Treg Cell Population Research Articles

Abstract 1651: Profiling immune cells within the tumor microenvironment for optimal model selection for pre-clinical investigations

Abstract Infiltrating host immune cells can have a significant impact on the growth and outcome of many tumor types. Several leukocyte populations have been implicated in establishing an immune suppressive microenvironment (e.g. myeloid derived suppressor cells [MDSCs) and regulatory T cells [Treg cells]) and thus making it harder for anti-tumor immune effector cells to function (e.g. CD8 cytotoxic T cells). Immune-Mediated Therapy (IMT) is emerging as a key strategy for the treatment of cancer through by enhancing a patient's anti-tumor immune response. In order to maximise the potential of IMT strategies it is important to understand the immune status within the tumor microenvironment as well as systemically. Therefore, fully immune-competent preclinical models are valuable tools to enable the study of the immune system, both locally within the tumor and systemically, to understand the mechanism of action of IMT monotherapies and combination therapies in vivo. Consequently, we have profiled the immune status of a panel of syngeneic mouse tumor models to assist optimal model selection to aid these investigations. We developed multi-parameter flow cytometry techniques to identify specific immune subsets in both the tumor microenvironment and the periphery. These investigations revealed a number of tumor infiltrating immune cell end-points that can be used to correlate with response to IMT depending on the syngeneic model being used. For example, we observed differences in the MDSC and Treg cell populations across the models investigated, which will influence optimal model selection depending on the mechanism of action of the IMT being tested. Citation Format: Andrew Leishman, Olivia Harris, Jane Coates Ulrichsen, James Harper, Amy Popple, Marianna Papaspyridonos, Geoff Williams, Stefanie Mullins, Viia Valge-Archer, Ross Stewart, Richard Sainson, Michelle Morrow, Robert Wilkinson. Profiling immune cells within the tumor microenvironment for optimal model selection for pre-clinical investigations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1651. doi:10.1158/1538-7445.AM2014-1651

Abstract 2893: Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo

Abstract Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. Tumor-specific immunity, where present, is typically at low frequency and affinity with compromised effector function. By contrast, immunity against persistent herpesviruses in man is characterised by high affinity cytotoxic T-lymphocytes (CTL) at high frequency with potent effector function. Furthermore, the immunosubversive mechanisms employed by herpesviruses show striking parallels to tumors, yet the associated anti-viral immunity limits these to life-long asymptomatic infections. We reasoned that the delivery of immunodominant viral peptide epitopes to the tumor surface might facilitate passive-loading of peptides into empty MHC class-I molecules, effectively mimicking viral infection, rendering tumors susceptible to lysis by anti-viral immunity. To address this we developed a new class of targeting antibodies: APEC (Antibody Peptide Epitope Complexes) that are able to deliver an antigenic payload at the cell surface through proteolytic release of covalently-coupled peptide antigens. As a proof-of-concept we used clinically-validated antibodies cetuximab (anti-EGFR) and rituximab (anti-CD20) to develop APECs that are able to target human tumors. We screened 15 HLA-A*0201+ EGFR-expressing NCI-60 cell lines, CD20+ lymphoma cell lines, 20 primary CD20+ CLL tumor samples and four healthy B-cells against a library of 190 cetuximab-APECs (cAPEC) or rituximab (rAPEC) incorporating the immunodominant cytomegalovirus (CMV) pp65495-503 epitope and candidate protease cleavage sites following co-incubation with CMV-specific CTL (CMV-CTL). The most effective cAPEC and rAPEC were those incorporating MMP2, MMP9, Cathepsin B and Cathepsin D protease recognition domains. Very few (2/190) rAPEC were able to redirect CMV-CTL against healthy cells. Heterogeneity was observed for primary CLL tumors but a limited number of rAPEC were effective in all cases (5/190). Mechanistic studies demonstrated that: (i) peptide loading occurred at the cell surface, (ii) required the expression of target antigens at the cell surface and (iii) T-cell recognition could be inhibited by unconjugated antibody (92%) or by incubation with protease inhibitors (83%). T-cell specificity was examined using rAPEC treated tumor targets co-incubated with various HLA-matched effector T-cell populations. No activation of CD4+ was observed including CD4+CD25hi regulatory T-cell populations. Incubation with CD8+ T-cells revealed that only pp65495-503-specific CD8+ T-cells engaged with APEC-treated tumor cells. Lastly, xenograft studies using EGFR+ and CD20+ tumor cell lines demonstrated efficacy of both cAPEC and rAPEC to eliminate tumors in vivo by redirecting anti-viral CTL. These data indicate that APECs represent a powerful new approach to combat cancer. Citation Format: David G. Millar, Laura Morton, Manuela Carvalho Gaspar, Punam Mistry, Hugo De La Peña, Ricky Joseph, Sarah Penny, Oliver C. Goodyear, Margaret Goodall, Guy E. Pratt, Mark Cobbold. Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2014-2893

