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.     

Circulating precursors of human CD1c+ and CD141+ dendritic cells

Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c⁺ and CD141⁺ Clec9a⁺ cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L.Conventional DCs (cDCs) induce immunity or tolerance by capturing, processing, and presenting antigen to T lymphocytes (Banchereau and Steinman, 1998). In the mouse, cDCs are short-lived cells, whose homeostasis in lymphoid and nonlymphoid tissues is critically dependent on continual replenishment from circulating pre-CDC (Liu et al., 2007; Liu and Nussenzweig, 2010). Murine pre-CDCs are BM-derived cells that are present in very small numbers in the blood but increase in response to Flt3L injection (Liu et al., 2007, 2009). pre-CDCs have a very short dwell time in the blood, 65% of these cells leave the circulation within 1 min after leaving the BM (Liu et al., 2007, 2009). Upon leaving the circulation, pre-CDCs seed tissues where they differentiate to cDCs, which divide further under the control of Flt3L (Liu et al., 2007, 2009). Thus, in addition to the BM and blood, mouse pre-CDCs are also found in peripheral lymphoid organs and nonlymphoid tissues (Naik et al., 2006; Bogunovic et al., 2009; Ginhoux et al., 2009; Liu et al., 2009; Varol et al., 2009).Mouse cDCs can be divided into two major subsets, CD11b⁺ DCs and CD8⁺/CD103⁺ DCs that differ in their microanatomic localization, cell surface antigen expression, antigen-processing activity, and ability to contribute to immune responses to specific pathogens (Merad et al., 2013; Murphy, 2013). Despite these important differences, both CD11b⁺ and CD8⁺/CD103⁺ cDC subsets of mouse DCs are derived from the same immediate precursor (pre-CDC) that expresses CD135 (Flt3), the receptor for Flt3L, a cytokine that is critical to DC development in vivo (McKenna et al., 2000; Waskow et al., 2008).Similar to the mouse, humans have two major subsets of cDCs. CD141 (BDCA3)⁺Clec9a⁺ DCs (CD141⁺ cDC herein) appear to be the human counterpart of mouse CD8⁺/CD103⁺ DCs, expressing XCR1, Clec9a, IRF8, and TLR3 and producing IL-12 (Robbins et al., 2008; Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010; Haniffa et al., 2012). CD1c (BDCA1)⁺ cDCs appear to be more closely related to mouse CD11b⁺ DCs, expressing IRF4, inducing Th17 differentiation upon A. fumigatus challenge, and imprinting intraepithelial homing of T cells (Robbins et al., 2008; Crozat et al., 2010; Schlitzer et al., 2013; Yu et al., 2013). In the mouse, the superior ability of CD8⁺/CD103⁺ DCs to cross-present exogenous antigens to CD8⁺ T cells is attributed to both differential antigen uptake (Kamphorst et al., 2010) and to increased expression of proteins and enzymes that facilitate MHC class I presentation (Dudziak et al., 2007). Human CD141⁺ cDCs are more efficient than CD1c⁺ cDCs in cross-presentation (Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010), but this difference appears to result from differences in antigen uptake and cytokine activation rather than a specialized cell-intrinsic program (Segura et al., 2012; Cohn et al., 2013; Nizzoli et al., 2013).Both CD1c⁺ cDCs and CD141⁺ cDCs are present in human blood and peripheral tissues. Each subset in the blood resembles its tissue counterpart in gene expression but appears less differentiated (Haniffa et al., 2012; Segura et al., 2012; Schlitzer et al., 2013). These observations are consistent with the idea that less differentiated human cDCs travel through the blood to replenish the cDC pool in the peripheral tissues (Collin et al., 2011; Segura et al., 2012; Haniffa et al., 2013). Others have postulated the existence of a less differentiated circulating DC progenitor based on absence of CD11c, expression of CD123, and response to Flt3L (O’Doherty et al., 1994; Pulendran et al., 2000), but the progenitor potential of these putative precursors that produced large amounts of IFN-α was never tested directly and they appear to correspond at least in part to plasmacytoid DCs (Grouard et al., 1997; Siegal et al., 1999). Thus, whether there is an immediate circulating precursor restricted to human immature and mature CD1c⁺ and CD141⁺ cDCs is not known.Here, we report the existence of a migratory pre-CDC in humans (hpre-CDC) that develops from committed DC progenitors (hCDPs) in the BM (Lee et al., 2015) and is the immediate precursor of both CD1c⁺ and CD141⁺ cDCs, but not pDCs or monocytes. hpre-CDCs are present in BM, cord, and peripheral blood, as well as peripheral lymphoid tissues. In studies of human volunteers, Flt3L injection induces expansion of hpre-CDCs in the circulation. Thus, human cDC precursors constitute a dynamic circulating population whose homeostasis is regulated by Flt3L, a cytokine that is responsive to inflammatory and infectious agents.     

