On-going Mechanical Damage from Mastication Drives Homeostatic Th17 Cell Responses at the Oral Barrier.

Immuno-surveillance networks operating at barrier sites are tuned by local tissue cues to ensure effective immunity. Site-specific commensal bacteria provide key signals ensuring host defense in the skin and gut. However, how the oral microbiome and tissue-specific signals balance immunity and regulation at the gingiva, a key oral barrier, remains minimally explored. In contrast to the skin and gut, we demonstrate that gingiva-resident T helper 17 (Th17) cells developed via a commensal colonization-independent mechanism. Accumulation of Th17 cells at the gingiva was driven in response to the physiological barrier damage that occurs during mastication. Physiological mechanical damage, via induction of interleukin 6 (IL-6) from epithelial cells, tailored effector T cell function, promoting increases in gingival Th17 cell numbers. These data highlight that diverse tissue-specific mechanisms govern education of Th17 cell responses and demonstrate that mechanical damage helps define the immune tone of this important oral barrier.

Amphiregulin-producing γδ T cells are vital for safeguarding oral barrier immune homeostasis.

γδ T cells are enriched at barrier sites such as the gut, skin, and lung, where their roles in maintaining barrier integrity are well established. However, how these cells contribute to homeostasis at the gingiva, a key oral barrier and site of the common chronic inflammatory disease periodontitis, has not been explored. Here we demonstrate that the gingiva is policed by γδ T cells with a T cell receptor (TCR) repertoire that diversifies during development. Gingival γδ T cells accumulated rapidly after birth in response to barrier damage, and strikingly, their absence resulted in enhanced pathology in murine models of the oral inflammatory disease periodontitis. Alterations in bacterial communities could not account for the increased disease severity seen in γδ T cell-deficient mice. Instead, gingival γδ T cells produced the wound healing associated cytokine amphiregulin, administration of which rescued the elevated oral pathology of tcrδ-/- mice. Collectively, our results identify γδ T cells as critical constituents of the immuno-surveillance network that safeguard gingival tissue homeostasis.

Progression of carcinogen-induced fibrosarcomas is associated with the accumulation of naïve CD4+ T cells via blood vessels and lymphatics.

The tumor microenvironment comprises newly formed blood and lymphatic vessels which shape the influx, retention and departure of lymphocytes within the tumor mass. Thus, by influencing the intratumoral composition of lymphocytes, these vessels affect the manner in which the adaptive immune system responds to the tumor, either promoting or impairing effective antitumor immunity. In our study, we utilized a mouse model of carcinogen-induced fibrosarcoma to examine the composition of tumor-infiltrating lymphocytes during tumor progression. In particular, we sought to determine whether CD4(+) Foxp3(+) regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor-driven immunosuppression. This was not the case as the proportion of Tregs and effector CD4(+) T cells actually declined within the tumor owing to the unexpected accumulation of naïve T cells. However, we found no evidence for antigen-driven migration of these T cells or for their participation in an antitumor immune response. Our data support the notion that lymphocytes can enter tumors via aberrantly formed blood and lymphatic vessels. Such findings suggest that targeting both the tumor vasculature and lymphatics will alter the balance of lymphocyte subpopulations that enter the tumor mass. A consideration of this aspect of tumor immunology may be critical to the success of solid cancer immunotherapies.

Th17-to-Tfh plasticity during periodontitis limits disease pathology.

Th17 cell plasticity is crucial for development of autoinflammatory disease pathology. Periodontitis is a prevalent inflammatory disease where Th17 cells mediate key pathological roles, yet whether they exhibit any functional plasticity remains unexplored. We found that during periodontitis, gingival IL-17 fate-mapped T cells still predominantly produce IL-17A, with little diversification of cytokine production. However, plasticity of IL-17 fate-mapped cells did occur during periodontitis, but in the gingiva draining lymph node. Here, some Th17 cells acquired features of Tfh cells, a functional plasticity that was dependent on IL-6. Notably, Th17-to-Tfh diversification was important to limit periodontitis pathology. Preventing Th17-to-Tfh plasticity resulted in elevated periodontal bone loss that was not simply due to increased proportions of conventional Th17 cells. Instead, loss of Th17-to-Tfh cells resulted in reduced IgG levels within the oral cavity and a failure to restrict the biomass of the oral commensal community. Thus, our data identify a novel protective function for a subset of otherwise pathogenic Th17 cells during periodontitis.