B2 Cells Suppress Experimental Abdominal Aortic Aneurysms

Recent reports of rupture in patients with abdominal aortic aneurysm (AAA) receiving B-cell depletion therapy highlight the importance of understanding the role of B cells (B1 and B2 subsets) in the development of AAA. We hypothesized that B2 cells aggravate experimental aneurysm formation. The IHC staining revealed infiltration of B cells in the aorta of wild-type (C57BL/6) mice at day 7 after elastase perfusion and persisted through day 21. Quantification of immune cell types using flow cytometry at day 14 showed significantly greater infiltration of mononuclear cells, including B cells (B2: 93% of total B cells) and T cells in elastase-perfused aortas compared with saline-perfused or normal aortas. muMT (mature B-cell deficient) mice were prone to AAA formation similar to wild-type mice in two different experimental AAA models. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AAA formation (102.0% ± 7.3% versus 75.2% ± 5.5%; P<0.05) with concomitant increase in the splenic regulatory T cell (0.24% ± 0.03% versus 0.92% ± 0.23%; P<0.05) and decrease in aortic infiltration of mononuclear cells. Our data suggest that B2 cells constitute the largest population of B cells in experimental AAA. Furthermore, B2 cells, in the absence of other B-cell subsets, increase splenic regulatory T-cell population and suppress AAA formation.

Clinical significance of peripheral blood CD4+CD25+ regulatory T cell in patients with extranodal NK/T cell lymphoma, nasal type

To investigate the clinical significance of peripheral blood regulatory T cell (Treg) population in patients with extranodal NK/T cell lymphoma, nasal type (ENKL). The peripheral blood CD4+CD25+ Treg cell population was detected by flow cytometry in 41 newly diagnosed ENKL patients between March 2009 and December 2012. The proportion of CD4+CD25+ Treg cell population increased in ENKL patients compared to healthy donors [(9.64±4.96)% vs(7.31±3.02)%, P<0.05], and decreased significantly after treatment [(5.18±2.19)%, P<0.01]. Patients when got response had significantly lower proportion of Treg cells [(8.79±4.15)%] as compared with those without response [(14.57±6.73)%, P<0.05]. The proportion of Treg population was positively related to the serum lactate dehydrogenass level. The proportion of peripheral blood Treg cells may be helpful for predicting prognosis and therapeutic efficacy in ENKL patients.

Tc1-mediated contact sensitivity reaction, its mechanism and regulation

The contact hypersensitivity reaction (CHS) to haptens is a classic example of cell-mediated immune response. In the effector phase, two stages can be distinguished: an early component, that appears only 2 hours after subsequent contact with the hapten, and the late component that develops approximately 24 hours later which is mediated by TCRαβ+ cells. The effector lymphocytes may be CD4+ T helper 1 (Th1) cells or CD8+ T cytotoxic 1 (Tc1) cells, which depends on the employed hapten and/or mice strain. NKT lymphocytes play the crucial role in the CHS initiation, by supporting B1 cells in the antigen-specific IgM antibodies production. The development of an early component is essential for the recruitment of T effector (Teff) cells to the side of hapten deposition and for the complete expansion of inflammatory reaction. The CHS reaction is under T regulatory (Treg) cells control, both in the induction phase as well as in the effector phase. A new view of a negative regulation of the Tc1 mediated CHS response is based on the suppression induced by epicutaneous (EC) application of protein antigen. The DNP-BSA skin application, on a gauze patch, leads to a state of immunosuppression. This maneuver results in rising the population of Treg cells with TCRαβ+CD4+CD25+Foxp3+ phenotype. The mechanism of suppression requires direct contact between Treg cells and Teff cells and the participation of CTLA-4 molecule is also necessary. The described method of evoking immune tolerance via EC immunization may contribute to elaborate a new method of allergic contact dermatitis therapy. This is because of its effectiveness, ease of induction and non-invasive protein antigen application.

