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September 18, 2013

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 HighlightsBasic Immunology & Novel Therapies | ImmunphenotypingPDF Version |Previous Issues

Highlights from Recent Literature

Tumor Inflating CD8+ T Cells: The Solution or Another Part of the Problem?

A review of Spranger, et al. Up-Regulation of PD-L1, IDO and Tregs in the Melanoma Tumor Microenvironment is Driven by CD8+ T Cells. Science Translational Medicine, 5, 200ra116 (2013). PMID:3006504.

Despite recent advances in targeted and immune therapies, treatment options for patients with metastatic melanoma are limited and long-term survival is poor. In some patients, melanoma tumors are infiltrated by CD8+  T cells and yet the tumor is tolerated by the immune system. To address why this might occur, the authors studied metastatic melanoma lesions in both humans and mice.

  • Suppression of T cell responses within tumors can result from a number of inhibitory pathways in the tumor microenvironment, including suppression by FoxP3+ regulatory T cells (Tregs), metabolic dysregulation via tryptophan catabolism by indoleamine-2,3-dioxygenase (IDO) and engagement of the inhibitory receptor PD1 by its ligand PD-L1. (PD-L1).
  • The authors studied human metastatic melanoma specimens by transcriptional profiling and immunohistochemical studies. They found that tumors that contained activated T cells also had the highest expression of genes associated with all three of these distinct immunosuppressive pathways (Tregs, IDO and PD-L1).
  • Using a mouse model, the authors demonstrated that up-regulation of both IDO and PD-L1 within the tumor microenvironment was dependent on the presence of CD8+ T cells and the production of IFNγ.
  • Recruitment of Tregs to the tumor and the induction of proliferation of Tregs within the tumor also required the presence of CD8+ T cells, but was not dependent on IFNγ. Instead, Tregs recruitment was found to be dependent on the production of CCL22 by CD8+ T cells, which supported the recruitment of CCR4+ Tregs. The authors also demonstrated that human CD8+ T cells recruited human Tregs in vitro and in a xenograft melanoma model in mice.

In summary, the authors found that immunosuppressive conditions only developed after T cell infiltration of tumors, suggesting infiltrating immune cells may themselves induce immunosuppressive mechanisms. In mice, all three major mechanisms of immune suppression were induced or worsened by the presence of CD8+ T cells infiltrating the tumor. This study demonstrates that tumor infiltrating CD8+ T cells contribute to the development of an immunosuppressive environment that in turn blocks their function. Combined therapy with multiple agents to combat the separate mechanisms of immunosuppression may therefore be needed to overcome local immune suppression and enhance antitumor T cell responses.

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

An Abnormal Population of Gut Bacteria is Associated with HIV Progression

A review of Vujkovic-Cvijin I., et. al. Dysbiosis of the Gut Microbiota is Associated with HIV Disease Progression and Tryptophan Catabolism. Science Translational Medicine, 5, 193ra91 (2013). PMID:3006438.

Evidence from both animal and human studies suggests that the progression of HIV disease is driven by chronically activated T cells and systemic inflammation. The source of this inflammation is not known, but breakdown of the gastrointestinal mucosal barrier and increased transfer of immunostimulatory microbial products from the gut lumen into this circulation have been suggested to participate. Increased tryptophan catabolism through interferon-inducible enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is associated with disease progression, but how it contributes is unknown. In this study, authors examined the gastrointestinal microbiota of patients with HIV to determine the role that microflora may play in disease progression.

  • Using high resolution bacterial community profiling, the authors found that a particular abnormal microflora composition existed in some HIV-infected people and that this microflora was associated with evidence of mucosal immune disruption, T cell activation and chronic inflammation.
  • This abnormal gut microbiome was characterized by increased numbers of Proteobacteria and decreased numbers of Bacteroidia members.
  • This abnormal microbiota was observed even in patients on highly active anti-retro viral therapy (HAART) and its presence correlated with levels of tryptophan catabolism and plasma concentrations of IL-6, two markers that have been previously associated with HIV disease progression.
  • Bacteria with the ability to break down tryptophan into kynurenine were increased in the gastrointestinal tracts of patients with HIV, and the levels of these bacteria correlated with kynurenine levels in the blood.

The authors have demonstrated the first link between mucosal-adherent colonic bacteria and the chronic inflammatory state that is thought to weaken the immune system and lead to HIV disease progression. Moreover, they found that this abnormal gut microbiota can be present and contribute to pathology even in patients in whom HIV viral production is suppressed with HAART. These exciting results suggest that modulation of the gastrointestinal microbiota may be a useful approach for preventing or reversing the chronic inflammation associated with HIV disease progression

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

Uncovering Key T Cell Epitopes in Human Disease

A review of Newell E.W., et. al. Combinatorial Tetramer Staining and Mass Cytometry Analysis Facilitate T Cell Epitope Mapping and Characterization. Nature Biotech, 31. 623-631 (2013). PMID:23748502.

For T cells to become activated, their T cell receptors (TCRs) must recognize and bind peptide antigens on major histocompatability complex molecules (MHC; in humans referred to as Human Leukocyte Antigen or HLA). In natural responses, the peptide-MHC (or HLA) are displayed on antigen presenting cells. To study the antigenic specificity of human T cells, it is possible to use peptide-MHC multimer reagents in traditional flow cytometry, but this has been fairly inefficient to understand the depth and breadth of a natural immune response (since it is limited by the number of flurophors that can be distinguished from each other, even when used combinatorially). Previous studies have been able to study up to 64 TCR specificities simultaneously using multiple flurophor tagged pMHCs.

The application of cytometry by time of flight (CyTOF), also known as single cell mass spectrometry, allows the assessment of a given T cell using up to 40 easily distinguished single metal labeled reagents (including pMHC multimers), but was used in this study in a combinatorial fashion to allow simulataneous assessment of 120 reagents labeled with unique three metal combinations as tags (in this case, each pMHC tetramer is conjugated to three different metal-tagged streptavidins, mixed and used as a single reagent).

This study applied this technique to uncover T cell epitopes for rotavirus, as a model system for the application of their technique to any infectious disease (or, arguably, to many forms of human disease).

Methods: Leukocyte reduction system cones from platelet apheresis donors were obtained (and for some experiments, selected HLA-A*0201+). In addition, deidentified proximal jejunum tissues from bariatric surgery were collected. HLA-A*0201 molecules were generated and refolded with various peptides and biotinylated and mixed with metal labeled streptavidins.