Kidney resident macrophages have distinct subsets and multifunctional roles.

BACKGROUND: The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. METHODS: Flow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5-/- (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. RESULTS: In wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5-/- (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. CONCLUSIONS: We identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

Avidity of influenza-specific memory CD8+ T-cell populations decays over time compromising antiviral immunity.

Decline of cell-mediated immunity is often attributed to decaying T-cell numbers and their distribution in peripheral organs. This study examined the hypothesis that qualitative as well as quantitative changes contribute to the declining efficacy of CD8(+) T-cell memory. Using a model of influenza virus infection, where loss of protective CD8(+) T-cell immunity was observed 6 months postinfection, we found no decline in antigen-specific T-cell numbers or migration to the site of secondary infection. There was, however, a large reduction in antigen-specific CD8(+) T-cell degranulation, cytokine secretion, and polyfunctionality. A profound loss of high-avidity T cells over time indicated that failure to confer protective immunity resulted from the inferior functional capacity of remaining low avidity cells. These data imply that high-avidity central memory T cells wane with declining antigen levels, leaving lower avidity T cells with reduced functional capabilities.

Hematopoietic stem and progenitor cells are present in healthy gingiva tissue.

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.

Tissue-resident macrophages in the intestine are long lived and defined by Tim-4 and CD4 expression.

A defining feature of resident gut macrophages is their high replenishment rate from blood monocytes attributed to tonic commensal stimulation of this site. In contrast, almost all other tissues contain locally maintained macrophage populations, which coexist with monocyte-replenished cells at homeostasis. In this study, we identified three transcriptionally distinct mouse gut macrophage subsets that segregate based on expression of Tim-4 and CD4. Challenging current understanding, Tim-4+CD4+ gut macrophages were found to be locally maintained, while Tim-4-CD4+ macrophages had a slow turnover from blood monocytes; indeed, Tim-4-CD4- macrophages were the only subset with the high monocyte-replenishment rate currently attributed to gut macrophages. Moreover, all macrophage subpopulations required live microbiota to sustain their numbers, not only those derived from blood monocytes. These findings oppose the prevailing paradigm that all macrophages in the adult mouse gut rapidly turn over from monocytes in a microbiome-dependent manner; instead, these findings supplant it with a model of ontogenetic diversity where locally maintained subsets coexist with rapidly replaced monocyte-derived populations.

Low-Dose Cyclophosphamide Induces Antitumor T-Cell Responses, which Associate with Survival in Metastatic Colorectal Cancer.

Purpose: Anticancer T-cell responses can control tumors, but immunosuppressive mechanisms in vivo prevent their function. The role of regulatory T cells (Tregs) in metastatic colorectal cancer is unclear. We have previously shown depletion of Tregs enhances colorectal cancer-specific effector T-cell responses. Low-dose cyclophosphamide targets Tregs in animal models and some human studies; however, the effect of cyclophosphamide in metastatic colorectal cancer is unknown.Experimental Design: Fifty-five patients with metastatic colorectal cancer were enrolled in a phase I/II trial and randomly assigned to receive 2-week-long courses of low-dose (50 mg twice a day) cyclophosphamide or not. The absolute number, phenotype, and antitumor function of peripheral blood-derived lymphocyte subsets were monitored throughout treatment, as well as during 18-month follow-up.Results: Initially, cyclophosphamide reduced proliferation in all lymphocyte subsets; however, a rapid mobilization of effector T cells overcame this decrease, leading to increased absolute T-cell numbers. In contrast, a reduction in proportional and absolute Treg, B-cell, and NK-cell numbers occurred. The expansion and subsequent activation of effector T cells was focused on tumor-specific T cells, producing both granzyme B and IFNγ. Cyclophosphamide-treated patients demonstrating the most enhanced IFNγ+ tumor-specific T-cell responses exhibited a significant delay in tumor progression [HR = 0.29; 95% confidence interval (CI), 0.12-0.69; P = 0.0047), compared with nonresponders and no-treatment controls.Conclusions: Cyclophosphamide-induced Treg depletion is mirrored by a striking boost in antitumor immunity. This study provides the first direct evidence of the benefit of naturally primed T cells in patients with metastatic colorectal cancer. Our results also support the concept that nonmutated self-antigens may act as useful targets for immunotherapies. Clin Cancer Res; 23(22); 6771-80. ©2017 AACR.