Evaluation of Peripheral Blood CD4+CD25+/-CD39+/- T Cell Populations in Patients With Renal Failure and During Acute Allograft Rejection.

Regulatory T cells (Treg) are important in mediating immune tolerance and outcomes of allotransplantation. We have shown that four distinct CD4+ T cell populations can be defined using the cell surface markers CD25 and CD39: CD25+CD39+ (memory/regulatory T cells; Treg); CD25+CD39- (pro-T helper; Th); CD25-CD39+ (memory/effector T cells; Tmeff) and CD25-CD39- T cells (naive T cells). Furthermore, the ratio of regulatory and pathogenic T cell populations has been shown to be altered in various states of inflammation. Aim: Examine expression of CD25 and CD39 within the CD4+ T cell compartment and the suppressive capacity of Treg in patients with end stage renal failure awaiting transplantation and in kidney transplant recipients and correlate these with disease. Methods: Peripheral blood leucocytes were isolated by density gradient centrifugation, enriched for CD4+ T cells and analysed for CD25 and CD39 expression by flow cytometry and Treg suppressive capacity. Results: CD4+CD25+/-CD39+/- T cell subsets can be tracked longitudinally in the peripheral blood of patients following renal transplantation (n=6). Patients with acute T cell mediated rejection diagnosed on renal biopsy post transplantation (n=4) had decreased CD4+CD25+CD39+ Treg and CD4+CD25-CD39+ Tmeff cells in the peri-transplant period compared with patients without rejection (n=6, p<0.01), three of which had undergone biopsy for an unexplained rise in creatinine. CD4+CD25+CD39+ Treg in transplant patients with stable graft function one year post-transplant (n=3) displayed more potent suppressive capacity compared with controls (n=4) (87.3% vs 71.0%, Tresponder:Treg ratio 4:1, p<0.05 and 88.0% vs 45.7%, Tresponder:Treg ratio 8:1, p<0.01). Conclusion: CD4, CD25 and CD39 identify Treg, Tmeff and Th cell populations in peripheral blood which can be tracked in renal transplant patients. CD4+CD25+CD39+ Treg suppressive function is sustained in patients with stable renal function post-transplant. Determining these T cell subsets may help to identify patients with, or at high risk of, renal allograft rejection.

Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease.

Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.

Antigen-Pulsed CpG-ODN-Activated Dendritic Cells Induce Host-Protective Immune Response by Regulating the T Regulatory Cell Functioning in Leishmania donovani-Infected Mice: Critical Role of CXCL10.

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a systemic infection of reticulo-endothelial system. There is currently no protective vaccine against VL and chemotherapy is increasingly limited due to appearance of drug resistance to first line drugs such as antimonials and amphotericin B. In the present study, by using a murine model of leishmaniasis we evaluated the function played by soluble leishmanial antigen (SLA)-pulsed CpG-ODN-stimulated dendritic cells (SLA–CpG–DCs) in restricting the intracellular parasitic growth. We establish that a single dose of SLA–CpG–DC vaccination is sufficient in rendering complete protection against L. donovani infection. In probing the possible mechanism, we observe that SLA–CpG–DCs vaccination results in the significant decrease in Foxp3+GITR+CTLA4+CD4+CD25+ regulatory T cells (Treg) cell population in Leishmania-infected mice. Vaccination with these antigen-stimulated dendritic cells results in the decrease in the secretion of TGF-β by these Treg cells by possible regulation of the SMAD signaling. Moreover, we demonstrate that a CXC chemokine, IFN-γ-inducible protein 10 (IP-10; CXCL10), has a direct role in the regulation of CD4+CD25+ Treg cells in SLA–CpG–DC-vaccinated parasitized mice as Treg cells isolated from IP-10-depleted vaccinated mice showed significantly increased TGF-β production and suppressive activity.

Highly heterogeneous, activated, and short-lived regulatory T cells during chronic filarial infection.

