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December 19, 2014

Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital

Highlights in Recent Literature | Clinical Immunology Highlights | Basic Immunology & Novel Therapies | ImmunphenotypingPDF VersionPrevious Issues

Highlights from Recent Literature

Metformin a Surprising New Ally in the War Against Drug-resistant Tuberculosis

A review of Singhal A., et al. Metformin as adjunct antituberculosis therapy. Sci. Transl. Med. 6, 263ra159 (2014).Sci Transl Med 6, 246ra99 (2014). PMID: 25411472

Tuberculosis (TB) remains one of the biggest killers worldwide and the emergence of drug-resistant strains has complicated therapy. Novel agents that control TB by enhancing host immune responses are urgently needed. In this study, authors report that the anti-diabetic medication metformin enhanced anti-TB immune responses via multiple pathways and may be a powerful new weapon in the war against drug-resistant tuberculosis.

  • Metformin is known to have multiple effects including activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK), an energy-sensing molecule that regulates cell growth and survival, alteration of host derived mitochondrial reactive oxygen species (ROS) production and modulation of autophagy mediated by mTOR signaling.

  • The authors studied the effect of various medications to inhibit intracellular growth of the TB vaccine strain BCG and pathogenic strains of TB.

  • Metformin was found to inhibit the growth of vaccine strains of TB, pathogenic strains of TB, and even multi-drug-resistant TB strains in vitro. This antibacterial function depended on AMPK activity and metformin also increased mitochondrial ROS production and enhanced autophagy.

  • Metformin was also effective in a mouse model of acute and chronic TB. Gene expression analyses determined that metformin down regulated genes associated with inflammation, including interferons, and acted to balance the immune response.

  • The authors then studied patients with both type II diabetes and TB and found that metformin use was associated with reduced severity of pulmonary lesions and with increased survival.

The authors found that metformin modulates several aspects of host immune responses to TB, acidifying the phagosome to enhance microbial killing, inducing autophagy and reducing overt inflammation which helps to ameliorate tissue damage. This comprehensive study, including in vitro analyses, animal models and retrospective analysis of patient responses, sets the stage for adoption of metformin as a new and powerful adjuvant therapy for TB.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


MicroRNA and Skewing of the T Cell Polarization in Asthmatic Lung

A review of Simpson LJ., et al. Epigenomic analysis of primary human T cells reveals enhancers associated with Th2 memory cell differentiation and asthma susceptibility. Nature Immunology. (2014) 15: 777. doi: 10.1038/ni.3026. PMID: 25362490

Asthma is a chronic inflammatory disease characterized by episodes of airflow limitation and bronchial hyperreactivity and is the most common chronic disease of childhood. While there is an increasing appreciation that there are multiple phenotypes within asthma, T helper 2 cells play a clear role in a subset of patients. In this study, Simpson et al profile miRNA expression in human airway T cells in asthma patients versus healthy controls.

SUMMARY

  • They isolated CD4+ T cells from bronchoalveolar lavage (BAL) fluid from 8 healthy controls and 12 steroid naive asthmatic patients and 21 ‘steroid using’ asthmatic patients and profiled 190 miRNAs, of which nearly 90 were present in at least 60% of subjects.

    • miRNA-19a (part of the miR-17~92 cluster) was the most correlated with asthma (both steroid naïve and steroid using)

    • The miR-17~92 cluster positively regulated secretion of type 2 cytokines IL-13, IL-4 and IL-5. This effect was found to be cell-intrinsic.

  • Rather than a brief requirement for miR-19a during Th2 differentiation, miR-19a was found to be needed throughout Th2 polarization. 

    • Using inhibitors and mimics of miR-19a in murine and human T cells, they show that altering miR-19a alone is sufficient to affect IL-13

  • In order to determine the relevant targets of miR-19a in Th2 polarization, they used siRNAs to inhibit the 38 previously known target genes of miR-19a and measured the effect on IL-4 and IL-13. They also used miR 17~92 deficient cells and showed that rescue of the phenotype was possible with inhibition of PTEN, SOCS1 and TNFAIP3.

    • All three are negative regulators of signaling, but for three separate signaling pathways (PI3K, Jak/STAT and NFkB respectively).

  • Finally, they demonstrated in mice that the reduction in Th2 cytokine production found in miR 17~92 T cells leads to a decrease in Th2 inflammation in vivo using an OVA/OT-II TCR transgenic model of airway hyperreactivity. They found that with antigen specific cells that lacked miR17~92 there was a decrease in eosinophilia in BAL fluid.