The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development

A key role of the thymic medulla is to negatively select autoreactive CD4⁺ and CD8⁺ thymocytes, a process important for T cell tolerance induction. However, the involvement of the thymic medulla in other aspects of αβ T cell development, including the generation of Foxp3⁺ natural regulatory T cells (nT_reg cells) and the continued maturation of positively selected conventional αβ T cells, is unclear. We show that newly generated conventional CD69⁺Qa2⁻ CD4 single-positive thymocytes mature to the late CD69⁻Qa2⁺ stage in the absence of RelB-dependent medullary thymic epithelial cells (mTECs). Furthermore, an increasing ability to continue maturation extrathymically is observed within the CD69⁺CCR7^−/loCCR9⁺ subset of conventional SP4 thymocytes, providing evidence for an independence from medullary support by the earliest stages after positive selection. In contrast, Foxp3⁺ nT_reg cell development is medullary dependent, with mTECs fostering the generation of Foxp3⁻CD25⁺ nT_reg cell precursors at the CD69⁺CCR7⁺CCR9⁻ stage. Our results demonstrate a differential requirement for the thymic medulla in relation to CD4 conventional and Foxp3⁺ thymocyte lineages, in which an intact mTEC compartment is a prerequisite for Foxp3⁺ nT_reg cell development through the generation of Foxp3⁻CD25⁺ nT_reg cell precursors.In the thymus, positive selection of CD4⁺8⁺ thymocytes recognizing self-peptide/MHC on cortical thymic epithelial cells (TECs) triggers the entry of CD4/CD8 single-positive (SP) T cells into the thymic medulla, a process essential for tolerance induction (Kurobe et al., 2006). Additionally, the medulla is also considered a key site of differentiation that supports thymocyte maturation after positive selection, including stages defined by loss of CD24/CD69 and acquisition of CD62L/Qa2 (McCaughtry et al., 2007; Li et al., 2007).Although the medulla also contains SP4 Foxp3⁺ natural regulatory T cells (nT_reg cells; Liston et al., 2008), its role in nT_reg cell generation remains unclear, with both medullary TECs (mTECs) and DCs being implicated (Aschenbrenner et al., 2007; Proietto et al., 2008; Spence and Green, 2008; Wirnsberger et al., 2009; Hinterberger et al., 2010). Importantly, nT_reg cell development is a multistage process, with TCR–MHC (Lio and Hsieh, 2008) and CD28–CD80/86 interactions (Lio et al., 2010; Vang et al., 2010; Hinterberger et al., 2011) driving the generation of Foxp3⁻CD25⁺ nT_reg cell precursors that give rise to Foxp3⁺CD25⁺ nT_reg cells (Lio and Hsieh, 2008). However, the role of mTECs during Foxp3⁻CD25⁺ nT_reg cell precursor generation is unknown.Here, we define steps in both conventional and nT_reg SP4 thymocyte maturation, mapping their requirements for a RelB-dependent mTEC compartment (Burkly et al., 1995; Weih et al., 1995; Heino et al., 2000). We show that conventional SP4 thymocytes can complete their maturation in the absence of RelB-dependent mTECs, with evidence of thymic independence occurring by the CD69⁺CCR7^−/loCCR9⁺ SP4 thymocyte stage. In contrast, Foxp3⁺ nT_reg cells require an intact thymic medulla, with a requirement for RelB-dependent mTEC mapping to the generation of Foxp3⁻CD25⁺ nT_reg cell precursors at the CD69⁺CCR7⁺CCR9⁻ stage. Collectively, our data reveal the differential importance of the thymic medulla during SP4 thymocyte development and highlight a specific role for mTECs in Foxp3⁻CD25⁺ precursor generation.     

