Induction of Tumor cell senescence by TH1 cytokines IFN-g and TNF

interferon betaInterferons, including type I (IFNα/β) and type II (IFNγ) are known to be critical for mediating multiple aspects of tumor immunity, by targeting both immune cells for activation, and cancer cells for expression of MHC and genes associated with growth arrest and apoptosis.  Thus, expression of cytokines such as IFNγ by immune cells has been shown to be critical in anti-tumor immune responses.  IFNγ is one of the major effector cytokines of CD4+ TH1 cells and cytotoxic CD8+ T cells.  However, the full effects of IFNγ as well as other TH1 cytokines in mediating anti-tumor effects have not been fully elucidated.

Previous observations were made that tumor-specific CD4+ TH1 cells, IFNγ, and TNF were required for controlling tumor growth.  However, tumor growth arrest induced by IFNγ and TNF occurred without either significant T cell infiltration into the tumor, or appreciable tumor cell destruction (Müller-Hermelink et. al, 2008).  Thus, the anti-tumor mechanisms of the combined action of IFNγ and TNF produced by TH1 cells remained to be defined.  In an interesting follow-up study by the same group published in the February 21, 2013 issue of Nature, Braumüller et. al, further explore the mechanisms by which IFNγ and TNF produced by TH1 cells induce direct tumor cell growth arrest.

Culturing of pancreatic β-cancer cells with IFNγ plus TNF directly induced tumor cell growth arrest in the G1/G0 phase.  Interestingly, even following removal of IFNγ and TNF, tumor cells remained growth arrested for at least two weeks in vitro, indicating IFNγ and TNF induced cellular senescence.  Both IFNγ and TNF were required to induce senescence as either alone was not sufficient.

Induction of the p16INK4a gene by the combined actions of IFNγ-STAT1 and TNF-TNFR1 pathways was found to mediate this effect via consequential hypo-phosphorylation of the p16–retinoblastoma protein (Rb), thus maintaining its activated state.   Rb mediates senescence growth arrest by suppressing E2F, a transcription factor that promotes expression of cell cycle progression genes.  The role for these pathways was further validated by short hairpin (sh)-RNA knockdown of p16INK4a and p19 which inhibited tumor cell senescence by IFNγ and TNF.  The authors further demonstrated this phenomenon of TH1 cell – IFNγ and TNF induced tumor cell senescence in multiple cancer cell types as well as in an in vivo pancreatic cancer model.  Tumor cells rendered senescent by IFNγ and TNF in vivo remained arrested, even in the absence of T cells, B cells, and NK cells, following implantation into NOD–SCID/IL2rγ−/− mice.

Thus these studies define a mechanism of tumor-growth inhibition by the direct actions of the CD4+ TH1 cytokines IFNγ and TNF in mediating tumor cell senescence through activation of the Rb pathway.

The role of IFNγ in mediating tumor-immune responses is increasingly complex. IFNγ production has been negatively correlated with effective anti-tumor CD8+ T cell responses in some models (Gattinoni et. al) and also has been shown to induce expression of the immune inhibitory receptor PD-L1 (Lyford-Pike et. al).  Thus, these studies highlight that the contextual roles of immune cell effector cytokines are critical in their functions for regulation of tumor immunity and direct effects in tumor cells themselves.

Further Reading:

T-helper-1-cell cytokines drive cancer into senescence.  Braumüller H, Wieder T, Brenner E, Aßmann S, Hahn M, Alkhaled M, Schilbach K, Essmann F, Kneilling M, Griessinger C, Ranta F, Ullrich S, Mocikat R, Braungart K, Mehra T, Fehrenbacher B, Berdel J, Niessner H, Meier F, van den Broek M, Häring HU, Handgretinger R, Quintanilla-Martinez L, Fend F, Pesic M, Bauer J, Zender L, Schaller M, Schulze-Osthoff K, Röcken M. Nature. 2013 Feb 21;494(7437):361-5. doi: 10.1038/nature11824.

TNFR1 signaling and IFN-gamma signaling determine whether T cells induce tumor dormancy or promote multistage carcinogenesis.  Müller-Hermelink N, Braumüller H, Pichler B, Wieder T, Mailhammer R, Schaak K, Ghoreschi K, Yazdi A, Haubner R, Sander CA, Mocikat R, Schwaiger M, Förster I, Huss R, Weber WA, Kneilling M, Röcken M. Cancer Cell. 2008 Jun;13(6):507-18.

Cellular senescence: when bad things happen to good cells. Campisi, J. & d’Adda di Fagagna, F. Nature Rev. Mol. Cell Biol. 8, 729–740 (2007).

Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells.  Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, Finkelstein SE, Theoret MR, Rosenberg SA, Restifo NP. J Clin Invest. 2005 Jun;115(6):1616-26.

Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma.  Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, Bruno TC, Richmon JD, Wang H, Bishop JA, Chen L, Drake CG, Topalian SL, Pardoll DM, Pai SI. Cancer Res. 2013 Jan 3.

photo credit: AJC1 via photopin cc

Development of Anti-viral CD4+ T cells in unexposed individuals

HIVIn a study published in the February 2013 issue of Immunity, Su. et. al., characterized the CD4+ T cell repertoire from adult human peripheral blood mononuclear cells (PBMC) and made a fascinating observation: that memory T cells can develop against pathogens the host has never been exposed to, including HIV-1, cytomegalovirus (CMV), and herpes simplex virus (HSV).

To determine the baseline frequencies of pathogen and self-antigen-specific CD4+ T cell populations in naive individuals by flow cytometry, PBMCs were stained with major histocompatibility complex (MHC)-peptide tetramers for HLA-DR4 -restricted epitopes.  The MHC-peptides assessed included peptides from viral pathogens including HIV-1 (gag p24), CMV (pp65), and HSV (VP16), as well as self-peptides: the melanoma-associated antigen gp100, the arthritis-associated antigen fibrinogen, and the diabetes-associated antigen preproinsulin. For both self and pathogen-associated antigens, the frequency of tetramer-positive cells was found to be one to ten cells per million CD4+ T cells, in individuals previously unexposed to these viruses.  Prior exposure to viral pathogens was determined by highly sensitive serological tests.

