Naive tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma

In vitro differentiated CD8⁺ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4⁺ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naive tumor/self-specific CD4⁺ T cells naturally differentiated into T helper type 1 cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8⁺, B, natural killer (NK), and NKT cells. Proper activation of CD4⁺ T cells in vivo was important for tumor clearance, as naive tumor-specific CD4⁺ T cells could not completely treat tumor in lymphopenic common gamma chain (γ_c)–deficient hosts. γ_c signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4⁺ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4⁺ T cells.Adoptive cellular therapy (ACT) of cancer using in vitro differentiated CD8⁺ T cells is a powerful treatment against established cancer in humans and mice. In recent years, great progress has been attained in the understanding of the mechanisms involved in enhancing treatment of large established tumors (Gattinoni et al., 2006). Lymphodepletion before adoptive therapy greatly enhances ACT in humans and mice through the creation of cytokine sinks, removal of regulatory T cells (T reg cells), and the release of toll-like receptor agonists (Gattinoni et al., 2005a; Paulos et al., 2007; Dudley et al., 2008). Recent evidence suggests that irradiation also enhances the expression of ICAM and VCAM in the tumor vasculature allowing tumor-reactive T cells to enter more readily (Quezada et al., 2008). Although CD8⁺ T cells are potent mediators of antitumor immunity, there has been little focus on tumor-specific CD4⁺ T cells. CD4⁺ Th cells are important in immunity because in the absence of help, CD8⁺ T cells can be deleted or lose the capacity to develop into memory CD8⁺ T cells upon rechallenge (Janssen et al., 2003; Antony et al., 2005; Williams et al., 2006). Therefore, the use of tumor/self-reactive CD8⁺ T cells in the adoptive immunotherapy of cancer may face similar fates because T cells must remove tumor antigen in the context of persisting self-antigen, which in some cases leads to autoimmunity (Gattinoni et al., 2006; Rosenberg et al., 2008). Adoptive cell therapies that incorporate CD4⁺ T cells are far superior to therapies that only use CD8⁺ T cell clones (Dudley et al., 2002). Therefore, one theoretical means of improving immunotherapy to self may involve the provision of tumor-reactive or self-reactive CD4⁺ T cells (Nishimura et al., 1999; Marzo et al., 2000; Antony et al., 2005), but a more direct role for CD4⁺ T cells in tumor immunity remains unclear (Ho et al., 2002; Muranski and Restifo, 2009).Recently, adoptive transfer of in vitro differentiated tumor-specific CD4⁺ T cells in humans and mice has shown promise against cancer as a therapy (Nishimura et al., 1999; Perez-Diez et al., 2007; Hunder et al., 2008; Muranski et al., 2008). This has rekindled the idea of using antigen-specific CD4⁺ Th during immunotherapy because CD4⁺ Th cells can mediate the proper signals required in vivo to activate CD8⁺ T cells and other cells of the innate immune system (Kahn et al., 1991; Hung et al., 1998; Nishimura et al., 1999; Antony et al., 2006; Williams et al., 2006). In fact, several preclinical and clinical trials have shown the importance of CD4 help during immunotherapy of cancer (Nishimura et al., 1999; Antony et al., 2006; Dudley et al., 2008). However, isolation of tumor-specific CD4⁺ T cells has been difficult (Wang, 2001) and only a few MHC class II vaccines have been produced as a result of the lack of knowledge of how to generate vaccines that specifically activate Th cells instead of tumor-specific Foxp3⁺ T reg cells (Rosenberg, 2001; Vence et al., 2007). In addition, lack of appropriate mouse models to study tumor-specific CD4⁺ T cell responses to self-antigens has hindered progress in our understanding of the role of CD4⁺ T cells in maintaining immunity to cancer.Now, with a better understanding of CD4⁺ T cell biology, the use of cytokines to differentiate and expand T cells in vitro has led to a panoply of CD4 lineages with specific in vivo functions (Weaver and Rudensky, 2009). For example, in vitro differentiated CD4⁺ Th17 tumor-specific T cells have shown superiority over CD4⁺ Th1 differentiated T cells in the adoptive immunotherapy of cancer in a mouse model of melanoma (Muranski et al., 2008). IL-2 and IL-7 in vitro expanded NY-ESO-1–specific CD4⁺ T cells in humans have also shown clinical promise in one patient who had not received prior lymphodepleting conditioning or a vaccine (Hunder et al., 2008). Although these are promising studies, the mechanisms involved in the direct therapy of cancer by CD4⁺ T cells remain elusive. Likewise, methods for enhancing adoptive immunotherapy without prior in vitro manipulation that may lead to the terminal differentiation of T cells also remain unclear (Gattinoni et al., 2005b, 2009; Klebanoff et al., 2005). Although such manipulations can lead to vaccine independence (Klebanoff et al., 2009), long-term benefits from in vivo differentiation may outweigh in vitro stimulation because the in vivo environment may provide the correct signals that cannot be attained in a culture dish.To test a direct role for CD4⁺ T cells in the immunotherapy of cancer, we used a gp75/tyrosinase-related protein (TRP) 1–specific CD4⁺ TCR transgenic (Tg) mouse that produces class II–restricted T cells that recognize mouse TRP-1 in the context of I-A^b (Muranski et al., 2008). TRP-1 is expressed in malignant melanoma and in the skin and eyes of mice and humans; therefore, this model mimics the human condition as closely as possible. Surprisingly, we found that adoptive transfer of naive TRP-1–specific CD4⁺ T cells into lymphopenic animals bearing large established melanoma caused tumor regression and depigmentation independent of vaccination, cytokine administration, and CD8⁺, B, NK, and NKT cells. This therapy was dependent on common gamma chain (γ_c) signaling in the host for survival and differentiation of CD4⁺ T cells in vivo. These data provide a better understanding for the design of immunotherapies that incorporate tumor/self-reactive CD4⁺ T cells.     

Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium

Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4⁺ T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.Inducing specific immunity and maintaining tolerance requires cells of the mononuclear phagocyte lineage. This lineage is comprised of three closely related cell types: DCs, monocytes, and macrophages (Shortman and Naik, 2007; Geissmann et al., 2010a,b; Liu and Nussenzweig, 2010; Yona and Jung, 2010; Chow et al., 2011). DCs are essential to both immunity and tolerance (Steinman et al., 2003); however, the role monocytes and macrophages play in these processes is not as well defined (Geissmann et al., 2008).In mice, DCs and monocytes arise from the same hematopoietic progenitor, known as the macrophage–DC progenitor (MDP; Fogg et al., 2006). Their development diverges when MDPs become either common DC progenitors (CDPs) that are Flt3L-dependent, or monocytes, which are dependent on CSF1 (M-CSF; Witmer-Pack et al., 1993; McKenna et al., 2000; Fogg et al., 2006; Waskow et al., 2008). CDPs develop into either plasmacytoid DCs or preDCs that leave the bone marrow to seed lymphoid and nonlymphoid tissues, where they further differentiate into conventional DCs (cDCs; Liu et al., 2009). In contrast, monocytes circulate in the blood and through tissues, where they can become activated and develop into several different cell types, including some but not all tissue macrophages (Schulz et al., 2012; Serbina et al., 2008; Yona et al., 2013).Despite their common origin from the MDP, steady-state lymphoid tissue cDCs can be distinguished from monocytes or macrophages by expression of cell surface markers. For example, cDCs in lymphoid tissues express high levels of CD11c and MHCII, but lack the expression of CD115 and F4/80 found in monocytes and macrophages, respectively. However, this distinction is far more difficult in peripheral tissues, like the intestine or lung, or during inflammation when monocytes begin to express many features of DC including high levels of MHCII and CD11c (Serbina et al., 2003; León et al., 2007; Hashimoto et al., 2011).The function of cDCs in immunity and tolerance has been explored extensively using a series of different mutant mice to ablate all or only some subsets of cDCs (Jung et al., 2002; Liu and Nussenzweig, 2010; Chow et al., 2011). In contrast, the methods that are currently available to study the function of monocytes and macrophages in vivo are far more restricted and less specific (Wiktor-Jedrzejczak et al., 1990; Dai et al., 2002; MacDonald et al., 2010; Chow et al., 2011). For example, Ccr2^−/− and Ccr2^DTR mice (Boring et al., 1997; Kuziel et al., 1997; Serbina and Pamer, 2006; Tsou et al., 2007) have been used to study monocytes (Boring et al., 1997; Peters et al., 2004; Hohl et al., 2009; Nakano et al., 2009). However, CCR2 is also expressed on some subsets of cDCs, activated CD4⁺ T cells, and NK cells (Kim et al., 2001; Hohl et al., 2009; Egan et al., 2009; Zhang et al., 2010). Thus, it is challenging to dissect the precise role of monocytes as opposed to other cell types in immune responses in Ccr2^−/− or Ccr2^DTR mice. Inducible DTR expression in CD11c^Cre x CX₃CR1^LsL-DTR mice is far more specific (Diehl et al., 2013), but restricted to a small subset of mononuclear phagocytes.Here, we describe a genetic approach to targeting monocytes and macrophages that spares cDCs and lymphocytes, and we compare the effects of monocyte and macrophage ablation to cDC depletion on the adaptive immune response to intestinal infection with Citrobacter rodentium.     

Absence of MHC class II on cDCs results in microbial-dependent intestinal inflammation