This group was able to use profiling of miRNA in airway infiltrating T cells to identify a miRNA, miR-19a, that plays a mechanistic role in encouraging Th2 polarization of T cells, which may contribute to pathogenesis in a subset of asthmatics. While this is fascinating, it will require additional confirmation and mechanistic studies to further elucidate the details of this process (including possible roles in using this mediator to predict prognosis, define phenotype and even possibly provide a therapeutic target for asthmatics with Th2 predominant disease).

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


Using Systems Analysis to Investigate Pathogen Susceptibility in Primary Immunodeficiency

A review of Alsina L., et al. A narrow repertoire of transcriptional modules responsive to pyogenic bacteria is impaired in patients carrying loss-of-function mutations in MYD88 or IRAK4. Nature Immunology. (2014) 15: 1134-42. PMID: 25344726

Many components of innate and adaptive immunity have been implicated in primary immunodeficiency syndromes, including selective pathogen associated molecular pattern recognition abrogation due to Toll-like receptor (TLR) pathway dysfunction due to MyD88 and IRAK4 loss of function mutations. Of the 10 TLRs in humans, all but one signals via MyD88, and IRAK4 is recruited by MyD88 to signal downstream of those receptors. Interestingly, patients with these defects do not have the broad increased susceptibility to pathogens that might be expected, and instead have a relatively narrow range of pathogens that they are susceptible to (i.e. pyogenic gram positive and gram negative bacteria) and they show clinical improvement in adolescence. Rather than use a traditional panel approach, this team compares responses of patients to purified TLR ligands and whole pathogens using transcriptional readouts and cutting edge systems analysis.

SUMMARY

  • They began by characterizing the effect of purified TLR agonists and PMA-ionomycin on whole blood transcriptome analysis in 14 healthy controls. Transcripts with consistent changes were retained. Pathway analysis was undertaken and shared programs (i.e. inflammatory response) were examined.

  • They next assessed the responses of (n=4) IRAK4 and (n=4) MyD88 deficient patients to purified TLR agonists.

    • While responses to PMA/ionomycin were preserved and LPS responses were only partially reduced, transcriptional response to LPS, PAM3, PAM2, flagelln IL-1b and IL-18 and R-848 were dramatically reduced

    • No differences seen between IRAK4 and MyD88 deficient patients

  • Interestingly, when patient samples were stimulated instead with heat killed whole bacteria (S. pneumonia or S. aureus) they retained the ability to have > 50% of the transcriptional activity of healthy control cells responding to these infections.

    • In addition, MyD88 deficient patients had consistently lower responses versus IRAK4 deficient patients

  • To better understand the exact effect of the IRAK4 or MyD88 deficiency, the group clustered their genome-wide data and were able to use clustering to define modules of transcriptional change in the setting of infection. The 1088 transcripts yielded 66 modules, which were then arranged into 7 larger clusters. These included a cluster (5) which contained genes that were associated with bacterial infection but not part of the TLR pathway (consistent with compensatory processes being available to protect the host from these pathogens), including the inflammasome.

    • They also demonstrated strain specific susceptibility to bacteria in some patients

    • In addition, responses to fungi and viruses ranged from normal (Candida) to impaired (HSV) in these patients

This group is able to use whole transcriptome analysis of responses of whole blood from IRAK4 or MyD88 deficient patients to demonstrate that while responses to purified TLR ligands is blocked, as expected, they retain the ability to respond to selective whole pathogens (even to the pathogens that they are clinically susceptible to), likely secondary to compensatory inflammatory responses (e.g. inflammasome). This application of genome wide transcriptional analysis to individualized pathogen susceptibility in primary immunodeficiency is interesting and could be broadened to the more detailed assessment of responses to immune stimuli in other patient groups (i.e. autoimmunity or malignancy) or other infectious agents. If feasible for broader application, it might yield a more nuanced view of the mechanisms underlying susceptibility in a given patient and potentially more individualized prophylaxis or treatment regimens in those patients.

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


Naughty or Nice? Potential Role of Tumor-associated Neutrophils in Early Stage Human Lung Cancer

 A review of Erusalnov EB., et al. Tumor-associated neutrophils stimulate T cell responses in early stage human lung cancer. J. Clin. Invest. 2014 Dec. 124(12)5466-5480. PMID:25384214

While several recent studies have highlighted the role of adaptive immunity and the value of enhancing cytotoxic T cell function with checkpoint inhibitors such as those targeting PD1 or CTLA4 in oncogenesis, the role of the innate immune system in modulating human malignancy is less well characterized. A recent study by Eruslanov, Signhal, and colleagues sheds new light on a potential role for tumor-associated neutrophils (TANs) in the cancer microenvironment. Most prior studies employing immunohistochemistry have suggested that TAN infiltrates are associated with a poor prognosis in human cancers. However, studies in murine models have suggested that TANs could have both pro-tumor as well as anti-tumor effects. Moreover, in human gastric cancer, neutrophils were associated with a good prognosis. To address these conflicting results, the authors employed both immunohistochemical as well as functional studies of TANs in early stage human lung cancer. Contrary to the investigators initial hypothesis, TANs surprisingly appear to have an anti-tumor effect by stimulating T cells in early stage lung cancer. There are several intriguing findings from this work.