The mechanisms underlying the increase in the numbers of regulatory T (Treg) cells in chronic infection settings remain unclear. Here we have delineated the phenotype and transcriptional profiles of Treg cells from 18 filarial-infected (Fil(+) ) and 19 filarial-uninfected (Fil(-) ) subjects. We found that the frequencies of Foxp3(+) Treg cells expressing CTLA-4, GITR, LAG-3, and IL-10 were significantly higher in Fil(+) subjects compared with that in Fil(-) subjects. Foxp3-expressing Treg-cell populations in Fil(+) subjects were also more heterogeneous and had higher expression of IL-10, CCL-4, IL-29, CTLA-4, and TGF-β than Fil(-) subjects, each of these cytokines having been implicated in immune suppression. Moreover, Foxp3-expressing Treg cells from Fil(+) subjects had markedly upregulated expression of activation-induced apoptotic genes with concomitant downregulation of those involved in cell survival. To determine whether the expression of apoptotic genes was due to Treg-cell activation, we found that the expression of CTLA-4, CDk8, RAD50, TNFRSF1A, FOXO3, and RHOA were significantly upregulated in stimulated cells compared with unstimulated cells. Taken together, our results suggest that in patent filarial infection, the expanded Treg-cell populations are heterogeneous, short-lived, activated, and express higher levels of molecules known to modulate immune responsiveness, suggesting that filarial infection is associated with high Treg-cell turnover.

Mesenchymal stem cell population derived from human pluripotent stem cells displays potent immunomodulatory and therapeutic properties.

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however, loss of potency and inconsistent quality severely limit their use. To overcome these issues, we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking, scraping, and sorting that other hESC-MSC derivation methods require. Moreover, unlike previous reports on hESC-MSCs, we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected, they dynamically secrete a range of bioactive factors, display enzymatic activity, and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However, they also display unique immunophenotypic properties, as well as a smaller size and >30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition, this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models, including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources.

Understanding the effect of early-life pulsed antibiotic treatment on mucosal T-lymphocyte populations (MUC4P.827)

Abstract Broad-spectrum antibiotics are frequently prescribed for children. The gut microbiota have functional roles in the development and differentiation of the host immune system. Early-life antibiotic use may have dynamic effects on the host microbiota, promoting long-term immunologic dysregulation and subsequent allergic and autoimmune pathology. Using a model of early-life antibiotic use, we hypothesize that early-life pulsed antibiotic treatment (PAT) -induced perturbation of the intestinal microbiota leads to alterations in tissue-specific and systemic T-cell populations. To test this hypothesis, we characterized splenic and ileal T-cell populations in control and PAT-treated C57BL/6 mice. Flow cytometric analysis demonstrated that early-life PAT significantly (p&amp;lt;0.05) decreased the frequency of splenic CD4+ and CD8+ T-cells and ileal CD4+ IL-17A+ Th17 populations. In contrast, ileal CD4+ Tbet+ (Th1) T cells and CD4+ Foxp3+ regulatory T-cell populations were significantly increased in PAT-treated mice. Alterations in immune cell populations were associated with microbial compositional changes, including significantly higher relative abundance of Enterobacteriaceae and Akkermansia muciniphila in PAT-treated mice. These results provide evidence that early-life perturbation of the intestinal microbiota by PAT modulates systemic and mucosal T-cell populations, phenotypes that may persist after microbial recovery, promoting life-long consequences to host immunity.

Abstract 110: B2 Cells Suppress Abdominal Aortic Aneurysms and Increase Peripheral Regulatory T Cells

Objective: Recent reports have documented abdominal aortic aneurysm (AA) rupture in patients receiving B cell depletion therapy. This highlights the importance of understanding the role of B cells (B1 and B2 subtypes) in the development of AA. We hypothesized that B2 cells are abundant in AA and aggravate experimental aneurysm formation. Methods: AAs were induced in WT (C57BL/6) mice by elastase perfusion of the abdominal aorta and harvested at 14 days. Aortic B1 and B2 cells were quantified, and, intracellular cytokine synthesis was determined using flow cytometry. Next, AAs were induced in B cell deficient (muMT) mice using either elastase perfusion or topical elastase application to determine the impact of loss of B cells in AA. Finally, adoptive transfer experiments were performed on muMT mice by tail vein injection of 25x10^6 isolated splenic B2 cells or saline. Regulatory T cell (Treg) population was identified as CD4+ T cell expressing Foxp3 (forkhead box P3). Results: B cells appeared in abdominal aortas at day 7 after elastase perfusion in WT mice and persisted at least till day 21. Compared to saline perfused or normal aortas, elastase perfused aortas had greater infiltration of mononuclear cells including B cells (3631±578 in elastase vs 1476±524 in saline, p=0.02) and T cells. B2 cells constituted 93% of total B cells in AA. Furthermore, B cells isolated from elastase and saline perfused aortas displayed high, but similar levels of intracellular IL-6 and IL-10 expression. Interestingly, in both experimental models of AA, muMT mice were prone to AA formation similar to WT mice as evidenced by elastin degradation or infiltration of T cells, neutrophils and macrophages. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AA formation (102.0±7.3% vs 75.2±5.5%, p=0.01) with concomitant increase in Treg cells in spleen (0.24±0.03% vs 0.92± 0.23%, p=0.02) and aorta (0.37±0.09% vs 1.09±0.38%, p=0.08), and decrease in aortic infiltration of mononuclear cells. Conclusions: Our data suggest that B2 cells constitute the largest population of B cells in experimental AA and participate in protecting aorta from aneurysm formation at least in part by increasing the Treg cell population.

