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Background: T lymphocytes are involved in the pathogenesis of nonallergic asthma. The objective of this study was to characterize the subset distribution and pattern of chemokine receptor expression in circulating T lymphocyte subsets from nonallergic asthma patients. Methods: Forty stable nonallergic asthma patients and 16 sex- and age-matched healthy donors were studied. Twelve patients did not receive inhaled steroids (untreated patients), 16 received 50-500 μg b.i.d. of inhaled fluticasone propionate (FP) (standard-dose patients), and 12 received over 500 μg b.i.d. of inhaled FP (high-dose patients) for at least 12 months prior to the beginning of this study and were clinically well controlled. Flow cytometry was performed using a panel of monoclonal antibodies (4 colors). Results: Nonallergic asthma patients treated with high doses of inhaled FP showed a significant reduction in the percentages of CD3+ T lymphocytes compared to healthy controls. Untreated patients showed a significant increase in CCR6 expression in CD8+CD25+ and CD8+CD25+bright T cells compared to healthy controls. The results were similar for CXCR3 and CCR5 expression. In patients treated with standard doses of FP, CCR5 expression was significantly increased in CD3+ T lymphocytes relative to healthy controls. Conclusions: The different groups of clinically stable nonallergic asthmatic patients showed distinct patterns of alterations in subset distribution as well as CCR6, CXCR3, and CCR5 expression on circulating T lymphocytes.

Background CD8 + T cell trafficking to the tumor site is essential for effective colorectal cancer (CRC) immunotherapy. However, the mechanism underlying CD8 + T cell infiltration in colorectal tumor tissues is not fully understood. In the present study, we investigated CD8 + T cell infiltration in CRC tissues and the role of chemokine–chemokine receptor signaling in regulation of T cell recruitment. Methods We screened chemokines and cytokines in healthy donor and CRC tissues from early- and advanced-stage patients using multiplex assays and PCR screening. We also utilized transcription factor activation profiling arrays and established a xenograft mouse model. Results Compared with tumor tissues of early-stage CRC patients, CD8 + T cell density was lower in advanced-stage tumor tissues. PCR screening showed that CXCL10 levels were significantly increased in advanced-stage tumor tissues. CXCR3 (the receptor of CXCL10) expression on CD8 + T cells was lower in the peripheral blood of advanced-stage patients. The migratory ability of CD8 + T cells to CXCL10 depended on CXCR3 expression. Multiplex arrays showed that IL-17A was increased in advanced-stage patient sera, which markedly downregulated CXCR3 expression via activating STAT3 signaling and reduced CD8 + T cell migration. Similar results were found after CD8 + T cells were treated with Th17 cell supernatant. Adding anti-IL-17A or the STAT3 inhibitor, Stattic, rescued these effects in vitro and in vivo. Moreover, survival analysis showed that patients with low CD8 and CXCR3 expression and high IL-17A levels had significantly worse prognosis. Conclusions CD8 + T cell infiltration in advanced-stage tumor was systematically inhibited by Th17 cells via IL-17A/STAT3/CXCR3 axis. Our findings indicate that the T cell infiltration in the tumor microenvironment may be improved by inhibiting STAT3 signaling.

Tumor-infiltrating cytotoxic T lymphocytes (TILs), including CD8 TILs, have been associated with favorable clinical outcomes in multiple tumor types. Tumor-infiltrating CD8 T cells and major histocompatibility complex (MHC) class I expression in urothelial carcinoma (UC) have not been previously reported. Most immune responses are mediated by local cytotoxic lymphocytes (CD8 T cells), which can eradicate tumor cells by recognizing tumor-associated antigens presented by MHC class I molecules. Here we analyzed the presence of intratumoral CD8 T cells, the expression of MHC class I antigen, and the expression of the NY-ESO-1 tumor antigen in UC samples and correlated our findings with clinical outcome. Immunohistochemical staining for intratumoral CD8 T cells in tissue samples from 69 patients with UC showed that patients with advanced UC (pT2, pT3, or pT4) and higher numbers of CD8 TILs within the tumor (>=8) had better disease-free survival (P < 0.001) and overall survival (P = 0.018) than did patients with similar-staged UC and fewer intratumoral CD8 TILs. We conclude that the extent of intratumoral CD8 TILs is an important prognostic indicator in advanced UC.