  • Using both immunohistochemistry as well as flow cytometry to analyze single cell populations, TANs constituted 5-25% of the cells isolated from 25 squamous cell carcinoma (SCC) and 45 adenocarcinoma (AC) stage 1 or 2 non-small cell lung cancer (NSCLC) samples. TANs were more prevalent in NSCLCs with a SCC phenotype. In both histologic subtypes, TANs were often co-localized with T cells. Interestingly, there was no correlation between the frequency of TANs and whether or not the patient was a smoker.

  • Compared with blood neutrophils or neutrophils obtained from non-malignant lung tissue from the same patient, TANs had a more activated phenotype (CD62LloCD54hi) with a distinct chemokine receptor repertoire (CCR5, CCR7, CXCR3, and CXCR4 high). TANs also failed to express inhibitory receptors or ligands such as PD-L1. These correlations were not impacted by histologic subtype or smoking history.

  • Relative to blood neutrophils or neutrophils from non-malignant sites, TANs produced increased quantities of proinflammatory cytokines upon stimulation, had high phagocytic activity in vitro, and produced high levels of reactive oxygen species (ROS). While the isolation process is an important caveat, these data suggest TANs are functionally active.

  • TANs stimulated antigen-nonspecific T cell proliferation and activation. Intriguingly, TANs from smaller tumors had a more robust impact than those isolated from larger tumors.

  • While the proportion of TANs in both SCC and AC did not correlate with tumor size, TANs obtained from larger tumors were associated with a lower capacity to augment T cell proliferation.

Naughty or nice? These findings suggest that TANs may potentially play a nice role in early stages of lung cancer by stimulating T cell responses to the tumor. While quite intriguing, additional work is clearly needed. Although the impact of TANs on T cells suggests an anti-tumor effect, it is conceivable that other TAN functions could simultaneously serve a pro-tumor function. For example, do TANs have a direct impact on tumor cell proliferation, metastasis, or other aspects of the tumor microenvironment such as angiogenesis or infiltration of other immune cell populations? It will also be important to test whether the functional properties of TANs change during tumor progression or at relapse to a more immunosuppressive phenotype and whether these findings are generalizable to other tumor subtypes. If this is the case, TANs could become another target in the immuno-oncology arsenal.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco


Naughty or Nice? Role of Tumor-infiltrating Myeloid Cells in Tumor Cell Senescence

A review of Di Mitri, et al. Tumor- infiltrating Gr-1+ myeloid cells antagonize senescence in cancer. Nature. 2014 (Nov 6) 515:134-137.  Nat. Med. 2014 Sep;20(9):1043-1049. Epub 2014 Aug 17. PMID:25156255

Cellular senescence is the induction of permanent loss of the ability of a cell to divide. It can be induced by a variety of mechanisms including aberrant activation of oncogenes or loss of tumor suppressor genes such as Pten or exposure to chemotherapy. In this regard, stable cell growth arrest due to cellular senescence is an important anti-cancer mechanism for the host. Recent work by Di Mitri, Tosso, Alimonti, and colleagues addresses the question of whether tumor-infiltrating immune cells can modulate whether tumor cells become senescent. The authors had previously demonstrated that loss of Pten in the mouse prostate epithelium induces the formation of benign tumors characterized by a strong senescence response that opposed tumor progression. Over time, these tumors overcame this senescence and adopted an aggressive and invasive phenotype. In the current work, the authors address the hypothesis that immune cells might enable Pten deficient tumor cells to evade senescence in a non-cell autonomous manner. Using both mouse models and human prostate cancer samples, they find a new role for the innate immune system in regulating cellular senescence and chemotherapy resistance. There are several intriguing findings from this work.

  • Characterization of Pten deficient prostate (PtenPC) tumors revealed a large infiltration of CD45+CD11b+Gr-1+ myeloid cells. Intriguingly, these cells tended to co-localize with Ki-67 positive proliferating cells that appeared to have overcome the senescence induced by loss of Pten.

  • Adoptive transfer for GFP-labeled bone marrow precursors into PtenPC mice resulted in infiltration of GR-1+ cells into prostate tumors, again with a preference for close proximity to proliferating tumor cells, raising the possibility that these cells prevented senescence in a paracrine manner.