CD4 T-cell activation and reduced regulatory T-cell populations are associated with early development of cataracts among HIV-infected adults in Uganda

BackgroundCataracts contribute 12% of visual loss among HIV-infected adults in Uganda. Immuno-pathogenesis of cataracts may differ among HIV-infected individuals; thus the need for innovative therapeutic interventions among HIV-infected adults. MethodsIn a laboratory based case-control study, nested in a clinical/surgical community outreach camp, 50 adults with cataracts eligible for surgery were selected consecutively. HIV testing was done for individuals with unknown HIV sero-status. Peripheral Blood Mononuclear Cells (PBMC) were collected from all HIV-positive-adults-with-cataracts (cases) and HIV-negative-adults-with-cataracts (comparative group) and age-matched HIV-negative and HIV-positive-adults-without-cataracts (comparative group). Treg were measured as CD3+CD4+FoxP3+CD25+Bright and immune activation as CD3+CD4+CD38+HALDR+ using a Facs Canto II flowcytometer. Mann Whitney test was used to compare expression among the four groups. ResultsOf 50 adults operated for cataracts, 24 (48%) were female, 25 (50%) were HIV-positive. HIV-positive-individuals had cataracts earlier [median; Inter-quartile Range (IQR); 49 (44–53) years] than HIV-negative [70 (IQR 59–75) years]; p=0.0005. Treg were lower among individuals with cataracts irrespective of HIV status; p=0.001; but comparable among younger HIV-positive and elderly HIV-negative with cataracts; p=0.301. Immune activation levels were comparable among HIV-positive and HIV-negative individuals with cataracts. However, HIV-positive-individuals with cataracts expressed higher levels of immune activation than HIV-positive-individuals without cataracts; p=0.012 and HIV-negative-individuals-with-cataracts expressed higher levels of immune activation that HIV-negative-without-cataracts; p<0.0001. ConclusionCD4 T-cell activation and reduced regulatory T-cell populations were associated with cataracts among adults aging with HIV. We recommend studies on clinical relevance of immune modulation in the prevention of early development of cataracts among adults aging with HIV in Africa.

Unique Effects of Mycophenolate Mofetil on Cord Blood T Cells

Hematopoietic stem cell transplantation (HSCT) is a common treatment for hematological diseases. Cord blood (CB) is increasingly used as a source of stem cells for HSCT. Prophylactic drugs, such as mycophenolate mofetil (MMF) and cyclosporine A (CsA), are often used together after HSCT to prevent graft-versus-host disease (GvHD), but so far little is known about their effects on CB mononuclear cells (CBMCs). As CB and peripheral blood (PB) have different cell compositions and characteristics, it was hypothesized that MMF and CsA might have different effects on CB and PB T cells. Using a combination of flow cytometry, ELISA, and quantitative PCR, the effects of MMF, CsA, and the combination of both drugs were studied on resting and activated CBMCs and peripheral blood mononuclear cells. MMF had a stronger effect on activated PB T cells than on activated CB T cells, which was consistent with the lower level of IMPDH2 mRNA expressed by PB T cells. Interestingly, only MMF could preserve the activated CB regulatory T-cell population. Activated CB T cells were more sensitive to CsA than activated PB T cells, which might be explained by the lower NFATc1 expression and cytokine secretion. These results may explain the lower GvHD incidence observed in recipients of CB transplants. This study provides valuable insight into the effects of immunosuppressive drugs used after HSCT on resting and activated T-cell subsets from PB but especially from CB.

HCV adaptations to altered CD8+ T-cell immunity during pregnancy.