Interestingly, on average across individuals, greater than 50% of these antigen-specific CD4+ T cells were found to be memory T cells based on CD45RO+ staining and expression of CD4+ memory T cell-associated genes.  Furthermore, viral antigen-specific CD45RO+ memory T cells secreted IFNg when stimulated with phorbol myristate acetate (PMA) plus ionomycin.  Sequencing of the TCR-beta chain from naïve and memory HIV-1-specific CD4+ T cells indicated that the antigen-specific memory phenotype but not naive CD4+ T cell populations had arisen from clonal expansion.  Thus the memory phenotype of these antigen-specific T cells indicates prior antigen experience and subsequent clonal expansion.

The question remained though, as to how individuals had developed CD4+ memory T cell responses against pathogens they had not apparently been exposed to.  One hypothesis for the existence of these CD4+ memory T cell populations is development via cross-reactivity to other antigens.  BLAST analysis of the HIV-1 peptide sequence identified several similar sequences present in gut and soil-resident bacteria, marine algae, and plants.  HIV-1-specific CD4+ memory T cell clones from various individuals showed cross-reactivity to several of these environmental microbial antigens as measured by production of IFNg and IL-2, and proliferation when stimulated with MHC-peptides.  Interestingly, antigen-specific CD4+ memory T cells elicited by vaccination of individuals with an H1N1 influenza virus vaccine were reciprocally cross reactive to several other microorganisms.

Overall, this study shows that pathogen-specific CD4+ memory T cells commonly arise in individuals through exposure to other environmental microbes containing similar peptide sequences.  The question remains however, as to whether these cross-reactive antigen-specific T cells are able to exhibit protective immune responses.

 

Further Reading:

Virus-Specific CD4 (+) Memory-Phenotype T Cells Are Abundant in Unexposed Adults.  Su LF, Kidd BA, Han A, Kotzin JJ, Davis MM. Immunity. 2013 Feb 5. pii: S1074-7613(13)00052-6.

Derivation and maintenance of virtual memory CD8 T cells.  A.D. Akue, J.Y. Lee, S.C. Jameson.  J. Immunol., 188 (2012), pp. 2516 – 2523

Nonrandom attrition of the naive CD8+ T-cell pool with aging governed by T-cell receptor:pMHC interactions. B.D. Rudd, V. Venturi, G. Li, P. Samadder, J.M. Ertelt, S.S. Way, M.P. Davenport, J. Nikolich-Žugich.  Proc. Natl. Acad. Sci. USA, 108 (2011), pp. 13694–13699.

Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans.  I. den Braber, T. Mugwagwa, N. Vrisekoop, L. Westera, R. Mögling, A.B. de Boer, N. Willems, E.H. Schrijver, G. Spierenburg, K. Gaiser et al.  Immunity, 36 (2012), pp. 288–297

Photo credit: Microbe World / Foter.com / CC BY-NC-SA

Identification of CD8+ TC1, TC2, and TC17 populations in human PBMC

Human peripheral blood mononuclear cells (PBMC) are composed of heterogeneous populations of various immune cell types.  CD4+ and CD8+ T cells are known to exist in various functional and differentiated states.  Following antigen experience, naïve T cells are thought to progressively differentiate along a path through central memory, effector memory, and terminally differentiated effector states. Markers for differentiating PBMC T cells into these subtypes using multiparametric flow cytometry include: CD3, CD4, CD8, CD45RA or CD45RO, and CCR7 or CD62L.

The cytokine milieu T cells are exposed to during antigen encounter directs differentiation into various subtypes that exhibit unique functional properties and gene expression programs including cytokines, transcription factors, and surface markers.  This is true for both CD4+ and CD8+ T cells. In a previous post, I discussed various markers that can be utilized by flow cytometry to identify CD4+ TH1, TH2, and TH17 populations in human PBMC.

CD8+ T cells can also differentiate into unique subsets similar to TH1, TH2, and TH17 CD4+ T cells.  The CD8+ versions of these subsets are referred to as TC1, TC2, and TC17 CD8+ T cells, respectively, and are defined by expression of the same characteristic cytokines as their CD4+ counterparts.

As previously discussed, expression of subset-specific surface markers is easily determined by flow cytometry. Identification of intracellular cytokine production in T cells can be assessed following 4-6 hours of TCR stimulation with anti-CD3 and anti-CD28 antibodies or the combination of Phorbol 12-Myristate 13-Acetate (PMA) and ionomycin in the presence of brefeldin A or monensin.  The cells are then fixed and permeabilized with buffers such as BD Biosciences’ Cytofix Cytoperm buffer set followed by antibody staining for cytokine expression and flow cytometry.

Like CD4+ TH1 cells, CD8+ TC1 cells characteristically produce IFNγ.  This population is by far the most common cytokine-producing CD8+ cell subset, and is very easy to identify using intracellular staining for IFNγ.

As with CD4+ TH2 cells, CD8+ TC2 cells can be identified by expression of IL-4, IL-5, and IL-13Cosmi et. al, found that the surface marker CRTH2 was a robust marker for identification of CD8+ and CD4+ cells producing IL-4, IL-5, and IL-13 expression but not IFNγ.  Expression of chemokine receptors CCR3 and CCR4 however, did not exclude IFNγ-producing cells.  Because expression of IL-4, IL-5, and IL-13 can be difficult to detect, CRTH2 may be the easiest of these markers for TC2 identification in human PBMC.

CD8+ TC17 cells are characterized by expression of the cytokine IL-17.  Expression of the chemokine receptors CCR5 and CCR6 were shown to enrich for IL-17 producing CD8+ cells.  However CCR5 and CCR6 expression are also associated with TC1 cells, and thus may not be useful to differentiate between these subsets.

CD8 Tc1 Tc2 Tc17 PMA resized 600

Figure: Expression of IFNγ, IL-17, and CRTH2 in CD8+ T cells from PBMC stimulated with for 4 hours with PMA/ionomycin.