  • Consistent with this notion, co-culture of Pten deficient mouse embryonic fibroblasts (MEFs) with conditioned media from GR-1+ cells prevented senescence in these cells. Using a variety of strategies, the authors identified interleukin-1 receptor antagonist (IL-1RA) as a potential GR-1+ cell secreted factor mediating this effect.

  • Validating a role for IL-1RA, Pten-loss induced senescence was enhanced in vivo when IL1ra deficient cells were adoptively transferred. Likewise, preventing migration of CD11b+ Gr1+ myeloid cells using a CXCR2 antagonist resulted in increased chemotherapy-induced senescence.

  • Finally, the authors evaluated a panel of human prostate cancer specimens. They found that patients with high levels of intratumoral IL-1RA levels had poor responses to chemotherapy and decreased progression-free survival. Importantly, high levels of IL-1RA correlated with increased numbers of tumor-infiltrating myeloid cells and aggressive phenotype. Conversely, low IL-1RA levels correlated with fewer infiltrating myeloid cells and increased expression of senescence markers in the tumor cells.

Are innate immune cells naughty or nice? In this context, they are naughty. Taken together, these data are consistent with a model whereby infiltrating innate immune cells are detrimental, promoting tumor progression by opposing senescence, perhaps via production of IL-1RA. It will be important to determine whether these findings can be expanded to other tumor types. If validated, these data suggest that blocking recruitment of Gr-1+ myeloid cells or inhibiting IL-1RA may be of therapeutic benefit.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco


New Bioinformatic Approaches for Neoepitope Identification

 A review of Duan, F., et al. Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity. J Exp Med. 2014; 211(11):2231-48. PMID:25245761

Cancer cells carry tens to hundreds of unique mutations. Each mutation has the potential to generate a neoepitope capable of being recognized by T cells and contributing to anti-tumor immunity; but in reality, only a small fraction of the total pool of mutations are immunoprotective. Despite the relative ease of identifying tumor-specific mutations using next-generation sequencing, predicting which mutations generate immunogenic neoepitopes that can drive protective antitumor responses has proven to be challenging. While NetMHC and other algorithms capable of predicting MHC:peptide binding affinities provide promising screening tools, they only tell part of the story. Screens using predicted MHC binding affinity as a sole metric omit contributions of the TCR, as well as tolerance mechanisms which may be preventing anti-tumor immunity. Duan et al. examine this problem by testing the efficacy of vaccines targeting putative neoepitopes selected based either on NetMHC scores alone (which predicts MHC:peptide binding affinities), or based on the authors’ newly proposed differential agretopicity index (DAI, which compares the MHC affinity of the mutant peptide to the wild-type peptide).

  • The authors first identify single nucleotide variants predicted to generate strong MHC binding peptides (identified using the NetMHC algorithm) from two chemically induced implantable murine tumor cell lines, CMS5 and Meth A. They identified 112 and 823 strong binding single nucleotide variants, respectively.

  • Next, the authors found that 56% of the top NetMHC predicted peptide neoepitopes had high MHC affinity (IC50 > 500nM) when measured experimentally. Despite this, when the authors vaccinated mice with these peptides, only 17% of the mutant peptides induced a specific T cell response (IFNγ secretion measured by FACS or ELIspot relative to the wild-type peptide). Furthermore, none of the highest NetMHC scoring peptides was able to protect mice from tumor challenge.

  • From these initial results, the authors conclude that the NetMHC score alone is insufficient to predict the immunogenicity or anti-tumor efficacy of a peptide. To identify peptides more likely to illicit a response, they created a new score called the differential agretopic index (DAI) which is calculated as the difference between the NetMHC scores for the wild-type and mutant peptides.

  • Neoepitope peptides identified based on DAI scores were much more successful in protecting mice from tumor challenge in a CD8-dependent manner, however the hit rates were still quite low (30% of peptides from CMS5 and 14% Meth A). The authors note that nearly all peptides identified by DAI bear anchor residue mutations.

  • Next, the authors performed structural analysis of the mutant and wild-type peptide:MHC complexes to determine if any physical properties correlated with immunogenicity. While no trend was observed in the overall magnitude of the structural differences induced by the mutation, peptides with greater structural stability (measured by root mean square fluctuation of the peptide's α carbons) were found to be more likely to induce an immune response.