Robust, polyfunctional CD8+ T-cell responses targeted to multiple viral epitopes have been associated with successful resolution of acute HCV infection. In most individuals, however, immunity fails and chronic viremia ensues as CD8+ T cells become functionally exhausted or select viral variants with escape mutations in class I epitopes. During pregnancy, dysfunctional HCV-specific CD8+ T-cell responses may be further impaired by maternal–fetal tolerance processes such as the expansion of regulatory T-cell populations or hormonal changes. Recent evidence suggests that HCV adapts to relaxed maternal CD8+ T-cell pressure in pregnancy by shedding unfit escape mutations in certain class I epitopes, resulting in the selection of viruses with more efficient replication. Emergence of HCV variants with enhanced replicative capacity during pregnancy has ramifications for mother-to-child transmission, the primary route of HCV infection in children, and could potentially be relevant to other important, vertically transmissible, persistent viruses such as HBV and HIV. HCV has infected over 185 million people worldwide and persists in approximately 75% of these individuals, predisposing to liver inflammation, cirrhosis and hepatocellular carcinoma [1]. More than 1 million children are born to HCV-infected mothers each year, and 3–5% acquire the infection in utero or at delivery, making vertical transmission the leading route of pediatric HCV infection in the developed world [2]. Here, we review the critical role of CD8+ T-cell immunity in HCV infection, its modulation in pregnancy and recent evidence that HCV takes advantage of the unique immunologic niche of pregnancy to improve replicative fitness and potentially promote vertical transmission.

Human chorionic gonadotropin and indolamine 2,3-dioxygenase in patients with GVHD

GVHD is a major complication following allogeneic hematopoietic SCT, and is associated with substantial morbidity and mortality. Based on the results of our previous clinical study with females treated with human chorionic gonadotropin (hCG) as preconditioning therapy for in vitro fertilization, we hypothesized that low-dose hCG stimulates indoleamine-2,3-dioxygenase (IDO), IL 10 and regulatory T cells (Treg), thereby suppressing clinical manifestations of chronic GVHD. Active chronic GVHD localized at skin, subcutaneous tissue, joints or gastrointestinal tract that was refractory or intolerant to glucocorticoid therapy improved substantially in 12 of 20 patients treated with hCG for 8 weeks (off-label), enabling a glucocorticoid dose reduction of 28% (average). Twelve of 19 patients with chronic GVHD of the skin responded to hCG therapy with a reduction of 25% (average) in their total skin score. HCG treatment increased IDO expression at median by sevenfold in peripheral mononuclear cells and IL10 levels in serum up to twofold at median from the pretreatment baseline. Further, an expansion of the Treg cell population was measured in one patient, which is also associated with the induction of tolerance. This novel application of low-dose hCG was well tolerated and is of clinical interest for GVHD treatment.

CCR6 supports migration and differentiation of a subset of DN1 early thymocyte progenitors but is not required for thymic nTreg development

T-cell selection and development occurs as precursor cells journey through the thymus and interact with stromal cells residing in distinct microenvironments. Although the chemokines CCL19, CCL21, CCL25 and CXCL12 are known to have major roles in intrathymic migration of thymocytes and thymocyte precursors, the significance of other chemokines such as CCL20, which is also expressed in the thymus, is unknown. This is of particular interest given that the thymus is the location of development of the natural regulatory T-cell (nTreg) population and that the CCL20 receptor CCR6 has an important role in peripheral tolerance via control of Treg cell migration. However, whether the CCL20/CCR6 axis has a role in the formation or migration of nTregs in the thymus is unknown. In this study, we addressed this by analyzing expression of CCR6/CCL20 within the thymus and assessing their role in thymocyte development using Ccr6(-/-) mice. CCL20 is predominately expressed in the thymic medulla and CCR6 expression is restricted to nTregs and a subset of early thymocyte progenitor double-negative 1 (DN1) cells (CD4(-)CD8(-)CD25(-)CD44(+)CD117(+)). Ex vivo chemotaxis assays indicated that these two subsets were apparently the sole subsets of thymocytes responsive to CCL20. The data indicate that nTreg frequencies and localization are unperturbed by deletion of Ccr6. However, in Ccr6(-/-) thymi, reduced frequencies of DN2 and DN3 cells, the thymocyte progenitor subsets that follow the DN1 stage, were apparent. Together, these data indicate that CCR6 has a supplementary role in coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for normal nTreg development.