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.     

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.     

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.     

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141⁺ DC subset. CD141⁺ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c⁺ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141⁺ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8⁺ cytotoxic T lymphocytes than poly I:C–activated CD1c⁺ DCs. Importantly, CD141⁺ DCs, but not CD1c⁺ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141⁺ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α⁺ DC subset. The data demonstrate a role for CD141⁺ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.The essential role of DCs in the induction and regulation of immune responses to pathogens, self-antigens (Ags), and cancers is now well established. All DCs excel at processing and presenting Ag and priming naive T cell responses, but the complexity of DC subsets and their individual specialized functions is just becoming apparent (MacDonald et al., 2002; Villadangos and Schnorrer, 2007; Naik, 2008). Promising DC-based therapeutic vaccines have been described to treat malignancies and infections (Vulink et al., 2008), but the majority of these use in vitro–generated monocyte-derived DC (MoDC), and the physiological standing of this DC subtype is currently unclear. Understanding the emerging complexities of human DC subset biology is therefore essential to develop new vaccines and therapeutics targeting DC.The characterization and function of human DC subsets has been confounded by their rarity, the lack of distinctive markers, and limited access to human tissues. Human blood DCs comprise ∼1% of circulating PBMCs and have been classically defined as Ag-presenting leukocytes that lack other leukocyte lineage markers (CD3, 14, 15, 19, 20, and 56) and express high levels of MHC class II (HLA-DR) molecules (Hart, 1997). These can be broadly categorized into two groups: plasmacytoid CD11c⁻CD123⁺ DC and conventional or myeloid CD11c⁺CD123⁻ DC. We have described three further phenotypically distinct subsets of CD11c⁺ DC, defined by their expression of CD16, CD1c (BDCA-1), and CD141 (BDCA-3; MacDonald et al., 2002). Gene expression profiling and hierarchical clustering data has indicated that plasmacytoid DC and CD16⁺ DC arise from separate precursor cells, whereas the CD1c⁺ DC and CD141⁺ DC subsets appear to have a common origin and represent two different stages of a similar subset (Lindstedt et al., 2005). However, CD1c⁺ and CD141⁺ DCs each have unique gene expression profiles distinct from monocytes and MoDC, and this predicts that they have different functions (Dzionek et al., 2000; MacDonald et al., 2002; Lindstedt et al., 2005).The concept of distinct DC subtypes with unique capabilities to influence immunological outcomes is exemplified by the mouse CD8α⁻ and CD8α⁺ conventional DC subsets that reside in the lymph nodes and spleen (Villadangos and Schnorrer, 2007; Naik, 2008). The CD8α⁻ DC subset appears to be most effective at inducing Th2 responses (Maldonado-López et al., 1999; Pulendran et al., 1999) and processing and presenting Ag to CD4⁺ T cells via the MHC class II pathway (Pooley et al., 2001; Dudziak et al., 2007; Villadangos and Schnorrer, 2007). In contrast, the CD8α⁺ DC subset has a unique ability to take up dead or dying cells and to process and present exogenous Ag on MHC class I molecules to CD8⁺ T cells (i.e., cross-presentation; den Haan et al., 2000; Iyoda et al., 2002; Schnorrer et al., 2006). There is now substantial evidence that the CD8α⁺ DC subset plays a crucial role in the induction of protective CD8⁺ CTL responses that are essential for the eradication of cancers, viruses, and other pathogenic infections (Dudziak et al., 2007; Hildner et al., 2008; López-Bravo and Ardavín, 2008; Naik, 2008). The identification of the human DC subset with similar functional capacity would be a significant advance and would enable translation of mouse DC biology into clinical practice.Correlation of the human and mouse DC subsets has been hampered by differences in their defining markers (human DCs do not express CD8α). Interestingly, computational genome-wide expression profiling clustered human CD141⁺ DC and CD1c⁺ DC with the mouse CD8α⁺ and CD8α⁻ conventional DC subsets, respectively (Robbins et al., 2008). Human CD141⁺ DC and mouse CD8α⁺ DC share a number of phenotypic similarities, including expression of Toll-like receptor (TLR) 3 (Edwards et al., 2003; Lindstedt et al., 2005), the novel surface molecule Necl2 (nectin-like protein 2; Galibert et al., 2005), and the C-type lectin CLEC9A (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). Thus, whether the human CD141⁺ DC subset is the human functional equivalent of the mouse CD8α⁺ DC subset has now become a major question for immunologists.CD141⁺ DCs constitute only ∼0.03% of human PBMCs and, although present in other human tissues, their low proportions and difficulties with aseptic human tissue access mean that they have never been isolated in sufficient quantity to study their function until now. We report the first detailed functional analysis of human CD141⁺ DCs in response to TLR3 stimuli and define their role in the induction of Th1 responses and cross-presentation.     