Duan et al. suggests that MHC:peptide affinities, predicted or calculated, are insufficient for predicting the immunogenicity of putative mutant neoepitopes. They identify two new principles which will likely guide future work in this field: first, the affinity of the mutant peptide must be considered relative to the wild type peptide; and second, increases in the structural rigidity of the peptide resulting from mutations may increase immunogenicity. These insights provide a foothold for the development of more reliable neoepitope prediction methods, which could have far reaching implications for the clinical development of mutant neoepitope-targeted immunotherapy.

Reviewed by Alexander Hopkins and Eric Lutz, PhD, Johns Hopkins University


Anti-CTLA-4 Therapy Broadens the Melanoma-Reactive CD8+ T Cell Response

A review of Kvistborg, P. et al., Anti-CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response, Science Translational Medicine 6, 254ra128–254ra128 (2014). PMID:25232180

Treatment of melanoma patients with the T cell checkpoint inhibitor ipilimumab (an anti-CTLA-4 antibody) has led to improved patient survival; however, it is unclear whether this benefit is derived from an increased magnitude of pre-existing T cell responses, or the induction of novel tumor-specific T cell responses, resulting in a broader tumor-specific CD8+ T cell repertoire. Kvistborg et al. show that patients with advanced-stage melanoma who received ipilimumab had an increase in the number of different T cell responses while the overall magnitude of pre-existing T cell responses was on average unchanged.

  • Peripheral blood mononuclear cells (PBMCs) from HLA-A*0201 positive, advanced-stage melanoma patients who received ipilimumab were probed for CD8+ T cell reactivity to peptide-major histocompatibility (pMHC) complexes for 145 defined melanoma epitopes as well as 3 viral epitopes (influenza A, CMV, and EBV). T cell reactivity was assessed from pre-treatment and several post-treatment samples, and revealed induction of novel melanoma epitope reactivity as soon as 3 days post-infusion of ipilimumab, while pre-existing responses generally did not change in magnitude.

  • CD8+ T cell responses were more commonly reactive against epitopes derived from cancer/germ-line and melanocyte differentiation antigens than epitopes from overexpressed antigens.

  • Comparisons of the kinetics of T cell response from time of diagnosis to treatment initiation and from the first 12 weeks of treatment demonstrated that T cell responses increased from 0.029 T cell responses per month prior to treatment to 0.321 T cell responses per month after the induction of ipilimumab therapy.

  • Melanoma epitope-specific T cell receptors that appeared after the induction of ipilimumab therapy were cloned and shown to recognize several melanoma tumor cell lines expressing the corresponding antigens, demonstrating the potential of the new T cell responses induced by anti-CTLA-4 to control tumor growth.

The T cell checkpoint inhibitor ipilimumab has shown a survival benefit for patients with advanced melanoma, but the mechanism by which targeting CTLA-4 confers a survival benefit remains to be determined. Kvistborg et al. demonstrate that while the magnitude of pre-existing melanoma-specific CD8+ T cell responses remains constant, new melanoma antigen-specific T cell responses arise shortly after the induction of ipilimumab therapy. Thus, these data suggest that anti-CTLA-4 works by priming new T cell responses and broadening the repertoire of the tumor-specific T cell response, rather than by amplifying pre-existing T cell responses. Likewise, other strategies capable of broadening the tumor-specific T cell response may induce similar antitumor effects as anti-CTLA-4.

Reviewed by Heather Kinkead and Eric Lutz, PhD, John Hopkins University

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Human Immunophenotyping Update

Phenotyping Assessment of Human Blood T Follicular Helper Cells by Flow Cytometry

Salah-Eddine Bentebibel and Hideki Ueno, MD, PHD, Baylor Institute for Immunology Research

T follicular helper (TFH) cells represent the subset of CD4+ T cells that helps B cells in germinal centers (GCs) and supports their differentiation into memory B cells or long-lived plasma cells. GC TFH cells express the chemokine receptor 5 (CXCR5), which guides their migration into B cell follicles. They express inducible co-stimulator (ICOS), a molecule essential for their development and function. GC TFH cells also express high levels of immune-regulatory molecule PD-1, signaling adaptor SLAM-associated protein (SAP), and the transcription repressor B cell lymphoma 6 (Bcl-6).

CXCR5+ CD4+ T cells are also found in human blood, and share functional properties with GC TFH cells in secondary lymphoid organs. Recent multiple lines of evidence shows that blood CXCR5+ CD4+ T cells are likely a memory compartment of TFH cells. Accordingly these cells have been termed as blood (or circulating) TFH cells (1). Blood TFH cells are defined as CD4+CD45RA-CXCR5+ and constitute 15-20% of memory CD4+ T cells in humans. The majority of these cells express CD62L, CCR7, and CD45RO and thus belong to central memory cells. In contrast to GC TFH cells, a vast majority of blood TFH cells do not express activation markers such as CD69 and ICOS, and express PD1 only at low levels. Blood memory TFH cells also lack the expression of Bcl-6. Thus, while TFH cells in secondary lymphoid organs are in active state, blood TFH cells are generally in resting state. Recent studies show that human blood memory TFH are composed of heterogeneous subsets with different phenotypes and functions. Currently, there are two main approaches to identify human blood TFH cell subsets.