In this study, we investigated the clinical relevance of CD274 (PD-L1) protein expression by tumor cells and tumor-infiltrating immune cells in colorectal cancer (CRC). To this end, 186 microsatellite instability-high (MSI-H) and 153 microsatellite stable (MSS) CRCs were subjected to immunohistochemistry (IHC) analysis for the expression of CD274 and mismatch repair proteins. CD274 expression was evaluated in tumor cells at the center (TC) and periphery (TP), and immune cells at the center (IC) and periphery (IP) of CRC. IHC slides stained for CD3 and CD8 were scanned using an Aperio ScanScope for precise calculation of tumor-infiltrating T cell density. Additionally, samples were screened for the B-Raf (BRAF)-V600E mutation using a Cobas 4800 System and IHC. In total, CD274 TC , CD274 TP , CD274 IC , and CD274 IP were observed in 43 (23.1%), 47 (25.3%), 107 (57.5%), and 102 (54.8%) of the MSI-H CRCs examined, and in three (2.0%), four (2.6%), 47 (30.7%), and 56 (36.6%) of the 153 MSS CRCs tested. Meanwhile, intratumoral heterogeneity of CD274 expression in tumor cells and immune cells was detected in 24 (12.9%) and 47 (25.3%) MSI-H CRCs, respectively. Notably, in both MSI-H and MSS CRC, CD274 IC and CD274 IP were independently associated with improved prognosis ( P  < 0.05), while BRAF mutation was associated with CD274 TP , poor differentiation, sporadic type, and hMLH1(−)/hMSH2(+)/hMSH6(+)/PMS2(−) in MSI-H CRC ( P  < 0.006). In conclusion, CD274 expression in tumor-infiltrating immune cells was an independent factor for improved prognosis in CRC patients. A deeper understanding of CD274 status may yield improved responses to future CRC immunotherapies.

The mouse CD8alpha+ DC subset excels at cross-presentation of antigen, which can elicit robust CTL responses. A receptor allowing specific antigen targeting to this subset and its equivalent in humans would therefore be useful for the induction of antitumor CTLs. Here, we have characterized a C-type lectin of the NK cell receptor group that we named DC, NK lectin group receptor-1 (DNGR-1). DNGR-1 was found to be expressed in mice at high levels by CD8+ DCs and at low levels by plasmacytoid DCs but not by other hematopoietic cells. Human DNGR-1 was also restricted in expression to a small subset of blood DCs that bear similarities to mouse CD8alpha+ DCs. The selective expression pattern and observed endocytic activity of DNGR-1 suggested that it could be used for antigen targeting to DCs. Consistent with this notion, antigen epitopes covalently coupled to an antibody specific for mouse DNGR-1 were selectively cross-presented by CD8alpha+ DCs in vivo and, when given with adjuvants, induced potent CTL responses. When the antigens corresponded to tumor-expressed peptides, treatment with the antibody conjugate and adjuvant could prevent development or mediate eradication of B16 melanoma lung pseudometastases. We conclude that DNGR-1 is a novel, highly specific marker of mouse and human DC subsets that can be exploited for CTL cross-priming and tumor therapy.

Mouse spleens contain three populations of conventional ($CD11c^{high}$) dendritic cells (DCs) that play distinct functions. The CD8⁺ DC are unique in that they can present exogenous antigens on their MHC class I molecules, a process known as cross-presentation. It is unclear whether this special ability is because only the CD8⁺ DC can capture the antigens used in cross-presentation assays, or because this is the only DC population that possesses specialized machinery for cross-presentation. To solve this important question we examined the splenic DC subsets for their ability to both present via MHC class II molecules and cross-present via MHC class I using four different forms of the model antigen ovalbumin (OVA). These forms include a cell-associated form, a soluble form, OVA expressed in bacteria, or OVA bound to latex beads. With the exception of bacterial antigen, which was poorly cross-presented by all DC, all antigenic forms were cross-presented much more efficiently by the CD8⁺ DC. This pattern could not be attributed simply to a difference in antigen capture because all DC subsets presented the antigen via MHC class II. Indeed, direct assessments of endocytosis showed that CD8⁺ and CD8⁻ DC captured comparable amounts of soluble and bead-associated antigen, yet only the CD8⁺ DC cross-presented these antigenic forms. Our results indicate that cross-presentation requires specialized machinery that is expressed by CD8⁺ DC but largely absent from CD8⁻ DC. This conclusion has important implications for the design of vaccination strategies based on antigen targeting to DC.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing intracellular enzyme of the L-kynurenine pathway, causes preneoplastic cells and tumor cells to escape the immune system by inducing immune tolerance; this mechanism might be associated with the development and progression of human malignancies. In the present study, we investigated the role of IDO in diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using IDO-knockout (KO) mice. To induce hepatocellular carcinoma (HCC), hepatic adenoma, and preneoplastic hepatocellular lesions termed foci of cellular alteration (FCA), male IDO-wild-type (WT) and IDO-KO mice with a C57BL/6J background received a single intraperitoneal injection of DEN at 2 weeks of age. The mice were sacrificed to evaluate the development of FCA and hepatocellular neoplasms. HCC overexpressed IDO and L-kynurenine compared to surrounding normal tissue in the DEN-treated IDO-WT mice. The number and cell proliferative activity of FCAs, and the incidence and multiplicity of HCC were significantly greater in the IDO-WT than in the IDO-KO mice. The expression levels of the IDO protein, of L-kynurenine, and of IFN-γ, COX-2, TNF-α, and Foxp3 mRNA were also significantly increased in the DEN-induced hepatic tumors that developed in the IDO-WT mice. The mRNA expression levels of CD8, perforin and granzyme B were markedly increased in hepatic tumors developed in IDO-KO mice. Moreover, Foxp3-positive inflammatory cells had infiltrated into the livers of DEN-treated IDO-WT mice, whereas fewer cells had infiltrated into the livers of IDO-KO mice. Induction of IDO and elevation of L-kynurenine might play a critical role in both the early and late phase of liver carcinogenesis. Our findings suggest that inhibition of IDO might offer a promising strategy for the prevention of liver cancer.