In my own studies, I have utilized TCR or PMA/ionomycin stimulation of PBMCs to successfully identify IFNγ (TC1) and IL-17 (TC17) expressing cells, and CRTH2 expression to identify TC2 cells, as these may be the most robust markers for identification of these unique CD8+ T cell populations. Note also that these same markers reliably detect CD4+ TH1, TH17, and TH2 cells, respectively. Thus, these markers are useful to quantitate and study the function of both CD8+ and CD4+ subsets in human PBMC.

 

Additional Reading

Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles.  Croft M, Carter L, Swain SL, Dutton RW. J Exp Med. 1994 Nov 1;180(5):1715-28.

CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.  Cosmi L, Annunziato F, Galli MIG, Maggi RME, Nagata K, Romagnani S.  Eur J Immunol. 2000 Oct;30(10):2972-9.

Cutting edge: Phenotypic characterization and differentiation of human CD8+ T cells producing IL-17.  Kondo T, Takata H, Matsuki F, Takiguchi M. J Immunol. 2009 Feb 15;182(4):1794-8.

Functional expression of chemokine receptor CCR6 on human effector memory CD8+ T cells.  Kondo T, Takata H, Takiguchi M. Eur J Immunol. 2007 Jan;37(1):54-65.

Unique anti-tumor functions of IFNg vs. IL-17 producing CD8 cells

interferon gammaThe anti-tumor effectiveness of IFNγ-producing CD4+ TH1 cells and CD8+ TC1 cells is well accepted.  However, the role of IL-17 producing CD4+ (TH17) and CD8+ T cells (TC17) in promoting or inhibiting tumor growth remains unclear, as various studies have shown both tumor-inhibiting and tumor-promoting functions of these cell types.  Thus, context is a key determinant factor in the role of IL-17 producing T cell subsets in tumor immune responses.

In the current February 15, 2013 issue of The Journal of Immunology, Yu. et. al. compared the role of adoptively transferred tumor-specific TC1 and TC17 cells in controlling tumor growth.  In this murine model, anti-gp100 T cells from Pmel-1 TCR transgenic mice were polarized ex-vivo into TC1 or TC17 phenotypes, and adoptively transferred into luciferase-expressing B16F10 melanoma lung metastatic tumor-bearing mice that had undergone total body irradiation.

The efficacy of anti-tumor TC1 or TC17 cells in mediating tumor regression was monitored by measuring tumor burden by luciferase luminescence and overall survival.  By these measures, both TC1 and TC17 cells exhibited anti-tumor activity, however TC1 cells were superior.  TC1 cells completely inhibited tumor growth, while TC17 cells delayed tumor growth but were ultimately unable to control the tumor.

Adoptively transferred TC1 cells were found to produce only IFNγ but not IL-17, while TC17 cells expressed high levels of IL-17 and could also differentiate into IFNγ-producing cells, as is known for these cell subsets.  In a fascinating observation, the authors found that eliminating IFNγ-responsiveness in tumor cells completely reversed the relative efficacy of TC1 and TC17 cells. IFNγ-responsiveness in tumor cells was required for TC1 mediated anti-tumor activity, indicating a critical role for IFNγ-responsive genes in promoting tumor cell recognition by TC1 cells and/or growth inhibition or sensitivity to apoptosis. However, when tumor cells could no longer respond to IFNγ, TC17 cells were now able to induce complete tumor regression. In cytokine-neutralization assays, in vivo IFNγ was required however, for the anti-tumor effectiveness of TC17 cells while IL-17 was not.  This indicates that in the context of TC17 cell therapy, IFNγ is required for modulation of non-tumor cells in the tumor microenvironment, while IFNγ signaling in the tumor cells themselves is puzzlingly detrimental.

Within the first few days of adoptive transfer, TC1 cells proliferated faster in vivo than TC17 cells.  However, after two and four weeks, in vivo levels of TC17 cells were higher or similar in the spleen and lungs compared with TC1 cells in mice bearing wild-type as well as IFNγ-nonresponsive tumors.  Thus, the superior in vivo persistence of TC17 cells may be a factor in TC17 cell-elicited anti-tumor responses.  IFNγ elicits its effects through activation of the STAT1 transcription factor, while IL-17 signals through an alternate ACT1 pathway to activate NF-ĸBIL-22, also released by TC17 cells activates STAT3.  Thus an interaction between these pathways in tumor cells may mediate the differential requirement for IFNγ-responsiveness in TC1 vs. TC17 mediated anti-tumor effects.

While it remains unclear why TC17 cells in this model were able to effectively control IFNγ-nonresponsive tumor cells but not wild-type tumor cells, and TC1 cells exhibited the opposite propensity, these observations have important implications for future enactment of adoptive cell transfer for tumor therapeutics.  Because of their inherent stem-like propensity for long term in vivo persistence and demonstrations of highly effective anti-tumor functions, TH17 cells have been proposed to be a superior CD4+ cellular subset for adoptive anti-tumor T cell therapy in combinations with CD8+ T cells.  However, the observations in this study suggest that different cytokine-producing CD8+ TC and CD4+ TH subsets will vary in their effectiveness depending on factors such as the tumor’s ability to respond to cytokines including IFNγ.

 

Further Reading:

Adoptive Transfer of Tc1 or Tc17 Cells Elicits Antitumor Immunity against Established Melanoma through Distinct Mechanisms.  Yu Y, Cho HI, Wang D, Kaosaard K, Anasetti C, Celis E, Yu XZ. J Immunol. 2013 Feb 15;190(4):1873-81.

Tumor-specific Th17-polarized cells eradicate large established melanoma.  Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A, Paulos CM, Palmer DC, Touloukian CE, Ptak K, Gattinoni L, Wrzesinski C, Hinrichs CS, Kerstann KW, Feigenbaum L, Chan CC, Restifo NP. Blood. 2008 Jul 15;112(2):362-73.

Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments.  Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, Huang E, Finlayson E, Simeone D, Welling TH, Chang A, Coukos G, Liu R, Zou W. Blood. 2009 Aug 6;114(6):1141-9.

Structure and signalling in the IL-17 receptor superfamily.  Sarah L. Gaffen.  Nat Rev Immunol. 2009 August; 9(8): 556.