  • They began by predicting epitopes that would bind to the relevant HLA molecule from viral peptides and selected candidate peptides conserved across rotavirus strains (132 candidate peptides).
  • Each candidate peptide was assessed for binding to soluble HLA-A*201 (in competition with a peptide known to bind to the HLA molecule). Only those that met a binding threshold were kept, leaving 77 candidate peptides.
  • They generated a set of 109 peptide-MHC tetramers, including 77 with rotavirus epitopes and 32 with either negative or positive control peptides (including epitopes from influenza, CMV, EBV).
  • Magnetically separated CD8+ T cells (and whole) patient samples (from 17 healthy blood donors and nine bariatric surgery patient jejunal tissue samples; chosen since rotavirus is a gastrointestinal virus) were stained with the panel of MHC tetramers and 23-27 additional surface markers (to assist in determining the cell subtype and activation status). Of note, patients with known CMV, influenza or EBV negative status were used to verify that the analysis techniques used showed that those samples had appropriately low levels of T cells that could bind epitopes they had not been exposed to. Of note, they verified these results with two different mixtures of three-metal labeled tetramers.
  • They compared the cell surface marker phenotypes (i.e. naïve versus effector memory versus central memory CD8+ T cells) of the T cells responding to the positive control epitopes and found them to be consistent with prior studies. 
  • They focused on the rotavirus specific T cells and found that only T cells that recognized epitopes from VP3 (a viral peptide) had an effector phenotype and that only those cells were found in the jejunal biopsies. They also identified non-VP3 epitope specific T cells.

Applying the powerful single cell mass spectrometry technique, this paper describes a method for starting from a known pathogen (here an infectious disease that has been targeted by multiple vaccine programs which led to a vaccine given to infants at 2, 4 and 6 months of age), predicting and validating candidate pathogen epitopes and then assessing the contribution of those epitopes to the immune responses of a healthy human population, in both PBMC and gut, the target tissue of this infection. Harnessing combinatorial tagging of peptide-MHC complexes, in tandem with deep characterization of surface markers, has the potential to allow to further rationalize our approach to both preventative and therapeutic vaccine design, as well as augmenting our understanding of the factors that determine key epitopes underlying immune responses in human diseases.

Reviewed by Sarah Henrickson, MD, PhD, Boston Children’s Hospital and Harvard Medical School

The Early Bird Gets The Worm: Antigenic Seniority and Influenza A Virus

A review of Miller M.M., et. al. Neutralizing Antibodies Against Previously Encountered Influenza Virus Strains Increase Over Time: A Longitudinal Analysis. Science Translational Medicine, 5, 198ra107 (2013). PMID:3006637.

Over time, influenza A viruses (IAVs) undergo both antigenic shift and drift, challenging both vaccine design and our immune systems. To truly understand how our cumulative lifetime exposure to slightly (or significantly) different IAVs each year affects our ability to respond to the next strain we encounter, a large-scale study across a significant time span was necessary.  While cross-sectional studies can yield a great deal of information, there is dramatic power in watching the evolution of the immune response over time in multiple individuals. This study considers the role of antigenic seniority, a theory wherein the amplitude of the neutralizing antibody response to the first strain of a given subtype is the greatest (via an as-yet unknown mechanism).  In addition, the current focus on developing broadly universal influenza vaccines (that would not have to be given yearly) based on generating neutralizing antibodies to the conserved hemagglutinin (HA) stalk domain, requires a more detailed understanding of the optimal antigen exposures to yield desired humoral immune responses.

Methods: Samples were collected approximately every five years over two decades from 40 patients in the Framingham Heart Study who were born between 1917 and 1952 (to confirm that they had been exposed to both seasonal and three pandemic influenza A virus strains, 1957 H2N2, 1968 H3N2 and 1977 H1N1). Tests on these samples included HA inhibition (HAI) titers for both pandemic viruses (as above) and seasonal strains. Both IAV and HCMV titers were measured, at each time point.

  • Interestingly, HAI titers against a pandemic strain that disappeared more than 40 years ago have increased over the four study samples, a consistent pattern seen with other pandemic strains. They hypothesize that increases in titers well after exposure to the pandemic strains may be secondary to exposures to descendent viruses with conserved epitopes. This would then support the model of antigenic superiority, because the first strain in a given subtype would have its response boosted by each subsequent exposure to a similar virus, retaining a superior response for the patient’s lifetime.
  • How does this boosting of antibodies against historical pandemic IAVs affect responses to novel IAVs? By examining responses to drifted viruses, it is possible to assess the effect of boosting responses to historical exposures. While pandemic strains had an advantage in the absolute titers, when compared to drifted variants, the IAVs that had undergone antigenic drift still showed increases in titers across the four study time points.
  • How do periods of low antigenic drift affect development (and change) of neutralizing responses to the antigenically stable HA stalk of IAVs? This was examined in both group 1 HA (where periods with low antigenic drift still led to increases in HA stalk antibody titers) and group 2 HA (a more restricted group with a single dominant lineage in circulation; here they also demonstrate increase in titers over the course of the study). In agreement with the model that significant amplitudes of neutralizing anti-HA stalk antibodies require exposure to a set of viruses with antigenic diversity, the group 1 viruses, with greater antigenic drift, yielded greater amplitude HA stalk antibody titers.
  • Interestingly, they contrasted the rapidly evolving IAVs with the stable human cytomegalovirus. This herpes virus sets up lifelong latency with episodic reactivation. The viral antigens do not change over time and titers of HCMV neutralizing antibodies also remained stable throughout the study.

This study quantitates the changes in human immune responses to IAVs across adult lifetimes with likely exposures to both well studied pandemic IAVs and seasonal IAVs. With the breadth and depth of data gathered, this study has been able to assess the effects of sequential exposures to drifted and shifted IAVs, with implications for both the theories of antigenic superiority and the importance of exposure to antigenically diverse IAVs for generation of neutralizing antibodies to the HA stalk. The value of a human study that focuses on these complex and subtle issues (fundamental to human health) is clear. However, the challenges of uncontrolled natural infection of healthy humans versus controlled infection of animal models does dramatically increase complexity. In addition, while influenza vaccine development is a key issue, extending this study to infant vaccination or therapeutic vaccination would be fascinating.

Reviewed by Sarah Henrickson, MD, PhD, Boston Children’s Hospital and Harvard Medical School

How Do HLA Class II Alleles Affect HIV Viremia?

A review of Ranasinghe S., et al. Association of HLA-DRB1-Restricted CD4+ T Cell Responses with HIV Immune Control. Nature Medicine (2013). PMID:23793098. 

A challenge in the field of HIV research is understanding the mechanisms that dictate why some HIV- infected individuals are able to control their disease with minimal intervention while other progress rapidly. While a genetic association of HLA class I alleles with HIV control is well established, the role of HLA class II-restricted responses in control of HIV infection is more poorly defined. Previously published genetic studies have suggested that HLA class II alleles of the HLA-DRB1 locus may influence HIV viremia; however, the mechanistic basis by which they may do so remains unclear. Since HIV preferentially infects CD4 T cells, understanding the role of the HLA class II-restricted response in viremia control is of interest. To address this knowledge gap, Ranasinghe and colleagues explored the breadth of HIV peptide-specific responses for each of HLA-DRB1 alleles and characterized the avidity of the responses in HIV controllers and progessors. There are several intriguing observations from this work.