Conventional dendritic cells (cDCs) play an essential role in host immunity by initiating adaptive T cell responses and by serving as innate immune sensors. Although both innate and adaptive functions of cDCs are well documented, their relative importance in maintaining immune homeostasis is poorly understood. To examine the significance of cDC-initiated adaptive immunity in maintaining homeostasis, independent of their innate activities, we generated a cDC-specific Cre mouse and crossed it to a floxed MHC class II (MHCII) mouse. Absence of MHCII on cDCs resulted in chronic intestinal inflammation that was alleviated by antibiotic treatment and entirely averted under germ-free conditions. Uncoupling innate and adaptive functions of cDCs revealed that innate immune functions of cDCs are insufficient to maintain homeostasis and antigen presentation by cDCs is essential for a mutualistic relationship between the host and intestinal bacteria.Conventional DCs (cDCs) are specialized immune cells that link the innate and adaptive immune system. Innate features of cDCs allow them to recognize and respond to pathogens by producing essential cytokines such as IL-6, IL-12, IL-23, and TNF. These cytokines contribute to the activation of other immune cells, including T and B cells and cells of the innate immune system. For example, in the intestine, cDCs sense bacteria and produce IL-23, which induces type III innate lymphoid cells (ILC3s) to produce IL-22, which in turn stimulates production of antimicrobial peptides (AMPs; Sonnenberg et al., 2011; Kinnebrew et al., 2012; Satpathy et al., 2013; Bernink et al., 2015). In addition to their innate functions, cDCs initiate adaptive immune responses by ingesting, processing, and presenting antigens to T cells (Nussenzweig et al., 1980; Steinman et al., 2003). In the intestine, cDCs are responsible for transport of antigen to the draining mesenteric LNs (mLNs). Under physiological conditions, the capacity of cDCs to migrate from tissue to draining LNs distinguishes them from more sessile macrophages (Schreiber et al., 2013). The importance of cDCs in adaptive immune function is exemplified by the fact that cDC depletion during viral and bacterial infection results in impaired T cell immunity and increased susceptibility to infection (Jung et al., 2002; Kassim et al., 2006; Hildner et al., 2008; Satpathy et al., 2013; Schreiber et al., 2013).In mice, expression of Itgax (CD11c) is a hallmark of the DC lineage, and its expression has been used to label (CD11c^YFP), deplete (CD11c^DTR), and conditionally target (CD11c^Cre) cDCs (Jung et al., 2002; Lindquist et al., 2004; Caton et al., 2007; Stranges et al., 2007). However, CD11c is also expressed by plasmacytoid DCs (pDCs), activated monocytes, macrophages, and some NK cells, and therefore CD11c-based labeling and targeting strategies are not entirely cDC specific (Serbina et al., 2003; Hohl et al., 2009; Meredith et al., 2012; Schreiber et al., 2013). Higher levels of specificity can be achieved by deletion of genes that regulate the development of specific subsets of cDCs, such as Batf3, Irf4, and Notch2; however, these methods have yet to be applied to dissect the relative contributions of the innate and adaptive functions of cDCs to immune homeostasis (Caton et al., 2007; Hildner et al., 2008; Persson et al., 2013; Schlitzer et al., 2013).To investigate the adaptive functions of cDCs independent of innate activities, we produced a cDC-restricted Cre mouse wherein Cre expression is directed by the Zbtb46 gene (zDC^Cre) and used it to delete MHCII in cDCs in vivo. These mice exhibited profound intestinal inflammation that was directly related to the presence of intestinal bacteria, as antibiotic-treated or germ-free mice lacking MHCII on cDCs showed no signs of intestinal inflammation. Colonization of germ-free mice allowed us to monitor adaptive immune responses against intestinal bacteria and revealed that mice lacking MHCII on cDCs have a defect in inducing proper adaptive immune responses against commensals. Collectively, our studies reveal the importance of the adaptive function of cDCs in maintaining intestinal homeostasis.     

Profound CD4+/CCR5+ T cell expansion is induced by CD8+ lymphocyte depletion but does not account for accelerated SIV pathogenesis