Human TH17 cells are long-lived effector memory cells.  Kryczek I, Zhao E, Liu Y, Wang Y, Vatan L, Szeliga W, Moyer J, Klimczak A, Lange A, Zou W. Sci Transl Med. 2011 Oct 12;3(104):104ra100.

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Defining Human PBMC T cell activation markers. Part 2: CD71 and CD95

In a previous posting, I discussed the use of T cell activation markers as a strategy for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC). Following T cell receptor (TCR) activation, T cells will express a series of activation markers that include chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins. Understanding what these activation markers are, when they are expressed, and their role in T cell function during normal responses and disease states is important when selecting markers for assessing T cell biology for studies on human PBMC.

In the previous posting, I discussed two immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.  In this article, the second in this series, I will discuss two additional mid-early T cell activation markers that can be assessed by flow cytometry: CD71 and CD95.

CD71 (TFRC, Transferrin Receptor, TfR) is a cell surface iron transport receptor that is upregulated in proliferating cells by 24-48 hours following T cell activation and expression continues to rise and is maintained for several days.  Thus CD71 can be considered a mid-early activation marker as compared with late activation markers that are not appreciably upregulated until even later time points.  CD71 has been shown to associate with the TCRz chain and ZAP70 and may participate in TCR signaling, and is an essential factor for proliferating T cells.

The inability of CD71 to be upregulated following TCR activation may be associated with T cell dysfunction.  As was similarly discussed for CD69, Critchley-Thorne et. al, 2007 showed that PBMC T cells from metastatic melanoma patients had reduced CD71 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients.  Thus CD71 may be aberrantly expressed by T cells in human disease.

fas signalingCD95 (Fas, APO-1, TNFRSF6) is a member of the TNF-receptor superfamily and is best known for its role in mediating activation-induced cell death in activated T cells following binding to its ligand, CD95L/FasL induced on antigen-presenting cells (APCs).  However, CD95 can also play additional, non-apoptotic roles in the modulation of T cell function.  CD95 ligation has been shown to inhibit TCR signaling and activation of naïve T cells.  However, this negative co-stimulatory effect appears to be dose-dependent, as low doses of CD95 agonists had the opposite effect and strongly promoted activation and proliferation of T cells.  Like CD71, CD95 expression can be detected by 24 hours following T cell activation and continues to increase over the course of several days.

Due to its differential roles in regulation of T cell apoptosis and activation, dysregulated expression of CD95 or its ligand CD95L could be avenues for T cell dysfunction in various human diseases.  Indeed, Strauss et. al, showed that regulation of CD95L expression may play a role in immune evasion during viral infections. CD95L was upregulated in HIV-infected APCs, and led to suppressed T cell activation.  Interferons are known to enhance CD95 expression, and our group (Critchley-Thorne et. al, 2009) has shown reduced upregulation of CD95 in PBMC T cells from breast cancer patients following T cell activation in the presence of interferons, indicating the lack of full T cell activation under these conditions.

Thus both CD71 and CD95 are upregulated in the mid-early phase of T cell activation and dysfunctional expression may be useful measures of T cell dysfunction in various disease states. Thus, these may be useful markers when assessing the phenotype and function of human PBMCs.

 

Additional Reading:

Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.  Reddy M, Eirikis E, Davis C, Davis HM, Prabhakar U. J Immunol Methods. 2004 Oct;293(1-2):127-42.

Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Biselli R, Matricardi PM, D’Amelio R, Fattorossi A. Scand J Immunol. 1992 Apr;35(4):439-47.

Surface markers of lymphocyte activation and markers of cell proliferation.  Shipkova M, Wieland E.  Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.  Caruso A, Licenziati S, Corulli M, Canaris AD, De Francesco MA, Fiorentini S, Peroni L, Fallacara F, Dima F, Balsari A, Turano A.   Cytometry. 1997 Jan 1;27(1):71-6.

Transferrin receptor induces tyrosine phosphorylation in T cells and is physically associated with the TCR zeta-chain.  Salmerón A, Borroto A, Fresno M, Crumpton MJ, Ley SC, Alarcón B. J Immunol. 1995 Feb 15;154(4):1675-83.

Transferrin synthesis by inducer T lymphocytes.  Lum JB, Infante AJ, Makker DM, Yang F, Bowman BH. J Clin Invest. 1986 Mar;77(3):841-9.

Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma.  Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. PLoS Med. 2007 May;4(5):e176.

Pro- and anti-apoptotic CD95 signaling in T cells.  Paulsen M, Janssen O. Cell Commun Signal. 2011 Apr 8;9:7.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling.  Strauss G, Lindquist JA, Arhel N, Felder E, Karl S, Haas TL, Fulda S, Walczak H, Kirchhoff F, Debatin KM.  J Exp Med 2009, 206:1379-1393.

Impaired interferon signaling is a common immune defect in human cancer.  Critchley-Thorne RJ, Simons DL, Yan N, Miyahira AK, Dirbas FM, Johnson DL, Swetter SM, Carlson RW, Fisher GA, Koong A, Holmes S, Lee PP. Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):9010-5.

*Image courtesy of http://en.wikipedia.org/wiki/Fas_ligand*

T cell dysfunction in cancer: Anergy, Exhaustion, or Senescence?

Immune responses against cancer have been shown to be effective in eliminating tumors.  However anti-tumor immunity is limited by dysfunctional T cells which have been described in cancer patients.  Understanding how dysfunctional T cells arise in cancer and the potential mechanisms for restoring functionality are critical for developing effective immunotherapeutics.  In the current issue of Current Opinion in Immunology, Dr. Weiping Zou’s group, (Crespo. et. al.,) review the various types of T cell dysfunction that occur in the tumor microenvironment.

Anergy, exhaustion, and senescence are three different mechanisms underlying T cell hyporesponsiveness, which share distinguishing phenotypic features but arise by different mechanisms and under different experimental settings.  However, what is the difference between these three types of T cell dysfunction and which contribute to impaired T cell responses in the setting of cancer?

Anergy: T cell anergy generally refers to a hyporesponsive state in T cells induced by triggering the TCR either without adequate concomitant co-stimulation through CD28 or in the presence of high co-inhibitory molecule signaling.  Without both TCR and CD28 signals, IL-2 is not effectively transcribed and instead, anergy-associated genes such as GRAIL are expressed which contribute to impaired TCR signaling via negative feedback.