Memory CD8+ T cells exhibit increased antigen threshold requirements for recall proliferation

A hallmark of immunological memory is the ability of previously primed T cells to undergo rapid recall responses upon antigen reencounter. Classic work has suggested that memory T cells proliferate in response to lower doses of antigen than naive T cells and with reduced requirements for co-stimulation. In contrast to this premise, we observed that naive but not memory T cells proliferate in vivo in response to limited antigen presentation. To reconcile these observations, we tested the antigen threshold requirement for cell cycle entry in naive and central memory CD8⁺ T cells. Although both naive and memory T cells detect low dose antigen, only naive T cells activate cell cycle effectors. Direct comparison of TCR signaling on a single cell basis indicated that central memory T cells do not activate Zap70, induce cMyc expression, or degrade p27 in response to antigen levels that activate these functions in naive T cells. The reduced sensitivity of memory T cells may result from both decreased surface TCR expression and increased expression of protein tyrosine phosphatases as compared with naive T cells. Our data describe a novel aspect of memory T cell antigen threshold sensitivity that may critically regulate recall expansion.The ability of the adaptive immune system to respond more rapidly and effectively to pathogens that have been previously encountered is the basis of immunological memory. This attribute of CD8⁺ T cell memory is primarily due to an estimated 5–100-fold increase in the frequency of antigen-specific cells after memory formation over that found in naive individuals (Ahmed and Gray, 1996). Additionally, evidence suggests that clonal competition during the expansion phase of T cell priming may increase the affinity of the resulting antigen-specific effector and memory CD8⁺ T cell pool compared with the naive pool (Busch and Pamer, 1999; Zehn et al., 2009). Indeed, based on functionality, memory CD8⁺ T cells appear to be more sensitive to TCR-mediated stimulation than naive cells. Multiple studies have observed that resting memory but not naive CD8⁺ T cells can secrete cytokines and produce cytolytic effectors more rapidly than naive cells upon antigen encounter (Zimmermann et al., 1999; Veiga-Fernandes et al., 2000; Slifka and Whitton, 2001). Consistent with this ability, memory CD8⁺ T cells show epigenetic changes at cytokine gene loci that are consistent with more rapid gene expression (Kersh et al., 2006; Northrop et al., 2006). In addition, memory T cells redistribute their TCR into higher order oligomers that may increase antigen sensitivity (Kumar et al., 2011). Multiple phenotypic differences between naive and memory CD8⁺ T cells have also been described that may influence TCR reactivity including up-regulation of adhesion molecules and increased surface expression of the IL-2Rβ chain CD122 (Berard and Tough, 2002).However, the characteristics ascribed to naive and memory T cells may have been influenced by the experimental systems used to test them. For example, although memory CD8⁺ T cells reportedly proliferate in response to lower doses of antigen than naive T cells (Pihlgren et al., 1996; Curtsinger et al., 1998; London et al., 2000), little difference