The first approach consists of using the chemokine receptors CXCR3 and CCR6 (2). This approach permits the dissection of blood memory TFH cells into three major subsets: CXCR3+CCR6- cells, CXCR3-CCR6- cells, and CXCR3-CCR6+ cells. The CXCR3+CCR6- cells resembles to TH1 cells (hereafter called blood TFH1 cells). The CXCR3-CCR6- subset resembles to TH2 cells (hereafter called blood TFH2 cells). The CXCR3-CCR6+ cells resembles to TH17 cells (hereafter called blood TFH17 cells). An example of the analysis of peripheral blood mononuclear cells (PBMCs) from a healthy individual is shown in Figure 1. While TFH2 and TFH17 cells produce interleukin-21 (IL-21) upon interactions with naïve B cells, and are capable of inducing B cell differentiation into immunoglobulin-producing cells, TFH1 cells lack this capacity (2).

The second approach consists of using the immune-regulatory molecule PD1, the inducible co-stimulatory molecule ICOS, and the chemokine receptor CCR7. This approach permits the dissection of another three subsets, one activated subset (ICOS+PD-1++CCR7lo), and two quiescent subsets (ICOS-PD-1+CCR7int, and ICOS-PD-1-CCR7hi) (3, 4). ICOS expression defines blood memory TFH cells with activated phenotype that highly express PD-1 and the cell cycle marker Ki-67. ICOS- cells are further divided into PD-1+CCR7int cells and PD-1-CCR7hi cells, both of which lack the expression of Ki-67 and therefore are in a quiescent state.

We propose that human blood memory TFH cells are composed of nine distinct subsets (1), which are defined by combining the two approaches. It is feasible to construct a flow cytometry panel containing CD3, CD4, CD8, CXCR5, CD45RA, CXCR3, CCR6, ICOS, PD1, and CCR7. A damp channel to exclude any contamination of other cells such as CD8+ T cells, dendritic cells, and natural killer cells and a viability dye such as Live/Dead also can be added to the panel. In order to assess whether CXCR3 and CCR6 staining is properly working, we recommend to compare the expression of these two chemokine receptors between naïve CD4+ T cells and blood memory TFH cells, as naïve CD4+ T cells do not contain cells that highly express CXCR3 or CCR6. When a 10-color flow cytometer is not available, the panel can be simplified by removing CCR7. CCR6 can also removed from the panel as the usage of CXCR3 alone can separate TFH1 (non-efficient helpers) and non-TFH1 cells (efficient helpers).

An increasing number of studies have already demonstrated that analyses of human blood TFH cell subsets provide insights regarding the type and the magnitude of ongoing Tfh and antibody response in patients with autoimmune or infectious diseases, and in subjects after vaccinations. For instance, patients with juvenile dermatomyositis, a pediatric autoimmune disease, are found to display an alteration in the balance of blood memory TFH1, TFH2, and TFH17 subsets (less TFH1 and more TFH2 and/or TFH17), which correlates with disease activity and circulating plasmablast (2). Similar finding was reported in studies of adult SLE, Sjogren’s syndrome, and multiple sclerosis (1). In a study with a large cohort of HIV infected subjects, subjects with broadly neutralizing antibodies against HIV are found to display relatively higher frequency of ICOS-PD-1+ TFH2 and TFH17 cells among blood TFH cells (4). Influenza vaccine was found to induce the emergence of the ICOS+CXCR3+ TFH1 cells at 7 days post-vaccination, which positively correlates with the generation of protective antibody responses (5).
Currently the strategy to define subsets in human blood TFH cells is often different among laboratories. We believe that a standardized approach for the analyses and definition of blood Tfh cell subsets will have a great advantage to the field, because it will permit an integration of results from multiple studies performed on different diseases at different sites. Such effort might facilitate the process in the identification of new biomarkers associated with antibody responses in vaccinations, and of similarities and differences in the type of alterations in the TFH response in patient with autoimmune diseases.

Picture1

 

References

  1. Schmitt N, Bentebibel SE, Ueno H. Phenotype and functions of memory Tfh cells in human blood. Trends Immunol. 2014;35(9):436-42.

  2. Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, et al. Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity. 2011;34(1):108-21.