  • Using a panel of 201 DRB1-restricted HIV specific CD4 T cells recognizing 67 peptide specificities generated from 42 HIV-infected patients, they find that HLA-DR variants show a high degree of promiscuity with multiple peptides restricted by two or more HLA-DR variants. Moreover, detailed analysis of the OLP-41 peptide in Gag p24 demonstrated that promiscuous HLA-DRB1 variants share overlapping epitope-binding registers. The authors postulate that this promiscuity is mediated by the open confirmation of HLA class II.
  • Despite the high degree of overall HLA-DRBI binding promiscuity, there were marked differences in the number of peptides restricted by each DRB1 variant. DRB1*01:01 and 04:01 had only three peptide restrictions, while DRB1*13:01, 13:02 and 13:03 had 18, 11 and 8 HIV peptide restrictions respectively. Interestingly, the DRB1*13 variants were also associated with lower HIV viral load.
  • Analysis of the HLA-DRB1 allele expression and viral load in a cohort of 1,085 treatment-naïve HIV-infected patients of European ancestry revealed a hierarchy of more protective to more hazardous HLA-DRB1 alleles. HLA-DRB1*15:02 most strongly associated with low viremia, while HLA-DRB1*03:01 was significantly associated with high viremia.
  • Functional analyses of an independent cohort of 26 patients (17 controllers and nine progressors) revealed that HLA-DRB1 variants linked to low viremia (i.e., controllers) were more promiscuous, particularly in their ability to present a variety of Gag peptides. Moreover, Nef proteins were exclusively restricted by variants linked with low viremia.
  • CD4+ T cells from individuals with HLA-DRB1 alleles linked with low viremia had greater breadth of HIV-specific CD4+ T cell responses ex vivo. Surprisingly, despite the increased promiscuity of protective HLA-DRB1 alleles, these CD4+ T cells had functionally lower peptide binding avidity than those from individuals expressing hazardous alleles.

These data suggest that HLA-DRB1 alleles regulate control of HIV infection through their peptide-binding promiscuity and avidity. The authors propose that the increased promiscuity, but decreased avidity of protective HLA-DRB1 alleles may lower viral titers by reducing T cell activation and proliferation, thereby minimizing susceptibility to HIV infection or activation-induced cell death. While additional studies are required to validate this hypothesis, these data suggest a new approach for stratifying HIV-infected patients and a novel mechanism of CD4+ T cell-mediated viral control.

Reviewed by Si-Han V. Hai and Michelle L. Hermiston, MD, PhD, University of California, San Francisco

What Mediates Vaccine Non-Responsiveness?

A review of Garner-Spitzer, et al. Tick-Borne Encephalitis (TBE) and Hepatitis B Non-Responders Feature Different Immunologic Mechanisms in Response to TBE and Influenza Vaccination with Involvement of Regulatory T and B Cells and IL-10. Journal of Immunology (2013). PMID:23872054.

Depending upon the vaccine, 1-10% of individuals will fail to mount a sufficient protective antibody response after immunization. Mechanisms mediating vaccine non-responsiveness have been incompletely elucidated. Most studies have focused on the hepatitis B vaccine where risk factors for non-responsiveness include obesity, smoking, renal disease, HLA-DRB1 and HLA-DQB1 subtypes, and increased TGF-β and IL-10. A central question in this field is whether non-responsiveness is a generalizable phenomenon, suggesting a more global immunologic defect, or whether it reflects an antigen/vaccine specific mechanism. To address this issue, Garner-Spitzer, Wiedermann, and colleagues compared tick-borne encephalitis (TBE) and hepatitis B non-responders that were otherwise healthy. Individuals with high antibody responses served as a control group. All participants received a TBE booster and an influenza vaccine. Humoral and cellular immune responses were analyzed pre-vaccine and at one week, eight weeks, and six months post-immunization. There are several interesting findings from this work.

  • While TBE titers upon TBE booster remained low in TBE non-responders, both TBE high responders and hepatitis B responders had appropriate increases in their TBE titers.
  • The antibody response to influenza vaccine was similar among all three groups.
  • IL-2 and IFNγ production by PBMC’s stimulated with TBE ex vivo were significantly higher in TBE responders than TBE non-responders. However, both groups had equivalent cytokine production in response to stimulation with influenza antigens. Interestingly, hepatitis B non-responders had low IL-2 and IFNγ production in response to both TBE and influenza antigens despite producing adequate antibody titers. Hepatitis B non-responders also had high IL-10 levels at baseline.
  • Flow cytometric quantification of T cell subsets post booster revealed increased frequencies of CD4 T effector memory cells and FoxP3+ regulatory T cells in both TBE and hepatitis B non-responders relative to controls. TBE, but not hepatitis B, non-responders also had elevated frequencies of CD8 T effector memory cells.
  • Only hepatitis B non-responders showed expansion of CD19+CD24highCD38high transitional B cells, which are thought to be a precursor of regulatory B cells and a potential source of IL-10. Functional studies to evaluate regulatory B cell function in this subgroup were not performed.
  • Confirming published reports, hepatitis B non-responsiveness was associated with the HLA-DRB1 and HLA-DQB1 alleles. There was no correlation with TBE non-responsiveness to these or any other DR-B1, B3/4/5 or DQ-B1 subtypes.

Taken together, these data suggest that vaccine non-responsiveness is antigen specific and not reflective of overall immune dysfunction. The data also suggests that pathways leading to vaccine non-responsiveness can be distinct. While TBE non-responsiveness appears strictly antigen specific, the associations of HLA alleles, increased IL-10 production, and poor T cell proliferation to multiple antigens in hepatitis B non-responders suggest that defective antigen presentation and cellular immunity plays a more prominent role in the response to hepatitis B. Further studies addressing the functional aspects of vaccine non-responsiveness are warranted.

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

Lethal Cross-Reactivity Following Adoptive Therapy with Affinity Enhanced T Cells: Subtle Changes in T Cell Receptors can have Unpredictable Adverse Consequences

A review of Cameron B.J., et al. Identification of a Titin-Derived HLA-A1-Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells. Science Translational Medicine (2013). PMID:3006034.

Adoptive transfer of autologous T cells engineered to target and kill tumors is a promising strategy being developed for cancer therapy. However, there are major challenges facing this approach. First, the success of this approach relies on the identification of T cell receptors (TCRs) with high affinity for antigens that are specifically expressed by tumor cells. The problem is that TCRs like this are rarely isolated from natural T cell repertoires because the majority of antigens expressed by tumors are self-antigens also expressed by normal tissues; and therefore, most high affinity TCRs capable of recognizing them are eliminated during thymic development. Fortunately, lower affinity TCRs can be isolated, and their affinity for antigen can be enhanced through the mutation of specific residues within the complementarily-determining regions (CDRs) of the TCR, which are the regions that interact with and control antigen specificity. Although affinity enhancement of TCRs can overcome the first challenge, it does not overcome serious toxicity risks associated with this therapeutic approach, including both on-target and off-target toxicity. Two recent clinical studies attempted to minimize on-target off-tissue toxicity risks by targeting MAGE A3, a cancer-testis antigen that is expressed by several tumor types, but is only normally expressed in adult testes with affinity enhanced TCR-engineered T cells. Although extensive preclinical evaluation suggested that there were no off-target antigen recognition concerns; in each study, the first patient treated died due to treatment-related cardiac toxicity. In this study, Brian Cameron and colleagues show that the lethal toxicity caused by transferring affinity enhanced MAGE A3-specific T cells was not a result of on-target off-tissue toxicity as was observed in a prior study using a TCR targeting a different MAGE antigen, but rather potent cross-reactivity against titin, a self antigen expressed in cardiomyocytes. Titin cross-reactivity was a direct effect of the CDR substitutions introduced to enhance the affinity for MAGE A3, and was not observed in T cells expressing the original non-mutated TCR. 