In the tumor microenvironment cancer, altered expression of B7 family members by APCs leads to an enhanced expression of B7 family co-inhibitory molecules including PD-L1 and a reduction in the B7 family co-stimulatory molecules CD80 and CD86.  Thus, T cell activation in this environment could lead to induction of anergy.

Exhaustion: T cell exhaustion occurs as a result of chronic over-stimulation, such as occurs in the settings of chronic viral infections including hepatitis C virus (HCV) and HIV, autoimmunity, and cancer.  Exhausted T cells progressively lose the ability to express effector cytokines including IL-2, IFNg, and TNFα.  They also express multiple inhibitory receptors including PD-1 and LAG-3, lose cytotoxic and proliferative potential, and may ultimately be driven to apoptosis.

Because anti-tumor T cells are persistently exposed to antigen in the tumor microenvironment, exhaustion is a likely mechanism contributing to T cell dysfunction in cancer patients.  As such, exhausted T cells have been described in patients with melanoma, ovarian cancer and hepatocellular carcinoma.

Senescence:  Senescence is thought to occur due to the natural life span, or aging of cells.  However, senescent T cells have been observed in the settings of chronic inflammation and persistent infection in young individuals, indicating other factors beyond a person’s age, such as DNA damage, drive acquisition of this state.  Senescent T cells are marked by deficient CD28 expression, telomere shortening, expression of regulatory receptors such as TIM-3 and KLRG-1, and inability to progress through the cell cycle.

Cells with features of senescence have been described in patients with lung cancer, head and neck cancer, hepatocellular carcinoma, melanoma and lymphoma.  Thus, senescence may also contribute to T cell dysfunction in cancer patients.

In conclusion, there is evidence that anergy, exhaustion, and senescence may all be contributing toward T cell dysfunction in cancer.  In the review, Crespo. et. al., make the point that distinguishing cells in these states may be complicated as they overlap phenotypically and in expression of various markers.  Furthermore, the mechanisms mediating establishment of these three states is not well defined.  However, it is important to clarify the mechanisms by which T cells gain and maintain dysfunction in cancer in order to best develop effective immunotherapeutics.

 

Further Reading:

 T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment.  Crespo J, Sun H, Welling TH, Tian Z, Zou W.  Curr Opin Immunol. 2013 Jan 5.

The three main stumbling blocks for anticancer T cells.  Baitsch L, Fuertes-Marraco SA, Legat A, Meyer C, Speiser DE. Trends Immunol. 2012 Jul;33(7):364-72.

Induction of T cell anergy: integration of environmental cues and infectious tolerance. P. Chappert, R.H. Schwartz.  Curr. Opin. Immunol., 22 (2010), pp. 552–559.

T cell exhaustion.  E.J. Wherry.  Nat Immunol, 12 (2011), pp. 492–499.

T-cell senescence: a culprit of immune abnormalities in chronic inflammation and persistent infection.  A.N. Vallejo, C.M. Weyand, J.J. Goronzy.  Trends Mol Med, 10 (2004), pp. 119–124.

Upcoming Immunology Conferences: March – June, 2013

antibodiesI previously posted about 2013 Conferences in Tumor Immunology and Cancer ImmunotherapyThis listing will include other upcoming Immunology-related conferences.

 

Keystone Symposium: Understanding Dendritic Cell Biology to Improve Human Disease

March 3 – 8, 2013.

Keystone, Colorado, USA.

Registration Deadline: March 3, 2013.

 

Gordon Research Conference: Cell Biology of Megakaryocytes & Platelets

March 10 – 15, 2013.

Galveston, Texas, USA

Application Deadline: February 10, 2013.

 

World Immune Regulation Meeting (WIRM) VII: Innate and Adaptive Immune Response and Role of Tissues in Immune Regulation

March 13 – 16, 2013.

Congress Center, Davos, Switzerland.

Registration is still open online.

 

Keystone Symposium: Host Response in Tuberculosis

This is a joint meeting with the Keystone meeting on Tuberculosis: Understanding the Enemy

March 13 – 18, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: March 3, 2013.

 

Keystone Symposium: Tuberculosis: Understanding the Enemy

This is a joint meeting with the Keystone meeting on Host Response in Tuberculosis.

March 13 – 18, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: March 3, 2013.

 

Keystone Symposium: Immune Activation in HIV Infection: Basic Mechanisms and Clinical Implications

April 3 – 8, 2013.

Breckenridge, Colorado, USA.

Registration Deadline: April 3, 2013.

 

Canadian Society for Immunology 26th Annual Spring Meeting.

April 5 – 8, 2013.

TELUS Whistler Conference Centre, Whistler, British Columbia, Canada.

Registration is open online.  Early registration ends March 1, 2013.

Abstract Submission Deadline: March 1, 2013.

 

Keystone Symposium: Immunopathology of Type 1 Diabetes

April 4 – 9, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: April 4, 2013.

 

Keystone Symposium: Advances in the Knowledge and Treatment of Autoimmunity

April l4 – 9, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: April 4, 2013.

 

Molecular Pattern Recognition Receptors

April 11 – 13, 2013.

Boston, Massachusetts, USA.

Early Registration Deadline: March 1, 2013.

 

Clinical Immunology Society Annual Meeting: Regulation and Dysregulation of Immunity

April 25 – 28, 2013.

Miami, Florida, USA.

Pre-Registration Deadline: April 3, 2013.

 

T cell Function and Modulation Meeting

April 28 – May 1, 2013.

Makena Beach & Golf Resort, Maui (Makena), HI.

Registration can be submitted online and is limited to the first 125 attendees.

 

IMMUNOLOGY 2013, AAI Annual Meeting and Centennial Celebration

May 3 – 7, 2013.

Hawaii Convention Center, Honolulu, Hawaii, USA.

Registration is open online.  Early registration ends March 18, 2013.

Abstract Submission Deadline: February 13, 2013.

 

Keystone Symposium: The Innate Immune Response in the Pathogenesis of Infectious Disease

May 5 – 10, 2013.