Effect of Modified Vaccinia Ankara-5T4 and Low-Dose Cyclophosphamide on Antitumor Immunity in Metastatic Colorectal Cancer: A Randomized Clinical Trial.

IMPORTANCE: The success of immunotherapy with checkpoint inhibitors is not replicated in most cases of colorectal cancer; therefore, different strategies are urgently required. The oncofetal antigen 5T4 is expressed in more than 90% of cases of metastatic colorectal cancer (mCRC). Preliminary data using modified vaccinia Ankara-5T4 (MVA-5T4) in mCRC demonstrated that it safely induced serologic and T-cell responses. OBJECTIVE: To determine whether antitumor immunity in mCRC could be increased using MVA-5T4, metronomic low-dose cyclophosphamide, or a combination of both treatments. DESIGN, SETTING, AND PARTICIPANTS: In this randomized clinical trial, 55 patients with inoperable mCRC and prior stable disease after standard chemotherapy were enrolled at a single center and randomized to watch and wait (n = 9), cyclophosphamide treatment only (n = 9), MVA-5T4 only (n = 19), and a combination of MVA-5T4 and cyclophosphamide (n = 18). Patients were enrolled and treated from July 9, 2012, through February 8, 2016, and follow-up was completed on December 13, 2016. Data were analyzed based on intention to treat. INTERVENTIONS: Patients randomized to a cyclophosphamide group received 50 mg twice daily on treatment days 1 to 7 and 15 to 21. Patients randomized to a MVA-5T4 group received an intramuscular injection at a dose of 1 × 109 50% tissue culture infectious dose on treatment days 22, 36, 50, 64, 78, and 106. MAIN OUTCOMES AND MEASURES: The predefined primary end point was the magnitude of anti-5T4 immune responses (5T4-specific T-cell and antibody levels) generated at treatment week 7. Secondary end points included analysis of the kinetics of anti-5T4 responses, progression-free survival (PFS), and overall survival (OS). RESULTS: Fifty-two patients (38 men and 14 women; mean [SD] age, 64.2 [10.1] years) were included in the study analysis. The 5T4-specific antibody immune responses were significantly increased in the MVA-5T4 (83.41 [36.09] relative units [RU]; P = .02) and combination treatment (65.81 [16.68] RU; P = .002) groups compared with no treatment (20.09 [7.20] RU). Cyclophosphamide depleted regulatory T cells in 24 of 27 patients receiving MVA-5T4, independently prolonging PFS (5.0 vs 2.5 months; hazard ratio [HR], 0.48; 95% CI, 0.21-1.11; P = .09). MVA-5T4 doubled baseline anti-5T4 responses in 16 of 35 patients, resulting in significantly prolonged PFS (5.6 vs 2.4 months; HR, 0.21; 95% CI, 0.09-0.47; P < .001) and OS (20.0 vs 10.3 months; HR, 0.32; 95% CI, 0.14-0.74; P = .008). No grade 3 or 4 adverse events were observed. CONCLUSIONS AND RELEVANCE: This initial randomized clinical immunotherapy study demonstrates a significant survival benefit in mCRC. Prior depletion of regulatory T cells by cyclophosphamide did not increase immune responses generated by MVA-5T4 vaccination; however, cyclophosphamide and MVA-5T4 each independently induced beneficial antitumor immune responses, resulting in prolonged survival without toxic effects. Larger clinical trials are planned to further validate these data. TRIAL REGISTRATION: isrctn.org Identifier: ISRCTN54669986.