  • Examination of cardiac tissue from each of the two subjects treated revealed marked T cell infiltration, and indicated the underlying cause of death was T cell-mediated acute cardiac injury. However, the authors showed that this could not have been the result of on-target off-tissue toxicity because MAGE antigen expression was not detected in cardiac tissue from either of the subjects.
  • Comparison of the frequency of T cells expressing the a3a MAGE A3 affinity-enhanced TCR to T cells expressing only the V 5.1β chain used by the a3a TCR, suggested that TCR mispairing was not responsible for the toxicity.  
  • Alloreactivity was ruled out because T cells expressing the a3a TCR were not activated by a panel of cells covering 95% of the HLA serotype population. In vitro activation only occurred in response to HLA-A1+ targets that expressed MAGE A3. 
  • T cells expressing the a3a TCR were found not to recognize 38 cardiac-derived primary cell lines. However, they did recognize and kill iCell cardiomyocytes, which are derived from induced pluripotent stem cells and more closely resemble heart tissue. iCell cardiomyocytes were shown not to express MAGE antigens, which suggested that a different peptide was recognized by the a3a TCR.  
  • The authors could not identify the cross-reactive epitope being recognized by the a3a TCR by blasting the human genome for similar sequences. Instead, they used alanine and glycine substitution to determine the amino acid motif recognized by the a3a TCR.  Using the ScanProsite tool, they were able to identify three candidate proteins containing the a3a TCR recognition motif.
  • They then screened each of the three candidate peptides and showed that the cross-reactive epitope being recognized by the a3a TCR was derived from the muscle protein titin and presented by HLA-A1. This peptide was not recognized by the parental non-mutated MAGE A3-specific TCR. 
  • Titin was shown to be expressed in iCell cardiomyocytes and normal cardiac tissue, but not primary cardiac cell lines. Furthermore, the titin peptide could be stripped from HLA-A1 molecules present on the surface of a B cell lymphoma line shown to express titin that could be recognized by the a3a TCR, indicating that the epitope could be naturally processed and presented.
  • Interestingly, the murine homologue peptide was not recognized by T cells expressing the a3a TCR, which suggests that evaluation of a3a TCR-expressing T cells in HLA-A1 transgenic mice would not have predicted the lethal cross-reactive toxicity observed in patients.

This study highlights the danger associated with transferring affinity-enhanced T cells as an approach for antitumor immunotherapy. Although the transferred cells have the potential to induce robust antitumor responses, they also have the potential to induce lethal toxicity. This study demonstrates that even in cases where on-target off-tumor toxicity are non-existent, which was reported to be the cause of fatal toxicity in a separate study, the possible presence of non-obvious peptide homologues must also be considered. The estimate is that a single TCR can recognize more than a million different peptides. As this study illustrates, modifying CDRs alters the affinity of the TCR for more than just the single epitope of interest, and can even lead to the recognition of new epitopes. Most importantly, this study has shown that we need improved methods for assessing off-target toxicity risks so similar adverse events can be avoided in future studies and this approach can be developed more safely.      

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

Microbial Metabolites in the Gut Send Messages to the Immune System

A Review of Smith P.M., et al. The Microbial Metabolites, Short-Chain Fatty Acids, Regulate Colonic Treg Cell Homeostasis. Science (2013). PMID:1241165.

Immune homeostasis in the gut requires a proper balance between pro- and anti-inflammatory pathways. The disruption of this balance leads to intestinal inflammatory diseases, and can lead to increased cancer occurrence. Maintenance of immune homeostasis is influenced by over 500 different species of bacteria residing in the human intestine. Whereas some bacteria in the gut microbiota, like Clostridia strains belonging to cluster IV and XIVa, suppress inflammation in the gut, other strains, like the closely related Clostridium cluster XI, promote inflammation. One way that particular strains of intestinal bacteria influence immune homeostasis is by affecting the recruitment, development and function of FoxP3+ regulatory T cells (Tregs), cells that are known to have a critical immunoregulatory role in the gut. Current efforts are aimed at determining how bacteria communicate with Tregs, and why the effects are unique to particular strains of bacteria. In this study, Patrick Smith and colleagues demonstrate that one of the signals between bacteria and Tregs is mediated through short-chain fatty acids (SCFAs), metabolites generated during the fermentation of complex carbohydrates, such as dietary fiber.   

  • The authors compared levels of SCFAs in specific pathogen-free (SPF) mice, gnotobiotic altered Schaedler flora (ASF)-colonized mice, and germ-free (GF) mice and found that GF mice had lower concentrations of acetic acid, propionic acid and butyric acid.
  • The authors hypothesized that lower concentrations of these SCFAs were responsible for the decreased frequencies of Tregs seen in colons of GF mice compared to SPF and ASF-colonized mice. 
  • They supplemented drinking water with SCFAs for three weeks and found that the SCFAs individually, or in combination, increased Treg frequencies and numbers in the colons, but not the spleens, mesenteric lymph nodes or thymuses of GF mice. The SCFAs did not significantly alter CD4+ TH1 or TH17 cells in the colon.
  • Microbiota-induced Treg development has been shown to be associated with increases in inducible Tregs (iTregs). However, the majority of the Tregs that were expanded by SCFAs expressed the transcription factor Helios, suggesting that SCFAs specifically induce natural Tregs (nTregs) in the colon rather than iTregs.
  • SCFAs were shown to induce FoxP3 and IL-10 expression in Tregs, but not TGFβ, suggesting that SCFAs can selectively alter Treg effector gene expression and function. SCFAs were also shown to enhance the proliferation and suppressive capacity of Tregs.
  • B. fragilis and Clostridium cluster XIV members known to affect Tregs in the gut were shown to produce higher levels of SCFAs than Clostidium clusters XI and XVII members that do not affect Tregs.
  • The authors showed that the GPCR43 surface receptor that binds SCFAs was specifically upregulated on Tregs in the colon, and that the upregulation of GPCR43 on Tregs was dependent on signals provided by the gut microbiota.
  • Using Ffar2 (the gene encoding GPCR43) knock-out mice, the authors demonstrated that the effects of SCFAs on Treg proliferation and function were mediated directly on Tregs through the GPCR43 receptor. 
  • The authors showed that SCFAs specifically reduced the expression of HDAC6 and HDAC9, two histone deacetylases that inhibit nTreg function, suggesting that the effects of SCFAs on Tregs is mediated through the inhibition of HDACs.
  • The authors demonstrated the therapeutic potential of their findings by showing that SCFAs could reduce the severity of colitis in a T cell adoptive transfer model of inflammatory bowel disease in a GPCR43-dependent manner.