Depletion of CD8⁺ lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8⁺ lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8⁺ lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4⁺ effector memory T (T_EM) cells and, to a lesser extent, transitional memory T (T_TrM) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4⁺/CCR5⁺ SIV “target” cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8⁺ lymphocytes in SIV⁻ RMs led to a sustained increase in the number of potential CD4⁺ SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4⁺ T_EM cell proliferation of CD8⁺ lymphocyte–depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4⁺ T_EM and T_TrM cell proliferation, it did not recapitulate the viral dynamics of CD8⁺ lymphocyte depletion. These data suggest that CD8⁺ lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.In the initial weeks of HIV infection of humans and pathogenic simian immunodeficiency virus (SIV) infection of Asian macaques, viral replication peaks, then declines to a quasiequilibrated set point of ongoing viral production and clearance, the level of which plays a major role in determining the subsequent tempo of disease progression (Mellors et al., 1996; Staprans et al., 1999). Outcomes range from an inability to substantially restrain viral replication from peak levels, leading to early immunological collapse and rapid progression to AIDS, to control of viral replication to undetectable levels and long-term nonprogression (Farzadegan et al., 1996; Picker et al., 2004; Deeks and Walker, 2007; Goulder and Watkins, 2008). However, the vast majority of infections manifest viral replication set points and progression rates between these two extremes (Munoz et al., 1989; Okoye et al., 2007). The mechanisms responsible for these different outcomes have not been precisely defined, although differences in adaptive immunity, innate immunity, and CD4⁺, CCR5⁺ target cell availability, susceptibility to infection, productivity (viral yield per infected cell), and dynamics have all been implicated (Goldstein et al., 2000; Seman et al., 2000; Zhang et al., 2004; Alter et al., 2007; Goulder and Watkins, 2008; Lehner et al., 2008; Mahalanabis et al., 2009).The HIV/SIV-specific CD8⁺ T cell response has been widely accepted as a major, if not dominant, contributor to this heterogeneity of outcomes based on the observations that (a) the appearance of these responses is temporally coordinated with the postpeak fall in viral replication (Koup et al., 1994), (b) vaccines that elicit strong CD8⁺ T cell responses can lower viral replication set points compared with unvaccinated controls (Wilson et al., 2006; Liu et al., 2009), (c) particular class 1 MHC alleles and their associated CD8⁺ T cell responses are strongly associated with postpeak control of viremia (Goulder and Watkins, 2008), (d) viral mutations facilitating escape from CD8⁺ T cell recognition can be associated with either loss of virologic control or a fitness cost that handicaps replication of escaped virus (Barouch et al., 2002; Goulder and Watkins, 2008), and (e) treatment of rhesus macaques (RMs) with depleting anti-CD8⁺ mAbs at the outset of SIV infection, transiently depleting CD8⁺ lymphocytes from blood and secondary lymphoid tissues, typically results in unrestrained viral replication and rapid disease progression (Matano et al., 1998; Schmitz et al., 1999; Kim et al., 2008; Veazey et al., 2008). On the other hand, there is considerable circumstantial evidence suggesting that the availability, susceptibility to infection, and cumulative per cell virus production of HIV/SIV target cells may also play a major role in determining acute-phase viral dynamics and subsequent viral load set points. In early acute SIV infection, the primary target cells are small, resting CD4⁺, CCR5⁺ T_EM and transitional memory T (T_TrM) cells in tissues; massive infection and destruction of these cells corresponds to the initial peak of viral replication and its subsequent decline (Picker et al., 2004; Li et al., 2005; Mattapallil et al., 2005). With the destruction of resting CD4⁺ target cells and the onset of infection-associated inflammation, the infection shifts to predominant replication in activated, proliferating CD4⁺ T_EM and T_TrM cells (Zhang et al., 2004; Haase, 2005). These observations suggest that in typical SIV infections, plateau-phase viral replication might depend on both the rate of new target cell production and the enhanced per cell virus production of activated target cells. Consistent with this, it has been well documented that both coinfection with other pathogens and other modes of immune activation in acute infection, which increase the number of activated target cells, are associated with increased levels of viral replication and rapid disease progression (Folks et al., 1997; Zhou et al., 1999; Sequar et al., 2002; Garber et al., 2004; Cecchinato et al., 2008).Determination of the interplay between cellular immune effector responses and target cell dynamics in the regulation of acute-phase HIV/SIV replication will be crucial to understand the potential impact of vaccines and other immunomodulators on HIV/SIV pathogenesis. However, experimental dissection of these processes in vivo is complicated by the fact that primary viral targets—CD4⁺ memory T cells—are an intrinsic and essential part of the adaptive immune response and are almost invariably coregulated with CD8⁺ lymphocyte–mediated effector responses. In this paper, we report on the use of CD8⁺ lymphocyte depletion in RMs, which effectively eliminates CD8⁺ effector lymphocyte responses in acute SIV infection (Matano et al., 1998; Schmitz et al., 1999; Kim et al., 2008; Veazey et al., 2008), to determine the importance of target cell expansion and activation on viral dynamics in acute SIV infection. We document that CD8⁺ lymphocyte depletion induces massive selective proliferation of CD4⁺, CCR5⁺ memory T cell targets, leading to profound expansion of this population in uninfected RMs and their increased consumption in acute SIV infection. Furthermore, we identify IL-15 as the major mediator of this proliferative response. Significantly, however, in vivo inhibition of IL-15 by administration of a neutralizing anti–IL-15 mAb during CD8⁺ lymphocyte depletion of acutely SIV-infected RMs blocked induction of CD4⁺ target cell proliferation, but did not reverse either the high viral replication or rapid disease progression in CD8⁺ lymphocyte–depleted, acutely infected animals. Moreover, administration of exogenous IL-15 during acute SIV infection in RMs with an intact CD8⁺ lymphocyte compartment stimulated CD4⁺ target cell proliferation with kinetics similar to CD8⁺ lymphocyte depletion, but did not recapitulate the early viral dynamics of CD8⁺ lymphocyte depletion. Collectively, these data provide compelling evidence that the relative effectiveness of adaptive and/or innate immune function of CD8⁺ lymphocytes plays a considerably more significant role in determining outcome of acute SIV infection than differences in target cell number and/or activation status.     

Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection

Leishmaniasis causes a significant disease burden worldwide. Although Leishmania-infected patients become refractory to reinfection after disease resolution, effective immune protection has not yet been achieved by human vaccines. Although circulating Leishmania-specific T cells are known to play a critical role in immunity, the role of memory T cells present in peripheral tissues has not been explored. Here, we identify a population of skin-resident Leishmania-specific memory CD4⁺ T cells. These cells produce IFN-γ and remain resident in the skin when transplanted by skin graft onto naive mice. They function to recruit circulating T cells to the skin in a CXCR3-dependent manner, resulting in better control of the parasites. Our findings are the first to demonstrate that CD4⁺ T_RM cells form in response to a parasitic infection, and indicate that optimal protective immunity to Leishmania, and thus the success of a vaccine, may depend on generating both circulating and skin-resident memory T cells.The development of effective vaccines for several intracellular microbial pathogens, such as Mycobacteria, Toxoplasma, Plasmodium, and Leishmania, remains an elusive goal. Despite substantial efforts to define the mechanisms required for resistance, to develop new adjuvants, and to identify protective antigens, the long-lived cellular immunity generated in response to infection has not been recapitulated by vaccination. To address this problem in leishmaniasis, we have focused on defining the memory T cells that mediate infection-induced immunity.C57BL/6 mice show robust immunity to reinfection after resolution of a primary Leishmania major infection (referred to here as immune mice), providing a useful model to interrogate the factors that might contribute to a successful vaccine. Previous studies have shown that immune mice contain circulating CD4⁺ T cells with effector, effector memory, and central memory phenotypes (Scott et al., 2004; Colpitts et al., 2009; Colpitts and Scott, 2010). Each of these T cell subsets likely plays a role in resistance to reinfection, with the effector subsets rapidly migrating into tissues to provide protection and central memory T cells proliferating in the draining lymph node to provide a pool of new effector cells. However, whereas adoptive transfer of either effector or central memory T cells to naive mice enhances immunity to reinfection (Zaph et al., 2004), neither subset alone or in combination provides the same level of protection as that seen in intact immune animals.In addition to circulating memory T cells, an additional memory T cell subset resides in the tissues as resident memory T cells (T_RM; Kim et al., 1999; Hogan et al., 2001a; Masopust et al., 2001; Clark et al., 2006; Gebhardt et al., 2009; Wakim et al., 2010; Hofmann and Pircher, 2011; Jiang et al., 2012; Mackay et al., 2012; Schenkel et al., 2013). Several studies have described T_RM cells that mediate immunity to acute viral infections, such as vaccinia, herpes simplex, influenza, and lymphocytic choriomeningitis virus (Gebhardt et al., 2009; Teijaro et al., 2011; Jiang et al., 2012; Schenkel et al., 2013). These T_RM cells can be found in the gut, brain, lung, and skin (Kim et al., 1999; Clark et al., 2006; Wakim et al., 2010; Teijaro et al., 2011), and their location allows them to respond immediately to control a challenge infection without the delay associated with the mobilization of circulating T cells. Additionally, T_RM cells can promote rapid recruitment of effector cells from the circulation (Schenkel et al., 2013) and induce antigen-independent innate immunity (Ariotti et al., 2014; Schenkel et al., 2014), thereby accelerating and amplifying resistance to infections.CD8⁺ T_RM cells are fairly well characterized, but less is known about CD4⁺ T_RM cells. Nevertheless, recent studies using Kaede-Tg mice to facilitate tracking of T cells in the skin indicate that a population of CD4⁺ T cells appear to be skin-resident under homeostatic conditions (Bromley et al., 2013). In addition, CD4⁺ T_RM cells in the lung and vaginal mucosa have been reported to enhance resistance to influenza and herpes simplex virus, respectively (Hogan et al., 2001b; Teijaro et al., 2011; Iijima and Iwasaki, 2014; Laidlaw et al., 2014), and a population of human tissue-resident CD4⁺ T cells remain in the skin after circulating T cells have been depleted (Clark et al., 2012; Watanabe et al., 2015). However, the potential role of CD4⁺ T_RM cells in establishing resistance to chronic parasitic infections, such as L. major, is virtually unknown.Here, we identify for the first time a tissue-resident population of CD4⁺ T cells that seed the skin after L. major infection. These cells are observed in skin sites far from the primary infection site, and persist long term in immune mice. They produce IFN-γ in response to stimulation with L. major and, during a secondary challenge, act as sentinels to rapidly recruit circulating memory cells, resulting in enhanced protection against reinfection. Thus, our results suggest that these previously unidentified memory CD4⁺ T cells are instrumental in protection against L. major parasites and should now be considered during vaccine development.     