Eliminating roles for T-bet and IL-2 but revealing superior activation and proliferation as mechanisms underpinning dominance of regulatory T cells in tumors.

Foxp3(+) regulatory T cells (Tregs) are often highly enriched within the tumor-infiltrating T cell pool. Using a well-characterised model of carcinogen-induced fibrosarcomas we show that the enriched tumor-infiltrating Treg population comprises largely of CXCR3(+) T-bet(+) 'TH1-like' Tregs which are thymus-derived Helios(+) cells. Whilst IL-2 maintains homeostatic ratios of Tregs in lymphoid organs, we found that the perturbation in Treg frequencies in tumors is IL-2 independent. Moreover, we show that the TH1 phenotype of tumor-infiltrating Tregs is dispensable for their ability to influence tumor progression. We did however find that unlike Tconvs, the majority of intra-tumoral Tregs express the activation markers CD69, CD25, ICOS, CD103 and CTLA4 and are significantly more proliferative than Tconvs. Moreover, we have found that CD69(+) Tregs are more suppressive than their CD69- counterparts. Collectively, these data indicate superior activation of Tregs in the tumor microenvironment, promoting their suppressive ability and selective proliferation at this site.

Escalating regulation of 5T4-specific IFN-γ+ CD4+ T cells distinguishes colorectal cancer patients from healthy controls and provides a target for in vivo therapy.

The relationship between the adaptive CD4+ T cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed on many human carcinomas, including colorectal cancer (CRC) cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4+ T cells in a proportion of CRC patients, and we extended this study to examine whether the quality or quantity of the T cell response reflects tumor stage. An overlapping peptide library spanning 5T4 was used as a target to enumerate cognate IFN-γ+CD4+ T-cells (measured as spot forming cells [SFC]/105 cultured T cells) in peripheral blood-derived lymphocytes following a 12-day in vitro culture period comparing patients pre-operatively (n = 27) to healthy controls (n = 17). Robust 5T4-specific T cell responses were present in 100% of healthy donors. There was a steady loss of T cell responses with advancing tumors with a significant negative correlation from stage I to III (P = 0.008). The predictability of the decline meant < 200 SFC/105 was only found in subjects with stage III CRC. The mechanism of loss of T cell response is independent of HLA-DR type or patient age, but does correspond to increases in Foxp3+ regulatory T cells (Tregs). Using low-dose cyclophosphamide to reduce the proportion of Tregs in vivo resulted in increased anti-5T4 T cell responses in CRC patients. The selective loss of 5T4-specific IFN-γ+CD4+ T cell responses implies a link between tumor stage and antitumor Th1 effector function; depleting Tregs can enhance such responses.

T-cell trafficking facilitated by high endothelial venules is required for tumor control after regulatory T-cell depletion.

The evolution of immune blockades in tumors limits successful antitumor immunity, but the mechanisms underlying this process are not fully understood. Depletion of regulatory T cells (Treg), a T-cell subset that dampens excessive inflammatory and autoreactive responses, can allow activation of tumor-specific T cells. However, cancer immunotherapy studies have shown that a persistent failure of activated lymphocytes to infiltrate tumors remains a fundamental problem. In evaluating this issue, we found that despite an increase in T-cell activation and proliferation following Treg depletion, there was no significant association with tumor growth rate. In contrast, there was a highly significant association between low tumor growth rate and the extent of T-cell infiltration. Further analyses revealed a total concordance between low tumor growth rate, high T-cell infiltration, and the presence of high endothelial venules (HEV). HEV are blood vessels normally found in secondary lymphoid tissue where they are specialized for lymphocyte recruitment. Thus, our findings suggest that Treg depletion may promote HEV neogenesis, facilitating increased lymphocyte infiltration and destruction of the tumor tissue. These findings are important as they point to a hitherto unidentified role of Tregs, the manipulation of which may refine strategies for more effective cancer immunotherapy.