The human gut harbors up to 1011 bacteria per gram consisting of over 500 different species. Recent studies have shown that different species in the gut microbiota have varying effects on immune homeostasis, and suggest that altering the composition of the gut microbiota through the introduction of particular strains may provide a therapeutic strategy for manipulating the gut immune environment. However, whether this approach can permanently change the repertoire of bacteria in the gut and result in long-term restoration of immune homeostasis remains unknown. This study demonstrates that SCFAs generated during the fermentation of complex carbohydrates, such as dietary fiber, provide a signal that enhances the proliferation and function of nTregs and suppresses inflammation in the gut; and may explain the benefits of dietary fiber and particular bacterial strains. Furthermore, this study shows that the identification of the specific molecules mediating signaling between bacteria and the immune system may provide novel therapeutic approaches for specifically controlling immune balance and treating or preventing inflammatory diseases in the gut without tampering with the composition of the gut micriobiota.  

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

Gut Bugs and Food Allergy

A Review of Rivas N., et. al. A Microbiota Signature Associated with Experimental Food Allergy Promotes Allergic Sensitization and Anaphylaxis. Journal of Allergy and Clinical Immunology (2013). PMID:2012.10.026.

In Rivas, et. al, the researchers were interested in profiling microbiome changes to observe whether oral sensitization to a food allergen resulted in a characteristic microbiota signature.  Prior to this study, it was hypothesized that the microbiome played a role in food allergy from studies that documented alterations in the signature before and after treatment. However, this study utilized oligonucleotide microarrays for 16S RNA to probe the complete microbiome profile, before and after therapy, as well as rescue experiments using regulatory T cells.

  • A mouse strain carrying a gain-of-function mutation for the IL-4 receptor that was susceptible to sensitization to the chicken egg protein, OVA, was used as a model for food allergy. 
  • Compared to wild-type mice, the mutant mice that were sensitized to OVA had decreased levels in Firmicutes family Erysipelotrichi, Erysipelotrichales, Erysipelotrichaceae and significant increase in the the Proteobacteria family Gammaproteobacteria, Enterobacteriales and Enterobacteriaceae. 
  • Furthermore, upon treating these sensitized mice with allergen-specific regulatory T cells, the microbiota signature was different from sensitized mice without regulatory T cell treatment, but also different from sham-sensitized wild-type mice, suggesting a distinctive microbial profile for induced tolerance from regulatory T cells.   
  • The researchers also performed experiments demonstrating that germ-free wild-type mice with flora derived from the OVA-sensitized mutant mice also experienced anaphylaxis and IgE-mediated allergic reactions to OVA.

In conclusion, these findings demonstrate that specific microbiota signatures play a role in the pathogenesis of food allergy in mice, suggesting potential therapeutic approaches targeting microbes. The experimental system may prove useful for research into the mechanisms through which gut flora influence the immune system.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

BACH Keeps the Immune System Composed

A Review of Roychoudhuri, R., et. al. BACH2 Represses Effector Programs to Stabilize Treg-Mediated Immune Homeostasis. Nature (2013). PMID:12199.

BACH2  is a x zinc-finger transcription factor previously shown to be required for B cell somatic hypermutation and class switching. Human genetic studies demonstrated polymorphisms in BACH2 are associated with several immune/inflammatory diseases, such as asthma, Crohn's disease, vitiligo, multiple sclerosis and Type 1 diabetes. Roychoudhuri et. al hypothesized that BACH2 plays a broad role in immune homeostasis and preventing inflammation. Using Bach2 knockout mice, these investigators provided evidence in support of that hypothesis.

  • By three months of age, Bach2 knockout mice developed diffuse inflammatory pathology, autoantibodies and activated CD4+ T cell infiltrates in various tissues, including lung and gut. 
  • Bone marrow chimeric mice with Bach2 KO marrow into recipient mice lacking recombination activating genes (RAG1) to prevent endogenous B and T cell differentiation, developed inflammatory disease, while co-transfer of Bach2 wild type bone marrow prevented disease. The protective effect of Bach2-wild type bone marrow was dependent upon FoxP3.
  • Thymic Treg development was impaired in Bach2 KO mice.
  • Additionally, far less adoptively transferred naïve Bach2 KO T cells differentiate into iTreg compared to transferred wild-type naïve T cells, but retroviral-mediated expression of BACH2 rescued iTreg differentiation in the Bach2 KO cells.
  • RNA sequencing of BACH2 deficient CD4+ T cells showed upregulation of many effector-lineage-associated genes, including genes known to represss Treg differentiation. Chromatin immunoprecipitation of wild-type T cells showed that that BACH2 bound to many of these genes that were derepressed in the BACH2 deficient CD4+ T cells.

In conclusion, these findings show that BACH2 functions to repress effector CD4+ T cell differentiation programs and to stabilize Tregs, thereby limiting inappropriate immune activation. Overall, from human genetic studies and this murine study, BACH2 appears to be of central importance in maintaining immune homeostasis and preventing autoimmune diseases. 

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine and Andrew Lichtman, Brigham and Women’s Hospital

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Highlights From Clinical Immunology, the Official Journal of FOCIS

New Insights into the Pathogenesis of Cirrhosis

A Review of Mchedlidze T., et al. Interleukin-33-Dependent Innate Lymphoid Cells Mediate Hepatic Fibrosis.  Immunity (2013). PMID:2013.07.018.

Hepatic fibrosis is a common complication of many different infectious and metabolic diseases of the liver and often leads to end stage cirrhosis with hepatic failure. Chronic inflammation is implicated in liver fibrosis, but the specific mediators that drive extracellular matrix deposition and tissue remodeling are not well understood. TH2 cells and the cytokines they produce, including IL-5, IL-9 and IL-13, have been implicated in fibrotic diseases of the lung and other tissues. Emerging data has recently implicated a subset of innate lymphoid cells, called ILC2, as important source of “TH2” cytokines that play roles in protection against infections and contribute to diseases (see review in Translational Immunology Update, 6/13). In this study, Mchedlidze and colleagues provide compelling evidence for a pathogenic pathway of liver fibrosis that depends on IL-33 secreted by hepatic cells and stimulates ILC2 cells in the liver to secrete IL-13, which in turn drives fibrosis. The major findings of this study are:

  • Patients with hepatic fibrosis have increased IL-33 in serum and liver compared to patients without hepatic fibrosis. Two murine models hepatic fibrosis were used in this paper, one induced by CCL4 administration and one by common bile duct ligation. IL-33 was increased in both of these models as well.
  • Within the context of the murine models, forced expression of IL-33 in the liver, by a vector or transgene increased liver fibrosis, and IL-33 deficiency caused significantly reduced liver fibrosis and reduced expression of extracellular matrix remodeling genes (collagen and TIMP genes).
  • IL-33 treatment prominently upregulated IL-13 gene expression in the liver, but IL-13 deficient mice, or mice deficient in either of the two chains of the IL-13 receptor (IL-4Rα and IL-13Rα1) were resistant to IL-33 induced fibrosis. Both these receptor chains were highly expressed in fibrotic areas of human cirrhotic liver. Bone marrow chimeric mice deficient IL-4Rα only in cells of hematopoietic origin remained susceptible to IL-33 induced liver fibrosis, showing that the target of profibrotic effect of IL-13 was an intrinsic tissue cell in the liver.
  • Hepatic stellate cells (perisinusoidal cells or Ito cells) are known to be key players in hepatic fibrosis. The authors isolated these cells from mouse liver, and showed they respond to IL-13 by STAT6 phosphorylation, proliferation and induction of profibrotic and chemokine genes.
  • Bone marrow chimeric mice deficient in the IL-33 receptor (Il1rl1-/-) only in cells of hematopoietic origin did not develop liver fibrosis, showing the IL-33 responsive cells were of hematopoietic origin.
  • Various data are presented supporting the conclusion that the source of IL-13 in the fibrotic livers was ILC2 cells. These cells were identified and shown to be increased in the affected livers, based on cell surface markers, dependence on RORα, and lack of T and B cell markers. In fact, hepatic fibrosis was maintained or worse in Rag1-/- mice but was diminished in Rag1-/- γc-/- mice, which lack ILC2 development, because it depends on IL-7, which signals though a ɣc receptor.
  • ILC2 cells were shown to mediate the profibrotic response to IL-13 by depletion studies, which diminished the response, and by adoptive transfer into IL-33 receptor deficient mice, which restored the fibrotic responses.

This paper contributes to the rapidly emerging knowledge base about the role of ILCs in disease, and provides new information about how liver fibrosis develops. The study raises the possibility that targeting ILC2 cells, IL-13 or IL-33 may be helpful to treat this very significant clinical problem. Some parts of this story require further work. The actual cellular source of the IL-33, and the precise signals that induce it are not defined in this study, nor is it proven that the Ito cells are the key responders to IL-13 in vivo. There is also some data in the recent literature that IL-33 may be protective in acute liver injury.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

Gamma Delta Dependent Hair

A Review of Gay, et al. Fgf9 from dermal γδ T Cells Induces Hair Follicle Neogenesis After Wounding. Nature Medicine (2013). PMID:3181.

T cells are implicated in skin wound repair in both mice and humans. In particular, epidermal γδ T cells produce a variety of growth factors, including insulin growth factor-1 (IGF-1) and fibroblast growth factor ( Fgf)7 and Fgf10, which promote keratinocyte proliferation and migration that contributes to wound healing. However, cutaneous wound repair in humans usually results in scars without hair follicle regeneration, while hair follicles are regenerated in wounded mouse skin. In this study, the authors show that hair follicle regeneration in mice depends on Fgf9 initially produced by dermal γδ T cells. The main experimental findings are:   

  • Using a full thickness excisional wound model of hair neogenesis, Fgf9 was found to be upregulated in the dermis before the detection of early stages of new hair follicle development.
  • The importance of Fgf9 in hair follicle neogenesis after wounding was demonstrated by showing that dermal injection of anti-Fgf9 reduced new hair follicle formation and overexpression of Fgf9 in the epidermis using an inducible keratin-promoter Fgf9 transgenic mice caused an increase in the number of neogenic hair follicles.
  • qRPC analysis of sorted populations of cells taken from mouse skin post wound day 12 showed that the primary source of Fgf9 was dermal Vγ4+ γδ T cells.
  • The importance of Fgf9 expressed by γδ T cells in induced hair follicle neogenesis was shown using Lck-Cre; Fgf9fl/fl mice. These mice lacking Fgf9 in T cells showed markedly fewer new hair follicles compared to single transgene controls, and qRT-PCR analyses showed that only γδ T cells, and not other T cells expressed Fgf9.
  • Uinsg Wnt-reporter mice (Axin2-LacZ heterozygotes), the authors showed that Fgf9 from γδ T cells induces Wnt expression and subsequent Wnt activation in wound fibroblasts, and this induction is markedly attenuated in Axin2-LacZ;Tcrd−/− that lack γδ T cells.
  • The authors confirmed previous works showing that there are very few dermal γδ T cells, in human skin, potentially explaining their inability to regenerate hair after wounding.

This paper is of interest because it shows how the immune system can contribute to different aspects of tissue regeneration in a highly specific manner. In this case, one sub-subset of the γδ subset of T cells is activated by wounding to express a single growth factor that is the major inducer of only one aspect of skin wound, namely hair follicle regeneration. The lack of Fgf9-producing γδ T cells in human dermis may explain why wounded human skin usually does not regenerate hair, and this may be of relevance to alopecia therapy.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

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Developments in Basic Immunology and Novel Therapies

Epigenetic Control of Helper T Cell Plasticity

Lai Wei, PhD, and Robert B. Nussenblatt, MD, National Eye Institute, National Institute of Health

Naïve CD4+ T cells differentiate into various subsets of effector helper or regulatory T cells when facing pathogenic attack, including T helper (Th) 1, Th2, Th17, Th9, Th22, T follicular help (Tfh) and regulatory T (Treg) cells. Appropriately activated effector and regulatory T cells subsets are critical for host defense, while disregulation of them leads to autoimmunity and inflammatory diseases. The differentiation process of helper T cells is tightly controlled by distinct cytokine milieus, the products of innate immune responses to diverse microbial pathogens. Importantly, the phenotypic and functional changes acquired by naïve CD4+ T cells during differentiation is heritable to daughter cells and do not involve DNA sequence changes. Therefore, the epigenetic mechanisms underlying the cell fate decision become a key area of interest in studying the regulation of the adaptive immune response.

Basic Concept of Epigenetics
Epigenetics studies the changes in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence. In another word, epigenetics studies how the body adjusts to the environment without changing its own DNA code. Currently, the main focus of epigenetic studies, however, is on the alteration of chromatin structure through adjusting the patterns of DNA methylation and histone modifications. One of the first well studied epigenetic phenomenon is the 5-methylcytosine of DNA methylation, which is generally associated with gene silencing. It occurs predominantly in the symmetric CG context, called CpG. Most of CG dinucleotides of the genome are normally methylated and tend to cluster together and form CpG islands. Recent studies revealed multiple forms of DNA methylation that may be the derivative of the DNA demethylation process, including 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. In addition to DNA methylation, the histone code, referring to over 100 types of post-translational covalent modifications of histones, plays a major role in epigenetic regulation of various cellular processes. The abundant forms of modifications on histone tails are acetylation, methylation, phosphorylation, ubiquitination, ADP-ribosylation, and sumolation. Recent studies also strongly suggest a crosstalk between DNA methylation and histone modifications in coordinating the epigenetic regulation of cellular functions. Therefore, cooperation of both DNA methylation and histone modifications critically decides the physiological and pathological outcomes of the organism.