A novel self-lipid antigen targets human T cells against CD1c+ leukemias

T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c⁺ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c⁺ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.CD1-restricted T lymphocytes recognize lipid antigens presented by the nonpolymorphic, MHC class I–related family of CD1 molecules (Porcelli and Modlin, 1999). CD1-restricted T cells can respond to lipid antigens derived from microbial cells and may exert protective roles during host infection (Moody et al., 2000, 2004; Amprey et al., 2004; Gilleron et al., 2004; Kinjo et al., 2005; Sriram et al., 2005; Wu et al., 2005; Montamat-Sicotte et al., 2011). A striking characteristic of many CD1-restricted T cells is autoreactivity against different types of APCs even in the absence of microbial antigens, implying that they can also recognize endogenous self-lipid molecules (Dellabona et al., 1993; Mattner et al., 2005; Vincent et al., 2005). Autoreactive T cells recognize different types of self-lipids present in cell membranes and synthesized within different cellular compartments (Shamshiev et al., 1999, 2000; Gumperz et al., 2000; Wu et al., 2003; De Libero et al., 2005). CD1a- and CD1c-autoreactive T cells are relatively abundant among circulating T cells in healthy individuals (de Jong et al., 2010; de Lalla et al., 2011) and might become activated by host antigens in autoimmune diseases and cancer. Lipid-specific T cells can control cancer cell growth in mouse models (Berzofsky and Terabe, 2009) as well as in human patients (Dhodapkar and Richter, 2011; Metelitsa, 2011), but it remains unknown whether they recognize unique lipids expressed by tumor cells.Acute leukemia comprises a heterogeneous group of hematological disorders characterized by blood and bone marrow accumulation of immature and abnormal cells derived from hematopoietic precursors (Pui et al., 2004; Rubnitz et al., 2008). Current therapy for acute leukemia is based on polychemotherapy and allogeneic hematopoietic stem cell (HSC) transplantation (HSCT). A major cause of treatment failure and area of substantial unmet need in HSCT is posttransplant regrowth of residual leukemia blasts that survive the conditioning regimen (Wingard et al., 2011). Donor-derived T cells transferred into patients may induce a beneficial graft versus leukemia (GVL) reaction capable of maintaining remission (Kolb, 2008), but grafted T cells are also capable of killing patient cells in nonhematopoietic tissues to induce detrimental graft versus host disease (GVHD; Socié and Blazar, 2009). A promising therapeutic strategy is the selective targeting of T cell responses against malignant hematopoietic cells, while maintaining hematopoietic capacity among grafted cells and preserving organ functions in recipient patients (Kolb, 2008). Because CD1 molecules are both nonpolymorphic and preferentially expressed by mature hematopoietic cells (Porcelli and Modlin, 1999; Brigl and Brenner, 2004), targeting tumor-associated lipid antigens presented by CD1 molecules might provide opportunities to improve the efficacy of HSCT. Immune recognition of tumor-associated lipid antigens may also complement ongoing antitumor responses mediated by protein antigens.Here we have identified the novel self-lipid antigen that stimulates CD1c autoreactive T cells to destroy tumor cell lines and primary human leukemia cells. We report that both group 1 CD1 molecules and a novel class of tumor-associated lipids are broadly expressed by different types of acute leukemia. In addition to killing CD1c⁺ leukemia cell lines and primary blasts in vitro, the CD1c-restricted T cells also displayed therapeutic efficacy in a mouse xenograft model of human leukemia. Our findings provide proof-of-concept evidence that T cell responses against lipids accumulated in acute leukemia could be exploited for leukemia immunotherapy.     

CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells

Recent studies have challenged the view that Langerhans cells (LCs) constitute the exclusive antigen-presenting cells of the skin and suggest that the dermal dendritic cell (DDC) network is exceedingly complex. Using knockin mice to track and ablate DCs expressing langerin (CD207), we discovered that the dermis contains five distinct DC subsets and identified their migratory counterparts in draining lymph nodes. Based on this refined classification, we demonstrated that the quantitatively minor CD207⁺ CD103⁺ DDC subset is endowed with the unique capability of cross-presenting antigens expressed by keratinocytes irrespective of the presence of LCs. We further showed that Y-Ae, an antibody that is widely used to monitor the formation of complexes involving I-A^b molecules and a peptide derived from the I-E α chain, recognizes mature skin DCs that express I-A^b molecules in the absence of I-E α. Knowledge of this extra reactivity is important because it could be, and already has been, mistakenly interpreted to support the view that antigen transfer can occur between LCs and DDCs. Collectively, these data revisit the transfer of antigen that occurs between keratinocytes and the five distinguishable skin DC subsets and stress the high degree of functional specialization that exists among them.Langerhans cells (LCs) constitute a subset of DCs. In their immature state, they reside in the stratified squamous epidermal layer of the skin and in the mucosal epithelia lining the ocular, oral, and vaginal surfaces (Iwasaki, 2007). LCs have long been regarded as the exclusive APCs of the skin, detecting pathogens that penetrate the skin barrier and, after undergoing a phase of maturation, conveying this information via lymphatic vessels to T cells present in cutaneous LNs (CLNs; Steinman and Nussenzweig, 2002; Larregina and Falo, 2005). Recent studies have shown, however, that LCs do not constitute the exclusive APCs of the skin. In addition to LCs, the skin contains a second type of DCs known as dermal DCs (DDCs). Epidermal LCs and DDCs migrate to CLNs under both steady-state and inflammatory conditions and constitute the direct precursors of the migratory LCs (mLCs) and migratory DDCs (mDDCs) found in CLNs, respectively. Some studies also suggested that migratory skin DCs play an indirect role in T cell priming, possibly by ferrying skin-derived antigens to those DCs that reside throughout their life cycle in CLNs and are denoted as lymphoid tissue–resident DCs to distinguish them from tissue-derived migratory DCs (Allan et al., 2003; Carbone et al., 2004; Allenspach et al., 2008).Langerin (CD207) is a C-type lectin originally thought to be specifically expressed in LCs (Valladeau et al., 2000; Kissenpfennig et al., 2005a). The use of Lang-EGFP mice that express an enhanced GFP (EGFP) under the control of the langerin gene showed that CD207 alone is not a reliable marker for the identification of LCs once they have migrated outside the epidermis (Kissenpfennig et al., 2005b) and led to the identification of three subsets of CD207⁺ DCs in steady-state CLNs (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007; Shklovskaya et al., 2008). A minor subset corresponds to lymphoid tissue–resident CD207^low CD8α⁺ DCs and represents ∼10% of the CD207⁺ DCs found in CLNs. The two other subsets account for ∼90% of the CD207⁺ cells present in CLNs and, consistent with their CD11c^{inter-to-high} MHCII^high phenotype, originate from the skin. They result from two independent developmental pathways that coexist in steady-state conditions. The first pathway gives rise to epidermal LCs and to their migratory derivatives found in CLNs, whereas the second pathway generates the CD207⁺ DCs that reside in the dermis and their CD207⁺ mDDC progeny (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007; Shklovskaya et al., 2008). LCs are radio resistant, and their numbers are maintained through continuous in situ proliferation (Merad et al., 2002; Tripp et al., 2004; Poulin et al., 2007). In contrast, the continuous renewal of DDCs and of lymphoid tissue-resident DCs depends on blood-borne radiosensitive BM precursors (Liu et al., 2009). As a consequence, in lethally irradiated mice reconstituted with BM transplants, LCs in the epidermis and their migratory counterparts in the dermis and CLNs remain of host origin, whereas other DC subsets are primarily repopulated by donor BM–derived cells (Merad et al., 2002).The role played by LCs and DDCs during skin immune responses remains controversial (Kaplan et al., 2008; Lee et al., 2009). Therefore, the present study intends to further analyze the phenotypic and functional complexity of the DC network present in the skin and of their migratory derivatives present in CLNs. Based on the expression of CD207, CD11b, and CD103, we identified five distinct skin DC subsets and evaluated whether some functional specialization exists among them. We examined the contribution of each of them to the presentation of keratinocyte- or LC-expressed antigens. We demonstrated that CD207⁺ CD103⁺ DDCs are endowed with the unique capability of cross-presenting a model antigen expressed by keratinocytes and showed that such a task can be accomplished irrespective of the presence of LCs. In contrast to a previous study (Ginhoux et al., 2007), we also demonstrated that DDCs do not have the capacity to capture a model antigen carried by mLCs en route to the CLNs.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells

In recent years, human dendritic cells (DCs) could be subdivided into CD304⁺ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c⁺, CD16⁺, and CD141⁺ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141⁺ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca²⁺ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141⁺ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8⁺ T cells when directly compared with CD1c⁺ DCs, CD16⁺ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141⁺ DCs correspond to mouse CD8⁺ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141⁺ DCs as professional antigen cross-presenting DCs in the human.The adaptive immune response is initiated through presentation of antigen to T cells by DCs. In the mouse, DCs can be broadly grouped into plasmacytoid DCs (pDCs) and conventional DCs (cDCs; earlier termed myeloid DCs). Mouse cDCs can be further subdivided into several DC types, which are apparently specialized for optimal antigen uptake, processing, and presentation to T cells in different body compartments (Steinman and Banchereau, 2007; Heath and Carbone, 2009; Segura and Villadangos, 2009). One particular type of antigen presentation is cross-presentation: in this case, extracellular antigen is not classically presented in the context of MHC-II but is instead shunted into the MHC-I presentation pathway (Bevan, 2006; Shen and Rock, 2006; Villadangos et al., 2007). CD8⁺ T cells can thus be activated by antigens taken up from the extracellular space and then differentiate into cytotoxic T cells. This mechanism is thought to be of major importance for the recognition of viral or bacterial antigens when DCs are not directly infected. In these instances, debris of cells that were infected and have subsequently undergone apoptosis as part of a cellular stress reaction is taken up and cross-presented by specialized DCs. Through this type of processing, the antigenic composition of the pathogen can become visible to the CD8⁺ T cell immune system. In the mouse, extensive experimentation has demonstrated that within cDCs, CD8⁺ DCs are the most effective in antigen cross-presentation (den Haan et al., 2000; Iyoda et al., 2002; Schulz and Reis e Sousa, 2002; Heath et al., 2004). Whether mouse pDCs play a significant role in antigen presentation and more so in antigen cross-presentation is controversial (Colonna et al., 2004; Liu, 2005; Villadangos and Young, 2008).We have recently shown in the mouse system that splenic CD8⁺ DCs (and their counterparts in other organs) are the only cells in the body expressing XCR1, a chemokine receptor with a unique ligand, XCL1 (Dorner et al., 2009). In vitro, XCL1 induces potent chemotaxis of XCR1⁺ CD8⁺ DCs. In vivo, XCL1 secreted by activated CD8⁺ T cells augments their expansion and differentiation into cytotoxic T cells when the antigen is cross-presented by CD8⁺ DCs in the context of MHC-I (Dorner et al., 2009). Collectively, these observations indicate that the XCL1–XCR1 communication axis optimizes the cooperation of antigen-specific CD8⁺ T cells with XCR1⁺ DCs, which cross-present antigen to them.Based on our studies in the mouse, we were interested to determine whether human DCs express XCR1. Human DCs have been extensively phenotyped in the past and subdivided again into pDC and into CD1c⁺ (BDCA-1⁺), CD16⁺, and CD141⁺ (BDCA-3⁺) cDC subsets (Dzionek et al., 2000; MacDonald et al., 2002; Piccioli et al., 2007; for review see Ju et al., 2010). Meticulous gene expression analyses of all human and mouse DCs have recently revealed a large gene expression program shared by human and mouse pDCs, and also led to the suggestion that human CD141⁺ DCs correspond to mouse CD8⁺ DCs (Robbins et al., 2008). In spite of this groundbreaking work on the subdivision of human DCs into subsets, information on the function of human primary DCs remained very scarce, apparently because of the limitations imposed by the very low frequencies of DCs in human blood (CD1c⁺ DCs, 0.31 ± 0.14% SD; CD16⁺ DCs, 0.75 ± 0.41%; CD141⁺ DCs, 0.04 ± 0.03%; pDCs, 0.29 ± 0.08%; n = 8; not depicted). Instead, antigen cross-presentation in the human system was essentially analyzed with DCs derived from monocytes in culture (Fonteneau et al., 2003), a system that may not reflect all of the functional properties of primary DCs.In the present study, we demonstrate that CD141⁺ DCs are the only population in human blood that expresses the chemokine receptor XCR1. Human CD141⁺ DCs react to the chemokine XCL1 by mobilization of intracellular Ca²⁺ ([Ca²⁺]_i) and by strong chemotaxis in vitro. More importantly, our experiments demonstrate that primary CD141⁺ DCs excel in cross-presentation of antigen when directly compared with CD1c⁺ DCs, CD16⁺ DCs, and pDCs from the same donors. Collectively, these functional data strongly indicate that human CD141⁺ DCs are the homologue of mouse CD8⁺ DCs. At the same time, the professional capacity of human CD141⁺ DCs to cross-present antigen is of major interest in the ongoing quest to develop vaccines capable of inducing antiviral or antitumor cytotoxicity in the human.     

Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4⁺ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1–expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4⁺ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4⁺ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4⁺ T cell expansion contributes to the exacerbation of T cell–mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4⁺ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.The surface of all mammalian cells is covered by complex carbohydrate structures termed glycans (van Kooyk and Rabinovich, 2008). The glycan structure is determined by the enzymatic processes that produce glycosidic linkages of saccharides to other saccharides, and the expression profile of these glycan-modifying enzymes is altered by several factors, such as cell differentiation and activation, inflammatory insults, and the environment (Marth and Grewal, 2008; van Kooyk and Rabinovich, 2008; Baum and Crocker, 2009; Rabinovich and Toscano, 2009). Consequently, there is a diverse and abundant repertoire of glycan structures on the cell surface, which is known as the glycome (Marth and Grewal, 2008; van Kooyk and Rabinovich, 2008; Rabinovich and Toscano, 2009). The importance of the glycome in immune responses has been highlighted by its role in the control of cell homing, apoptosis, and microbial recognition (Marth and Grewal, 2008; van Kooyk and Rabinovich, 2008; Baum and Crocker, 2009; Rabinovich and Toscano, 2009). In addition, a recent human genetic study provides support for an alteration of the glycome in B cell signaling as a protective factor in some autoimmune diseases (Surolia et al., 2010). Therefore, understanding the functional role of each glycome motif in the immune response can potentially open up a new avenue for the treatment of immune-mediated diseases (Rabinovich and Toscano, 2009).Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that develops in a genetically predisposed host. IBD is characterized by two major forms, Crohn’s disease (CD) and ulcerative colitis (UC), which are mediated by both common and distinct mechanisms (Xavier and Podolsky, 2007; Kaser et al., 2010; Mizoguchi and Mizoguchi, 2010). For example, Th1/Th17 responses have been implicated in the pathogenesis of CD, whereas UC has a significant contribution from Th2 cytokines (Xavier and Podolsky, 2007; Kaser et al., 2010; Mizoguchi and Mizoguchi, 2010). However, both diseases are characterized by a significant expansion of inflammatory memory CD4⁺ T cells in the inflamed intestine (Xavier and Podolsky, 2007; Kaser et al., 2010). Although much is known about the pathogenic effector mechanisms in these diseases, little information is currently available on how the carbohydrate structure of T cells contributes to these responses (Santucci et al., 2003; Hokama et al., 2004; Müller et al., 2006; Srikrishna et al., 2005). This is in contrast to the abundant amount of information available on the heavily O-glycosylated mucus that has been clearly demonstrated to play a protective role in IBD (An et al., 2007; Stone et al., 2009).We herein demonstrate a colitis-associated glycome (CAG) on CD4⁺ T cells, which is characterized by immature core-1 O-glycan and induced through down-regulation of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1 expression under intestinal inflammatory conditions. We also propose that the inducible glycome motif contributes to the exacerbation of colitis by enhancing the expansion of effector memory CD4⁺ T cells through stabilization of protein kinase C (PKC) θ activation.