A Universidade Federal de Ouro Preto, Ouro Preto, Brazil.

Early Registration Deadline: March 5, 2013.

 

Cell Symposia: Microbiome and Host Health

May 12 – 14, 2013.

Lisbon, Portugal.

Abstract Submission Deadline: February 8, 2013.

Early Registration Deadline: March 9, 2013.

 

30 Years of HIV Science: Imagine the Future

May 21 – 23, 2013.

The International Research Centre in Paris, the Institut Pasteur Conference Centre, Paris, France.

Abstract Submission Deadline: March 1, 2013.

Early Registration Deadline: March 20, 2013.

 

Abcam: Allergy & Asthma 2013

May 23 – 24, 2013.

Bruges, Belgium.

Oral Abstract Submission Deadline: February 22, 2013.

Poster Abstract Submission Deadline: March 25, 2013.

Early Registration Deadline: March 25, 2013.

 

ISIR 2013 – Building Bridges in Reproductive Immunology.

May 28 – June 1, 2013.

Boston Park Plaza Hotel, Boston, Massachusetts, USA.

Registration is open online.

Abstract Submission Deadline: February 15, 2013.

 

78th Cold Spring Harbor Symposium on Quantitative Biology: Immunity & Tolerance

May 29 – June 3, 2013.

Cold Spring Harbor Laboratory, New York, USA

Abstract and Registration Deadline: March 15, 2013.

 

6th International Singapore Symposium of Immunology.

June 5 – 6, 2013.

Matrix Level 2 Auditorium, Biopolis, Singapore.

Registration is open online.

Abstract Submission Deadline: April 5, 2013.

 

Cell Symposium: Immunometabolism: From Mechanisms to Therapy

June 9 – 11, 2013.

The Sheraton Centre Toronto Hotel, Toronto, Canada.

Abstract Submission Deadline: February 22, 2013.

Early Registration Deadline: April 5, 2013.

 

Gordon Research Conference: Mucosal Health & Disease

June 9 – 14, 2013.

Stonehill College, Easton, Massachusetts, USA.

Application Deadline: May 12, 2013.

 

Gordon Research Conference: Phagocytes

June 9 – 14, 2013.

Waterville Valley, New Hampshire, USA.

Application Deadline: May 12, 2013.

 

European Academy of Allergy & Clinical Immunology and World Allergy Organization: World Allergy & Asthma Congress

June 22 – 26, 2013.

Milan, Italy.

Early Registration Deadline: February 20, 2013.

 

Aegean Conference: 10th International Conference on Innate Immunity

June 23 – 28, 2013.

Kos, Greece.

Early Registration and Abstract Submission Deadline: March 15, 2013.

Gordon Research Conference: Apoptotic Cell Recognition & Clearance

June 23 – 28, 2013.

University of New England, Biddeford, Maine, USA.

Application Deadline: May 26, 2013.

 

Abcam: Inflammasomes in Health and Disease

June 24 – 25, 2013.

Boston, Massachusetts, USA.

Oral Abstract Submission Deadline: April 26, 2013.

Poster Abstract Submission Deadline: May 17, 2013.

Early Registration Deadline: April 26, 2013.

 

FOCIS 2013

June 27 – 30, 2013.

Boston, Massachusetts, USA.

Late-breaking Abstract Submission Deadline: April, 2, 2013.

Registration Deadline for Poster Presenters:  March 28, 2013.

Websites that list upcoming Conferences & Events in Immunology, Tumor Immunology, and Cancer Immunotherapy:

The American Association of Immunologists (AAI) Meetings and Events Calendar

Nature Reviews Immunology’s list of conferences

Cancer Immunity Journal’s List of Conferences

Human PBMC T cell immediate early activation markers: What are they and what do they do?

melanoma dividing cellsThere are many strategies for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC).  T cells that have recently been activated through their T cell receptor (TCR) will express a series of activation markers at different time points following activation.   Activation markers include receptors such as chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins.  Some of these molecules have established functions in T cell biology, while the relevance or function of others remains elusive.  Flow cytometry is the method of choice for evaluating various types of surface or intracellular markers that indicate the activation status of T cells.  However, what are these markers, what is their function in T cell biology, what T cell populations will express them, and when can they be assessed are key questions to address when deciding which markers are best for a given assay and question of interest.

In this article, the first of a short series, I will discuss two of the most commonly used immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.

Immediate Early Activation Markers:

CD69 (AIM, Leu23, MLR3) is a signaling membrane glycoprotein involved in inducing T cell proliferation. CD69 is expressed at very low levels on resting CD4+ or CD8+ T cells in PBMC (<5-10%), and is one of the earliest assessable activation markers, being rapidly upregulated on CD4+ or CD8+ T cells within 1 hour of TCR stimulation or other T cell activators such as phorbol esters via a protein kinase C (PKC) dependant pathway.  Expression of CD69 peaks by 16-24 hours and then declines, being barely detectable 72 hours after the stimulus has been withdrawn.

The inability to upregulate CD69 following TCR activation may be associated with T cell dysfunction.  For instance, Critchley-Thorne et. al, showed that PBMC T cells from metastatic melanoma patients with lower responsiveness to interferons had reduced CD69 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients.  Thus CD69 expression may be a measure of T cell dysfunction in human disease.

CD40L (CD154) is a member of the TNF-receptor superfamily that functions as a co-stimulatory molecule by binding CD40 which is constitutively expressed on antigen presenting cells (APCs).  The CD40L-CD40 ligation results in the activation of multiple downstream pathways including the MAPK (JNK, p38, ERK1/2), NF-ĸB, and STAT3 transcription factors.  CD40L expression is quickly upregulated within 1-2 hours after TCR stimulation via the transcription factors NFAT and AP-1.  CD40L expression peaks near 6 hours after stimulation, and declines by 16-24hrs. CD40L expression however is biphasic, and the addition of anti-CD28 or IL-2 along with TCR stimulation leads to sustained expression for several days (Snyder et. al., 2007).