Epigenetic Regulation of Signature Cytokines
Traditionally, effector T helper cell subsets are defined according to the signature cytokines they secrete. IFN-γ, IL-4, IL-17, IL-22, and IL-9 are broadly used as the markers of Th1, Th2, Th17, Th22, and Th9, respectively. In addition, another important character of Th subsets is the silencing of lineage-inappropriate cytokines. For example, fully polarized Th1 cells produce IFN-γ, while the expression of IL-4 and IL17 is effectively suppressed. Previous studies mapping the genome-wide histone modifications H3K4me3 (active marker) and H3K27me3 (silent marker) demonstrate that in fully polarized murine Th subsets, signature cytokine loci are selectively associated with H3K4me3 alone which  marks the chromosome structure permitting active gene transcription in their featured subsets, while they are associated with H3K27me3 alone which marks the suppressive chromosome structure preventing gene transcription in all other Th subsets in which they are not expressed.

However, recent reports have suggested that helper T cells are more plastic than the simple scenario (one signature cytokine in one subset) previously defined and have challenged the traditional view of exclusive expression of signature cytokines in their Th subsets. Expression of IFN-γ can be detected in a subset of fully polarized Th17 and Th2 cells in both in vitro cultures of human T cells as well as in vivo murine disease model systems. Therefore, the partially heritable epigenetic control of signature cytokine loci is also dynamically regulated by the microenvironment helper T cells reside in.

Epigenetic Regulation of Transcription Factors
The successful differentiation of helper T cells requires induction of multiple lineage-specific transcription factors that drive the programing of cell fate decision, such as Tbet in Th1, Gata3 in Th2, Rorc in Th17, Bcl6 in Tfh and FoxP3 in Treg. It is believed that the dynamic epigenetic regulation of these key transcription factors during the differentiation process promotes the plasticity of effector T cells. Genome-wide H3K4me3 and H3K27me3 data indicate that a bivalent domain is present on the promoters of several master regulators, including Tbx21 (encoded Tbet), Gata3 and Bcl6 in the T cell subsets in which these transcription factors are not highly induced. The bivalent domain represents genomic regions with both accessible H3K4me3 and repressive H3K27me3 marks and poises the downstream genes for flexible induction, which provides a mechanism for plasticity of gene regulation. Importantly, cells highly expressing Tbet and Gata3 as well as IFN-γ and IL-4 can be fully functional in host defense in vivo. Thus, helper T cell plasticity under epigenetic control of the key transcription factors may be critical for the generation of diverse cell mediated immune responses.  

Epigenetic Therapies
A growing body of evidence indicates critical role of epigenetic regulation in the immune system. Although the majority of DNA methylation and histone modification patterns remain unchanged and heritable during T cell differentiation and proliferation, the key cytokines and transcription factors defining the helper T cell subsets are all epigenetically regulated in response to  environmental stimuli, providing a potential application of epigenetic therapies in controlling helper T cell mediated inflammatory diseases, for example psoriasis, inflammatory bowel disease, uveitis, age-related macular degeneration et al. Recently, DNA methyltransferase inhibitors including 5-azanucleoside, azacitidine and decitabine, as well as histone deacetylase inhibitors such as Vorinostat and Romidepsin have been approved by the FDA for treatment of specific types of cancers. Many of these drugs can be tested for controlling inflammation through modulating the helper T cell differentiation/proliferation process. 

Conclusions and Prospects
Newly developed technologies have significantly advanced our ability to understand physiological and pathological responses in the immune systems. The comprehensive  understanding of the interaction between genetic and epigenetic regulation of helper T cell differentiation have expanded our potential approaches for distinguishing and controlling both health and disease. Importantly, targeting the epigenetic regulators may serve as a powerful new therapy for treating autoimmune and inflammatory diseases.


  1. Kanno Y., Vahedi G., Hirahara K., Singleton K., O'Shea J.J. Transcriptional and epigenetic control of T helper cell specification: molecular mechanisms underlying commitment and plasticity. Annu Rev Immunol. (2012) 30:707-31.     
  2. http://en.wikipedia.org/wiki/Epigenetics
  3. Wu H., Zhang Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev (2011) 25:2436-52.
  4. Cedar H., Bergman Y. Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet (2009) 10:295-304.
  5. Wei G., Wei L., Zhu J., Zang C., Hu-Li J., Yao Z., Cui K., Kanno Y., Roh T.Y., Watford W.T., Schones D.E., Peng W., Sun H.W., Paul W.E., O'Shea J.J., Zhao K. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity 30:155-67.
  6. Wei L., Vahedi G., Sun H.W., Watford W.T., Takatori H., Ramos H.L., Takahashi H., Liang J., Gutierrez-Cruz G., Zang C., Peng W., O'Shea J.J., Kanno Y. Discrete roles of STAT4 and STAT6 transcription factors in tuning epigenetic modifications and transcription during T helper cell differentiation. Immunity 32:840-51.
  7. Lee Y.K., Turner H., Maynard C.L., Oliver J.R., Chen D., Elson C.O., Weaver C.T. Late developmental plasticity in the T helper 17 lineage. Immunity. (2009) Jan 16;30(1):92-107.
  8. Baylin S.B., Jones P.A. A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer (2011) 11:726-34.
  9. Wei L., Liu B., Tuo J., et al. Hypomethylation of the IL-17RC promoter associates with age-related macular degeneration. Cell Rep (2012) 2:1151-8.
  10. Liu B., Wei L., Meyerle C., et al. Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration. J Transl Med (2011) 9:1-12.
  11. Kelly T.K., De Carvalho D.D., Jones P.A. Epigenetic modifications as therapeutic targets. Nat Biotechnol (2010) 28:1069-78.

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Human Immunophenotyping Update

Proficiency Testing Services: What's Available and for Whom

Holden T. Maecker, PhD, Stanford University School of Medicine

The idea of standardized services that allow a laboratory to test its proficiency at a particular assay is common in the clinical diagnostics world, but is a relatively new concept for research labs. For flow cytometry assays, the use of CD4+ T cell enumeration in HIV clinical monitoring drove the creation of several proficiency testing programs in the U.S. and U.K. These mostly use a stabilized blood sample that can be sent to participating labs, which perform the CD4 counts and return data for evaluation. Initial experiments of this kind showed the reproducibility between labs could be kept very tight.1 The ELISPOT assay, a test for enumerating cells secreting particular protein(s) such as cytokines, was one of the first to be subject to standardization among research labs. Because of its use in evaluating experimental HIV vaccines, a group of 11 vaccine testing labs led by Josephine Cox, then at the U.S. Military HIV Research Program, showed that qualitative concordance was possible, although precise quantitation varied between labs.2 Other groups have since claimed reproducible quantitation among labs3,4 by careful harmonization or standardization of protocols.

While CD4 counting has the longest history of flow cytometry proficiency testing programs, there are other assays for which such programs have also been developed. Notably, the Division of AIDS at the National Institute of Allergy and Infectious Diseases (NIAID) began a program in 2005 to test laboratories evaluating vaccine clinical trials.5,6 The target assay for this effort was intracellular cytokine staining (ICS), also used in the evaluation of experimental HIV vaccines.  This Quality Assurance Program eventually became an ongoing effort, and currently tests laboratories for proficiency in ICS and ELISPOT assays. It is overseen by a central laboratory, termed EQAPOL, for External Quality Assurance Program Oversight Laboratory (http://eqapol.dhvi.duke.edu). The EQAPOL lab is based at Duke University, under the direction of Thomas Denny. EQAPOL has also begun to administer a Luminex proficiency panel, which was developed in collaboration with Michael Kalos and the Cancer Immunotherapy Consortium (CIC).