in peptide sensitivity was observed in the absence of exogenous IL-2 (Curtsinger et al., 1998; Zimmermann et al., 1999). Thus, the increased sensitivity of memory T cells to cytokine may be responsible for their superior response. Additionally, although some in vitro studies have found that memory CD8⁺ T cells do not require CD28-mediated co-stimulation to initiate recall expansion (Flynn and Müllbacher, 1996; Bachmann et al., 1999), B-7 expression appears to be necessary for recall expansion in vivo (Borowski et al., 2007; Boesteanu and Katsikis, 2009). These inconsistent data may be attributable to comparison of in vitro and in vivo results or inadequate analysis of the contribution of distinct memory CD8⁺ T cell subsets. Extensive phenotyping of antigen-specific T cell responses has suggested that multiple markers may co-segregate with proliferative capacity. CD8⁺ central memory T cells expressing CD44^hi, CD62L^hi, CD27^hi, CXCR3^hi, CD43^lo, KLRG1^lo, and CD127^hi exhibit the most robust recall proliferation, whereas CD44^hi, CD62L^lo, CD27^hi, CXCR3^hi, CD43^hi, KLRG1^lo, and CD127^hi effector memory T cells exhibit sustained cytotoxicity but poorer recall expansion (Wherry et al., 2003; Sallusto et al., 2004; Hikono et al., 2007; Olson et al., 2013).Intriguingly, it has been reported that after clearance of acute influenza infection, residual viral antigen presentation can drive proliferation and expansion of naive but not memory CD8⁺ T cells of the same specificity (Belz et al., 2007; Khanna et al., 2008). This observation is in contrast to the expectation that memory T cells exhibit greater responsiveness than naive cells. It has been suggested that naive and memory T cells may respond to antigen presentation by distinct DC subsets or migrate to different areas of the lymph node (Belz et al., 2007; Kastenmüller et al., 2013). Currently, it remains unclear why residual, long-lived antigen does not stimulate memory T recall proliferation. To better understand the requirements for efficient proliferative recall expansion, we have compared the activation and proliferation of TCR transgenic CD62L^hiCD44^lo naive and CD62L^hiCD44^hi central memory CD8⁺ T cells in multiple models of noninfectious antigen presentation at limiting levels in vitro and in vivo. Although we found that both naive and central memory cells received a TCR stimulus and were activated by limiting levels of antigen presentation, only naive CD8⁺ T cells entered cell cycle and expanded. This effect appeared T cell intrinsic, as naive T cells preferentially proliferated in response to limiting levels of peptide presented by multiple DC subsets. Direct comparison of naive and memory T cells indicated that resting memory CD8⁺ T cells express more cyclin-dependent kinase inhibitor p27 and do not activate effectors of cell cycle progression or Zap70 upon low dose peptide stimulation, although no defect was observed at saturating concentrations. Additionally, we found that memory T cells expressed lower levels of surface TCR and higher levels of non-receptor tyrosine phosphatases involved in negative regulation of TCR signaling. Our data clearly indicates that, surprisingly, memory CD8⁺ T cells actually exhibit a higher antigen threshold than naive CD8⁺ T cells to stimulate cell cycle entry in vitro and in vivo.