specific, we generated tumor-specific T cells by genetic modification of mouse primary T cells with a chimeric receptor reactive with the human breast cancer-associated Ag erbB-2. These T cells killed breast cancer cells and secreted IFN-γ in an Ag-specific manner in vitro. We investigated their use against metastatic breast cancer in mice in an adjuvant setting, and compared their effectiveness with the commonly applied adjuvants doxorubicin, 5-fluorouracil, and herceptin. Mice were inoculated orthotopically with the human erbB-2-expressing spontaneously metastatic mouse breast cancer 4T1.2 in mammary tissue, and the primary tumor was surgically removed 8 days later. Significant metastatic disease was demonstrated in lung and liver at the time of surgery on day 8 with increased tumor burden at later time points. T cell adjuvant treatment of day 8 metastatic disease resulted in dramatic increases in survival of mice, and this survival was significantly greater than that afforded by either doxorubicin, 5-fluorouracil, or herceptin.