The CIC and the Association for Cancer Immunotherapy (CIMT) are the other major players in the development of quality assurance and proficiency testing programs.7 They have published the results of these programs together with major leaders in the field, including Pedro Romero and Cedrik Britten for T cell MHC multimer staining8 and Sylvia Janetzki for ELISPOT3. A current CIC effort is also underway to standardize gating of ICS assays, led by Lisa McNeil. This has produced a recent publication describing results from 110 participating labs, along with recommendations for best practices.9 

An interesting recent development has been the shift of proficiency testing for CIC panels to a commercial vendor, Immudex (http://www.immudex.com/proficiency-panels.aspx). The CIC ELISPOT and MHC multimer panels were developed to promote assay harmonization, by finding and controlling the major sources of variability. A natural evolution of such harmonization is the provision of on-going proficiency testing, which will be the role of Immudex.

So where should labs go to test their proficiency at flow cytometry assays, provided they are not already part of a required program such as EQAPOL?  For MHC multimer and ELISPOT assays, the obvious source is the CIC-Immudex collaboration. While the EQAPOL program is intended for laboratories in the NIAID DAIDS and CIC consortia, interested laboratories can contact the EQAPOL program to determine if their laboratory is eligible for participation. And, for those looking to harmonize assays other than those listed here, the CIC/CIMT would seem a natural group with which to work, given their history in this area.

Finally, what are the benefits of ongoing proficiency testing in a research lab setting? Obviously, an initial external comparison is desirable to determine whether a lab has truly optimized a particular assay. Thereafter, a minimum of annual (and possibly even quarterly) testing is desirable to show consistency and to validate the performance of new personnel or other changes that can drift into the lab environment over time. Proficiency testing participation is among the criteria called out by the MIATA initiative, as a reportable part of the lab environment.10-12 Along with the development of consensus protocols and panels, proficiency testing can perform a vital role in the standardization/harmonization of flow cytometry assays.

References and Suggested Reading:

  1. Reimann K.A., O'Gorman M.R., Spritzler J., Wilkening C.L., Sabath D.E., Helm K., Campbell D.E.: Multisite comparison of CD4 and CD8 T-lymphocyte counting by single- versus multiple-platform methodologies: evaluation of Beckman Coulter flow-count fluorospheres and the tetraONE system.The NIAID DAIDS New Technologies Evaluation Group, Clin Diagn Lab Immunol (2000), 7:344-351.
  2. Cox J.H., Ferrari G., Kalams S.A., Lopaczynski W., Oden N., D'Souza M.P.: Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus Type 1 vaccine trials, AIDS Res Hum Retroviruses (2005) 21:68-81.
  3. Janetzki S., Panageas K.S., Ben-Porat L., Boyer J., Britten C.M., Clay T.M., Kalos M., Maecker H.T., Romero P., Yuan J., Martin Kast W., Hoos A.: Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI), Cancer Immunol Immunother (2008) 57:303-315.
  4. Boaz M.J., Hayes P., Tarragona T., Seamons L., Cooper A., Birungi J., Kitandwe P., Semaganda A., Kaleebu P., Stevens G., Anzala O., Farah B., Ogola S., Indangasi J., Mhlanga P., Van Eeden M., Thakar M., Pujari A., Mishra S., Goonetilleke N., Moore S., Mahmoud A., Sathyamoorthy P., Mahalingam J., Narayanan P.R., Ramanathan V.D., Cox J.H., Dally L., Gill D.K., Gilmour J.: Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents, Clinical and vaccine immunology: CVI (2009) 16:147-155. 2643552.
  5. Maecker H.T., Rinfret A., D'Souza P., Darden J., Roig E., Landry C., Hayes P., Birungi J., Anzala O., Garcia M., Harari A., Frank I., Baydo R., Baker M., Holbrook J., Ottinger J., Lamoreaux L., Epling C.L., Sinclair E., Suni M.A., Punt K., Calarota S., El-Bahi S., Alter G., Maila H., Kuta E., Cox J., Gray C., Altfeld M., Nougarede N., Boyer J., Tussey L., Tobery T., Bredt B., Roederer M., Koup R., Maino V.C., Weinhold K., Pantaleo G., Gilmour J., Horton H., Sekaly R.P.: Standardization of cytokine flow cytometry assays, BMC Immunol (2005) 6:13.
  6. Jaimes M.C., Maecker H.T., Yan M, Maino V.C., Hanley M.B., Greer A., Darden J.M., D'Souza M.P.: Quality assurance of intracellular cytokine staining assays: Analysis of multiple rounds of proficiency testing, J Immunol Methods (2010).
  7. van der Burg S.H., Kalos M., Gouttefangeas C., Janetzki S., Ottensmeier C., Welters M.J., Romero P., Britten C.M., Hoos A.: Harmonization of immune biomarker assays for clinical studies, Sci Transl Med (2011) 3:108ps144.
  8. Britten C.M., Janetzki S., Ben-Porat L., Clay T.M., Kalos M., Maecker H., Odunsi K., Pride M., Old L., Hoos A., Romero P.: Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium, Cancer Immunol Immunother (2009) 58:1710-1713.
  9. McNeil L.K., Price L., Britten C.M., Jaimes M., Maecker H., Odunsi K., Matsuzaki J., Staats J.S., Thorpe J., Yuan J., Janetzki S.: A harmonized approach to intracellular cytokine staining gating: Results from an international multiconsortia proficiency panel conducted by the Cancer Immunotherapy Consortium (CIC/CRI), Cytometry Part A : the journal of the International Society for Analytical Cytology (2013) 83:728-738.
  10. Janetzki S., Britten C.M., Kalos M., Levitsky H.I., Maecker H.T., Melief C.J., Old L.J., Romero P., Hoos A., Davis M.M.: "MIATA"-Minimal Information about T Cell Assays, Immunity (2009) 31:527-528.
  11. Britten C.M., Janetzki S., Butterfield L.H., Ferrari G., Gouttefangeas C., Huber C., Kalos M., Levitsky H.I., Maecker H.T., Melief C.J., O'Donnell-Tormey J., Odunsi K., Old L.J., Ottenhoff T.H., Ottensmeier C., Pawelec G., Roederer M., Roep B.O., Romero P., van der Burg S.H., Walter S., Hoos A., Davis M.M.: T Cell Assays and MIATA: The Essential Minimum for Maximum Impact, Immunity (2012) 37:1-2.
  12. Janetzki S., Hoos A., Melief C.J., Odunsi K., Romero P., Britten C.M.: Structured reporting of T cell assay results, Cancer immunity (2013) 13:13. 3718734.

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Translational Immunology Update is the official publication of the Federation of Clinical Immunology Societies. Scope: Translational Immunology Update aims to provide a knowledge-sharing forum for basic, clinical and translational scientists around all aspects of human immunology including normal and cross-disease physiology, communication between laboratory-based and clinically-based scientists, the lack of negative results that are published and the difficulty for readers to keep up with important literature.

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