Tumor-associated antigen/IL-21-transduced dendritic cell vaccines enhance immunity and inhibit immunosuppressive cells in metastatic melanoma

Dendritic cell (DC)-based vaccine approaches are being actively evaluated for developing immunotherapeutic agents against cancers. In this study, we investigated the use of engineered DCs expressing transgenic tumor-associated antigen hgp100 and the regulatory cytokine interleukin-21, namely DC-hgp100/mIL-21, as a therapeutic vaccine against melanoma. Tumor-bearing mice were injected intratumorally with transgenic DCs followed by three booster injections. Transgenic DC-hgp100/mIL-21 showed significant reduction in primary tumor growth and metastasis compared with DC-hgp100 alone and DC-mIL-21 alone. In vivo depletion of specific immune cell types (CD8(+) T, CD4(+) T and Natural killer (NK)-1.1(+) cells) effectively blocked the protective effect of this combinational vaccine. In adoptive transfer experiments, a survival rate of nearly 90% was observed at 60 days post-tumor inoculation for the combinational vaccine group. In contrast, all mice in the DC-hgp100 and DC-mIL-21-only groups died within 43-46 days after tumor challenge. Considerably increased levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, granulocyte macrophage colony-stimulating factor (GM-CSF) and cytotoxic T lymphocytes (CTLs) were detected with the combination vaccine group compared with other individual treatment groups. In comparison with the DC-hgp100 or mIL-21 groups, the combinational DC-hgp100/mIL-21 vaccine also drastically suppressed the myeloid-derived suppressor cells (MDSCs) and T-regulatory (Treg) cell populations. Our findings suggest that a combinational DC- and gene-based hgp100 and mIL-21 vaccine therapy strategy warrants further evaluation as a clinically relevant cancer vaccine approach for human melanoma patients.

Partial dysfunction of Treg activation in sickle cell disease.

Sickle cell disease (SCD) is a chronic inflammatory disease associated with multiple organ damage, chronic anemia, and infections. SCD patients have a high rate of alloimmunization against red blood cells (RBCs) following transfusion and may develop autoimmune diseases. Studies in mouse models have suggested that regulatory T cells (Treg) play a role in alloimmunization against RBC antigens. We characterized the phenotype and function of the Treg cell population in a homogeneous cohort of transfused SCD patients. We found that the distribution of Treg subpopulations differed significantly between SCD patients and healthy blood donors. SCD patients have a particular Treg phenotype, with strong CTLA-4 and CD39 expression and weak HLA-DR and CCR7 expression. Finally, we show that this particular phenotype is related to SCD rather than alloimmunization status. Indeed, we observed no difference in Treg phenotype or function in vitro using autologous feeder cells between strong and weak responders to alloimmunization.

FoxA1 directs the lineage and immunosuppressive properties of a novel regulatory T cell population in EAE and MS

The defective generation or function of regulatory T (Treg) cells in autoimmune disease contributes to chronic inflammation and tissue injury. We report the identification of FoxA1 as a transcription factor in T cells that, after ectopic expression, confers suppressive properties in a newly identified Treg cell population, herein called FoxA1(+) Treg cells. FoxA1 bound to the Pdl1 promoter, inducing programmed cell death ligand 1 (Pd-l1) expression, which was essential for the FoxA1(+) Treg cells to kill activated T cells. FoxA1(+) Treg cells develop primarily in the central nervous system in response to autoimmune inflammation, have a distinct transcriptional profile and are CD4(+)FoxA1(+)CD47(+)CD69(+)PD-L1(hi)FoxP3(-). Adoptive transfer of stable FoxA1(+) Treg cells inhibited experimental autoimmune encephalomyelitis in a FoxA1-and Pd-l1-dependent manner. The development of FoxA1(+) Treg cells is induced by interferon-β (IFN-β) and requires T cell-intrinsic IFN-α/β receptor (Ifnar) signaling, as the frequency of FoxA1(+) Treg cells was reduced in Ifnb(-/-) and Ifnar(-/-) mice. In individuals with relapsing-remitting multiple sclerosis, clinical response to treatment with IFN-β was associated with an increased frequency of suppressive FoxA1(+) Treg cells in the blood. These findings suggest that FoxA1 is a lineage-specification factor that is induced by IFN-β and supports the differentiation and suppressive function of FoxA1(+) Treg cells.