  3. He J, Tsai LM, Leong YA, Hu X, Ma CS, Chevalier N, et al. Circulating Precursor CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T Cells Indicate Tfh Cell Activity and Promote Antibody Responses upon Antigen Reexposure. Immunity. 2013;39(4):770-81.

  4. Locci M, Havenar-Daughton C, Landais E, Wu J, Kroenke MA, Arlehamn CL, et al. Human Circulating PD-1(+)CXCR3(-)CXCR5(+) Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses. Immunity. 2013;39(4):758-69.

  5. Bentebibel SE, Lopez S, Obermoser G, Schmitt N, Mueller C, Harrod C, et al. Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination. Science Translational Medicine. 2013;5(176):176ra32.a

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Developments in Basic Immunology and Novel Therapies

Regulatory T Cells and Tolerance of the Fetus

Abul K. Abbas, MD, University of California San Francisco

The development of placentation in eutherian mammals enabled the fetus to mature and remain in a protected environment for much longer than was possible in less evolved species. But the free vascular connections between the fetus and the pregnant mother enabled the maternal immune system to readily access fetal tissues, including paternal antigens that were expressed in the fetus. These antigens are obviously foreign for the mother but have to be tolerated to prevent rejection of the fetus. The problem of tolerance of the fetus has fascinated immunologists for over fifty years, and has been addressed by a variety of experimental and clinical studies. Many explanations have been proposed, with varying degrees of experimental and clinical support. Recent discoveries implicating a central role of regulatory T cells promise to give meaningful answers that may not only solve a biological riddle but may also suggest approaches for elucidating the basis of spontaneous fetal loss in humans, and ultimately to provide therapies for this condition.

Mechanisms of fetal tolerance: the early findings
As the mechanisms of allograft rejection and acceptance were defined over the last twenty or so years, these same concepts were tested for their relevance to fetal tolerance. The principal mechanisms that have been proposed to explain fetal tolerance have been reviewed recently (1). These include: low expression of MHC molecules in the fetus and trophoblast; immaturity of antigen-presenting dendritic cells in the fetus; defects in accumulation of maternal dendritic cells in the decidua; imbalance of T cell activation, resulting in excess of anti-inflammatory Th2 over pro-inflammatory Th1 cells; production of the enzyme indoleamine 2, 3-dioxygenase (IDO) in the placenta, which generates tryptophan metabolites that are toxic to proliferating lymphocytes. Although all of these are plausible explanations, it has proved difficult to translate some of the experimental results to humans or, in some cases, to show that aberrations in these inhibitory pathways consistently result in fetal loss. Thus, the contribution of these mechanisms to fetal tolerance remains uncertain.

A role for Foxp3+ regulatory T cells: the evolutionary perspective
Regulatory T cells (Tregs) that express the Foxp3 transcription factor have emerged as a dominant mechanism of tolerance. It is now established that Tregs consist of multiple subsets. Some, called thymic Tregs (often referred to as “natural” Tregs) arise in the thymus upon recognition of self antigens expressed in this organ, and others, called peripheral Tregs (also called “adaptive” or “induced” Tregs) arise from mature CD4+ T cells upon recognition of newly encountered antigens in peripheral tissues, such as commensal microbes and tumors. One of the most intriguing clues about the role of Tregs in fetal tolerance came from a landmark study by Samstein et al (2). These authors had previously shown that the Foxp3 gene contained a regulatory region called the consensus noncoding sequence 2 (CNS2) that was required for the stable expression of Foxp3 in peripheral Tregs and, hence, for the development and maintenance of this population. They went on to demonstrate that during evolution, the CNS2 region appeared in eutherian mammals at precisely the same time as placentation, and it was not present in mammals such as wallabies and opossums that do not have a placenta or in non-mammalian species. The concordant appearance of two apparently unconnected features – the CNS2 genetic region in the canonical Treg gene Foxp3 and placentation – strongly implies a causal relationship between these. The authors went on to use a mouse model to show that T cells from CNS2-expressing mice developed into Tregs in a pregnant female and the fetus survived normally, but if the T cells lacked CNS2, T cells were activated and a significant fraction of the fetuses were resorbed. These biological experiments nicely complemented the evolutionary data and strongly suggested that peripheral Tregs induced by recognition of “foreign” antigens in the fetus were responsible for protecting the fetus from attack by the maternal immune system.