Expression of CD40L on resting PBMC CD4+ or CD8+ T cells from healthy donors is very low (<1%).  However this percentage has been shown to be significantly increased on up to 17% of CD4+ T cells and 21% of CD8+ T cells in patients with active SLE, and these differences between healthy and SLE patients were also seen following anti-CD3 stimulation of PBMCs (Desai-Mehta, et. al, 1996).  The review below by Daoussis et. al, discusses the role of CD40L expression in several other human diseases.

In summary, CD69 and CD40L are both rapidly induced following T cell activation and both exert important functions in T cell biology. Expressions of these markers have both been shown to be altered in various human diseases.  Understanding the biology of T cell activation markers will allow for the best application of these markers to specific experimental questions and assay types.

 

Additional Reading:

Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Biselli R, Matricardi PM, D’Amelio R, Fattorossi A. Scand J Immunol. 1992 Apr;35(4):439-47.

Surface markers of lymphocyte activation and markers of cell proliferation.  Shipkova M, Wieland E.  Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.  Caruso A, Licenziati S, Corulli M, Canaris AD, De Francesco MA, Fiorentini S, Peroni L, Fallacara F, Dima F, Balsari A, Turano A.   Cytometry. 1997 Jan 1;27(1):71-6.

T cell activation via Leu-23 (CD69).  Testi R, Phillips JH, Lanier LL. J Immunol. 1989 Aug 15;143(4):1123-8.

A whole-blood assay for qualitative and semiquantitative measurements of CD69 surface expression on CD4 and CD8 T lymphocytes using flow cytometry.  Lim LC, Fiordalisi MN, Mantell JL, Schmitz JL, Folds JD. Clin Diagn Lab Immunol. 1998 May;5(3):392-8.

Utility of flow cytometric detection of CD69 expression as a rapid method for determining poly- and oligoclonal lymphocyte activation.  P E Simms and T M Ellis.  Clin Diagn Lab Immunol. 1996 May; 3(3): 301–304.

Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma.  Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. PLoS Med. 2007 May;4(5):e176.

Direct inhibition of CD40L expression can contribute to the clinical efficacy of daclizumab independently of its effects on cell division and Th1/Th2 cytokine production.  Snyder JT, Shen J, Azmi H, Hou J, Fowler DH, Ragheb JA. Blood. 2007 Jun 15;109(12):5399-406.

Targeting CD40L: a Promising Therapeutic Approach.  D. Daoussis, A.P. Andonopoulos, and S. C. Liossis. Clin Diagn Lab Immunol. 2004 July; 11(4): 635–641.

Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J. Clin. Investig. 97:2063-2073. Desai-Mehta, A., L. Liangjun, R. Ramsey-Goldman, and S. Datta. 1996.

Photo credit: wellcome images / Foter.com / CC BY-NC-ND

New role for p53 in regulating the inflammatory tumor microenvironment

small 3316209998p53 (TP53), a transcription factor activated by various types of cellular stress, is one of the most well studied tumor suppressor proteins.  Stress signals such as various types of DNA damage lead to p53 activation and subsequent induction of genes involved in cell cycle arrest, DNA repair, and apoptosis.

In many types of cancers including colorectal cancer, loss of function mutations in p53 commonly occur, particularly in the DNA-binding domain.  Thus, transcriptional activity of p53 is altered or lost, and cells with damaged genomes maintain viability.  In colorectal cancer, p53 mutations commonly occur at early stages and are thought to allow for accumulation of DNA damage and survival of mutated cells during tumorigenesis.

In the January issue of Cancer Cell, Schwitalla et. al, demonstrate that p53 plays a role in not only its classically associated functions as a critical DNA-damage checkpoint in early stages of tumorigenesis, but during the progression stage, p53 plays an additional significant role in regulating the inflammatory tumor microenvironment.

To study the role of p53 during various stages of carcinogenesis, the authors established a colon tumor model in which azoxymethane (AOM), a promoter of Wnt-driven tumorigenesis that induces stabilizing mutations in β-catenin, was administered to mice with a p53-deficiency in intestinal epithelial cells.  A gene expression analysis of AOM-derived tumors from wild-type versus p53-deficient mice, found that p53 deficiency led to upregulation of genes involved in inflammation and epithelial-mesenchymal transition (EMT).  A large number of genes upregulated in p53-deficient tumors were found to be regulated by the inflammatory transcription factor NF-ĸB, and phosphorylated p65, a major transcriptional co-factor in the classical NF-ĸB pathway, was enhanced in p53-deficient tumors.  NF-ĸB regulated genes included the EMT gene Twist as well as myeloid cell chemokines CXCL1, CXCL2, and CCL2, indicating that loss of p53 may lead to EMT and recruitment of inflammatory myeloid cells via upregulation of the NF-ĸB pathway.

Reciprocal regulation of the NF-ĸB and p53 pathways has long been known, and these pathways play opposing roles in cell fate in response to stress. However, in this study, the authors demonstrated that p53-deficient intestinal epithelial cells were impaired in their epithelial barrier function: leakage of intestinal bacteria and LPS resulted in the observed NF-ĸB activation and NF-ĸB-regulated gene expression.  Interestingly, NF-ĸB signaling was required in both tumor cells and the myeloid cells for maximal tumor progression.  NF-ĸB signaling in intestinal epithelial cells was required for recruitment of myeloid cells to the tumor microenvironment and EMT induction. NF-ĸB signaling in hematopoietic cells was required for tumor cell proliferation, survival, and metastasis, by inducing expression of cytokines that activate STAT3 in the tumor cells.

Thus, in this model, p53 plays an additional role during tumor progression by regulating inflammation in the tumor microenvironment, via its effects on intestinal barrier function.  In my last blog post, I discussed another recent study demonstrating links between the TNFα/NF-ĸB, IL-6/STAT3, and SphK1/S1P pathways in chronic inflammation driven colorectal cancer.  Thus, this study adds another layer of links to consider in the complicated interactions between inflammatory cells and tumor cells in the tumor microenvironment that drive colorectal cancer progression: p53, NF-ĸB, and STAT3.