Inflammation initiates tendon healing and then normally resolves more or less completely. Unresolved inflammation might disturb the remodeling process. We hypothesized that suppression of inflammation during the early remodeling phase by systemic dexamethasone treatment can improve healing. 36 rats underwent Achilles tendon transection and were randomized to dexamethasone or saline on days 0–4 after surgery (early inflammatory phase), and euthanasia day 7. Another 54 rats received injections days 5–9 (early remodeling phase) and were euthanized day 12 for mechanical, histological and flow cytometric evaluation. Dexamethasone treatment days 0–4 reduced the cross-sectional area, peak force and stiffness by day 7 to less than half (p < 0.001 for all), while material properties (peak stress and elastic modulus) were not significantly affected. In contrast, dexamethasone treatment days 5–9 increased peak force by 39% (p = 0.002) and stiffness by 58% (p < 0.001). The cross-sectional area was reduced by 42% (p < 0.001). Peak stress and elastic modulus were more than doubled (p < 0.001 for both). Semi-quantitative histology at day 12 showed that late dexamethasone treatment improved collagen alignment, and flow cytometry revealed reduced numbers of CD8a + cytotoxic T cells in the tendon callus. These results suggest that downregulation of lingering inflammation during the early remodeling phase can improve healing.

Treg based immunotherapy is of great interest to facilitate tolerance in autoimmunity and transplantation. For clinical trials, it is essential to have a clinical grade Treg isolation protocol in accordance with Good Manufacturing Practice (GMP) guidelines. To obtain sufficient Treg for immunotherapy, subsequent ex vivo expansion might be needed. Treg were isolated from leukapheresis products by CliniMACS based GMP isolation strategies, using anti-CD25, anti-CD8 and anti-CD19 coated microbeads. CliniMACS isolation procedures led to 40-60% pure CD4(pos)CD25(high)FoxP3(pos) Treg populations that were anergic and had moderate suppressive activity. Such CliniMACS isolated Treg populations could be expanded with maintenance of suppressive function. Alloantigen stimulated expansion caused an enrichment of alloantigen-specific Treg. Depletion of unwanted CD19(pos) cells during CliniMACS Treg isolation proved necessary to prevent B-cell outgrowth during expansion. CD4(pos)CD127(pos) conventional T cells were the major contaminating cell type in CliniMACS isolated Treg populations. Depletion of CD127(pos) cells improved the purity of CD4(pos)CD25(high)FoxP3(pos) Treg in CliniMACS isolated cell populations to approximately 90%. Expanded CD127(neg) CliniMACS isolated Treg populations showed very potent suppressive capacity and high FoxP3 expression. Furthermore, our data show that cryopreservation of CliniMACS isolated Treg is feasible, but that activation after thawing is necessary to restore suppressive potential. The feasibility of Treg based therapy is widely accepted, provided that tailor-made clinical grade procedures for isolation and ex vivo cell handling are available. We here provide further support for this approach by showing that a high Treg purity can be reached, and that isolated cells can be cryopreserved and expanded successfully.

Gene targeting by homologous recombination is a powerful method to manipulate the genome precisely and could be exploited to correct genetic defects. Zinc finger nucleases are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease. Zinc finger nucleases were generated that stimulated gene targeting from half-site sequences from the human beta-globin gene and the human common gamma-chain gene. Zinc finger nucleases were also generated that stimulated gene targeting at full sites from the green fluorescent protein gene and the human CD8alpha gene. This work built on the prior zinc finger design work of others and in targeting these four genes had a 100% success rate at designing nucleases to the consensus half-site 5'-GNNGNNGNN-3' and the consensus full site 5'-NNCNNCNNCNNNNNNGNNGNNGNN-3', suggesting that zinc finger nucleases can be empirically designed to stimulate gene targeting in a large portion of the mammalian genome.