A role for Foxp3+ regulatory T cells: experimental studies
Many other experimental studies have explored the involvement of Tregs in fetal tolerance. In one elegant study (3), the investigators used transgenic technology to express a model antigen in male mice and, thus, in the fetus after breeding with normal females. Tregs specific for this antigen were detected in the pregnant females. When these Tregs were eliminated and replaced with Tregs from normal mice (not containing any cells specific for the paternal antigen), there was a high rate of fetal resorption. (The approach of replacing fetal antigen-specific Tregs with Tregs lacking this specificity was an important step because it prevented the development of systemic autoimmunity that invariably results from depletion of Tregs.) Thus, fetal antigens induce specific peripheral Tregs in the mother, and elimination of these cells results in fetal loss.

Other studies have indicated that even Tregs that develop in the thymus can protect the fetus from rejection (4). Since most (or all) thymic Tregs are specific for self antigens expressed in the thymus, these results imply that the fetus must contain some of the same self antigens that are present in the pregnant female – obviously a not unreasonable idea. However, these self antigens are unlikely to be targets of rejection by the mother’s immune system. Perhaps self recognition activates Tregs in the fetus that suppress responses not only against the self antigens but also against paternal antigens that are foreign to the mother, a form of “bystander suppression”.

A role for Foxp3+ regulatory T cells: observations in humans
A large number of studies have examined the frequencies of Tregs in the blood and trophoblast of individuals with normal pregnancies, pre-eclampsia, and pre-term fetal loss. These have been well reviewed recently (5). Despite considerable variations in the results, the bulk of the available evidence is consistent with the generation or expansion of Tregs in healthy pregnancies and quantitative or qualitative defects in this population in conditions associated with premature fetal loss. One of the major challenges in this field is to define the antigen specificity of the Tregs that may mediate fetal tolerance. This challenge reflects the technical difficulty of establishing the antigen specificity of all lymphocytes in humans.

Perspectives and future prospects
Recent data briefly reviewed above are leading to an increasing acceptance of Foxp3+ Tregs as a key mechanism responsible for tolerance of the fetus. These results have led to the hypothesis that thymic Tregs evolved to prevent harmful immune reactions against self antigens (many of which are expressed in the thymus) and peripheral Tregs evolved to prevent responses against commensal microbes and fetal antigens. Such a hypothesis is reasonable, but a role for thymic Tregs in fetal tolerance cannot be excluded.

Among the many questions that remain to be answered are how Tregs suppress immune responses against the fetus. Many mechanisms of Treg-mediated suppression of immune responses have been proposed. Of these, the ones that are supported by the most compelling evidence are blockade or elimination of B7 costimulators (CD80, CD86) on antigen-presenting cells by CTLA-4 expressed on Tregs; production of immunosuppressive cytokines, such as IL-10 and TGF-ß, by Tregs; and absorption of the T cell growth factor IL-2 by the high levels of IL-2 receptors expressed on Tregs. These mechanisms are, of course, not mutually exclusive, and at present there is little evidence to implicate or exclude any of them in Treg-mediated fetal tolerance.

Another important mechanistic question is why the trophoblast may favor the development of Tregs. It has been known for decades that while paternal antigens in the fetus are tolerated, the same antigens expressed in a tissue graft at a distant site are targets of rejection. Thus, the fetal environment is especially prone to activate mechanisms that suppress immune responses. Elucidating these mechanisms may provide new ideas about ways of inducing and harnessing Tregs to control harmful immune reactions at any location. A corollary to the postulated special role of the trophoblast is that Treg-mediated fetal tolerance is not associated with generalized immunosuppression.

Finally, the contribution of defective Treg generation or maintenance to recurrent abortions is a question of obvious clinical importance. As methods for inducing and expanding Tregs are entering clinical testing, this question becomes one of increasing significance. Preventing fetal loss by promoting Treg-mediated suppression without compromising protective immunity against infections remains a distant goal, but the recent basic research raises hopes that one day, the goal may be achieved.

References

  1. Erlebacher A. Mechanisms of T cell tolerance towards the allogeneic fetus. Nat Rev Immunol 13:23, 2013.

  2. Samstein RM, Josefowicz SZ, Arvey A, Treuting PM, Rudensky AY. Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell 150:29, 2012.

  3. Rowe JH, Ertelt JM, Xin L, Way SS. Pregnancy imprints regulatory memory that sustains anergy to fetal antigen. Nature 490:102, 2012.

  4. Chen T, Darrasse-Jeze G, Bergot AS, Courau T, Churlaud G, Valdivia K, Strominger JL, Ruocco MG, Chaouat G, Klatzmann D. Self-specific regulatory T cells protect embryos at implantation in mice. J Immunol 191:2273, 2013.

  5. Jiang TT, Chaturvedi, E,Ertelt JM, Kinder JM, Valent AM, Xin L, Way SS. Regulatory T cells: new keys for further unlocking the enigma of fetal tolerance and pregnancy complications. J Immunol 192:4949, 2014.

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