 

Further Reading:

Loss of p53 in Enterocytes Generates an Inflammatory Microenvironment Enabling Invasion and Lymph Node Metastasis of Carcinogen-Induced Colorectal Tumors.  Schwitalla S, Ziegler PK, Horst D, Becker V, Kerle I, Begus-Nahrmann Y, Lechel A, Rudolph KL, Langer R, Slotta-Huspenina J, Bader FG, Prazeres da Costa O, Neurath MF, Meining A, Kirchner T, Greten FR. Cancer Cell. 2013 Jan 14;23(1):93-106.

p53 and NF-κB: different strategies for responding to stress lead to a functional antagonism.  Ak P, Levine AJ. FASEB J. 2010 Oct;24(10):3643-52.

Alterations of the TP53 gene in gastric and esophageal carcinogenesis.  Bellini MF, Cadamuro AC, Succi M, Proença MA, Silva AE. J Biomed Biotechnol. 2012;2012:891961.

Transcriptional control of human p53-regulated genes. T. Riley, E. Sontag, P. Chen, A. Levine.  Nat. Rev. Mol. Cell Biol., 9 (2008), pp. 402–412.

Immunity, inflammation, and cancer.  S.I. Grivennikov, F.R. Greten, M. Karin.  Cell, 140 (2010), pp. 883–899.

photo credit: sc63 via photopin cc

Signal amplification loops in chronic inflammation and Colon Cancer

The inflammatory gastrointestinal diseases Crohn’s disease, inflammatory bowel diseases (IBD), and ulcerative colitis (UC), are all known to be key risk factors in the development of colorectal cancer.  TNFα and IL-6 are two pro-inflammatory cytokines that signal to activate the NF-ĸB and STAT3 transcription factors, respectively.  Both pathways are known to play a role in the persistent inflammation that drives development of colitis-associated colorectal cancer.

Sphingosine-1-phosphate (S1P) is a lysophospholipid that is metabolized from phosphorylation of sphingosine by the kinases, SphK1 and SphK2, and signals through the G-protein coupled receptor sphingosine-1-phosphate receptor-1 (S1PR1).  The S1P-S1PR1 signaling pathway has been shown to activate STAT3 and induce expression of both IL-6 and S1PR1, driving a positive feedback loop leading to persistent STAT3 activation in tumor cells (Lee et. al., 2010).  S1P and SphK1 have also been shown to play a critical role in the NF-ĸB signaling pathway.  S1P was required for TRAF2 E3 ubiquitin ligase activity and activation of NF-ĸB by TNFα independently from the S1PR1 receptor pathway (Alvarez et. al., 2010).  SphK1 has also been linked to development of colorectal cancer.  SphK1 was highly expressed in human colons cancers as compared with normal mucosa, and SphK1-/- mice were less susceptible to azoxymethane induction of colon cancer (Kawamori et.al., 2009).

In a study by Liang et al. in this month’s issue of Cancer Cell, the authors close these links between the TNFα/NF-ĸB, IL-6/STAT3, and SphK1/S1P pathways in chronic inflammation driven colorectal cancer.  In the dextran sodium sulfate (DSS) murine model of colitis driven tumorigenesis, deficiency of SphK2 led to a compensatory upregulation of SphK1 and S1P production, and exacerbated DSS driven colitis and subsequent tumorigenesis.  S1P-driven NF-ĸB activation was enhanced, as was production of TNFα, IL-6, and STAT3 activation in SphK2-/- mice, and the SphK1/S1P pathway primarily exerted its activities in colonic mucosa infiltrating immune cells.  FTY720, an antagonist of S1PR1 and SphK1, was able to inhibit the S1P positive-feedback loop and block persistent activation of STAT3 and NF-ĸB and production of IL-6.  Additionally, FTY720 inhibited the development of tumors in this colitis driven tumorigenesis murine model.

In summary, these studies show that SphK1 drives a feed-forward signal amplification loop that exacerbates inflammation and promotes tumorigenesis by production of S1P.  S1P then drives S1PR1 activation of STAT3 leading to further production of S1PR1, as well as activates NF-ĸB and induction of TNFα and IL-6 transcription, which further feed back on the NF-ĸB and STAT3 pathways, respectively.  Blocking this amplification loop by inhibiting S1PR1 and SphK1 may thus be a promising treatment strategy for colitis-associated colorectal cancer.

 

 

Further Reading:

Sphingosine-1-Phosphate Links Persistent STAT3 Activation, Chronic Intestinal Inflammation, and Development of Colitis-Associated Cancer.  Liang J, Nagahashi M, Kim EY, Harikumar KB, Yamada A, Huang WC, Hait NC, Allegood JC, Price MM, Avni D, Takabe K, Kordula T, Milstien S, Spiegel S. Cancer Cell. 2013 Jan 14;23(1):107-20.

Sphingosine 1-Phosphate Is a Missing Link between Chronic Inflammation and Colon Cancer.  Pyne NJ, Pyne S. Cancer Cell. 2013 Jan 14;23(1):5-7.

STAT3-induced S1PR1 expression is crucial for persistent STAT3 activation in tumors.  H. Lee H, Deng J, Kujawski M, Yang C, Liu Y, Herrmann A, Kortylewski M, Horne D, Somlo G, Forman S, Jove R, Yu H.. Nat. Med., 16 (2010), pp. 1421–1428.

Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2.  Alvarez SE, Harikumar KB, Hait NC, Allegood J, Strub GM, Kim EY, Maceyka M, Jiang H, Luo C, Kordula T, Milstien S, Spiegel S. Nature. 2010 Jun 24;465(7301):1084-8.

Role for sphingosine kinase 1 in colon carcinogenesis. T. Kawamori, T. Kaneshiro, M. Okumura, S. Maalouf, A. Uflacker, J. Bielawski, Y.A. Hannun, L.M. Obeid.  FASEB J., 23 (2009), pp. 405–414.

Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network.  Rosen H. and Goetzl EJ.   Nature Reviews Immunology 5, 560-570, July 2005.

 Immunity, inflammation, and cancer.  S.I. Grivennikov, F.R. Greten, M. Karin.  Cell, 140 (2010), pp. 883–899.

Intestinal inflammation and cancer.  T.A. Ullman, S.H. Itzkowitz.  Gastroenterology, 140 (2011), pp. 1807–1816.