OT-I Cells Research Articles

Spontaneous Mammary Tumors Differ Widely in Their Inherent Sensitivity to Adoptively Transferred T Cells

Immunotherapy of cancer can lead to the selection of antigen loss variants, which provides strong rationale to target oncogenes that are essential for tumor growth or viability. To investigate this concept, we tagged the HER2/neu oncogene with epitopes from ovalbumin to confer recognition by T-cell receptor transgenic CD8(+) (OT-I) and CD4(+) (OT-II) T cells. Transgenic mice expressing neu(OT-I/OT-II) developed mammary adenocarcinomas at 6 to 10 months of age. Adoptively transferred naive OT-I cells (with or without OT-II cells) proliferated vigorously on encountering neu(OT-I/OT-II)-expressing tumors. This was followed by the complete regression of 37% of tumors, whereas others showed partial/stable responses (40%) or progressive disease (23%). Those tumors undergoing complete regression never recurred. In mice with multiple primary tumors, simultaneous regressions and nonregressions were often seen, indicating that immune evasion occurred at a local rather than systemic level. The majority of nonregressing tumors expressed Neu(OT-I/OT-II) and MHC class I, and many avoided rejection through a profound block to T-cell infiltration. Thus, T cells directed against an essential oncogene can permanently eradicate a subset of spontaneous, established mammary tumors. However, in other tumors, local barriers severely limit the therapeutic response. To maximize the efficacy of immunotherapy against spontaneous cancers, predictive strategies that take into account the heterogeneity of the tumor microenvironment will be required.

Signal 3 Availability Limits the CD8 T Cell Response to a Solid Tumor

CD8 T cells need a third signal, along with Ag and costimulation, for effective survival and development of effector functions, and this can be provided by IL-12 or type I IFN. Adoptively transferred OT-I T cells, specific for H-2K(b) and OVA, encounter Ag in the draining lymph nodes of mice with the OVA-expressing E.G7 tumor growing at a s.c. site. The OT-I cells respond by undergoing limited clonal expansion and development of effector functions (granzyme B expression and IFN-gamma production), and they migrate to the tumor where they persist but fail to control tumor growth. In contrast, OT-I T cells deficient for both the IL-12 and type I IFN receptors expand only transiently and rapidly disappear. These results suggested that some signal 3 cytokine is available, but that it is insufficient to support a CTL response that can control tumor growth. Consistent with this, administration of IL-12 at day 10 of tumor growth resulted in a large and sustained expansion of wild-type OT-I cells with enhanced effector functions, and tumor growth was controlled. This did not occur when the OT-I cells lacked the IL-12 and type I IFN receptors, demonstrating that the therapeutic effect of IL-12 results from direct delivery of signal 3 to the CD8 T cells responding to tumor Ag in the signal 3-deficient environment of the tumor.

The CD8 T cell response to vaccinia virus exhibits site-dependent heterogeneity of functional responses

CD8 T cell responses to vaccinia virus (VV) and a virus-encoded ovalbumin peptide (OVAP) epitope were examined using adoptively transferred OT-I T cells. The results demonstrate that upon intra-peritoneal challenge with ovalbumin-expressing VV (VV-OVAP), OT-I T cell proliferation occurs initially in lymph nodes and spleens followed by migration of the divided cells to the peritoneal cavity. Massive clonal expansion occurs in response to both the virus and the virus-encoded ovalbumin (OVA) epitope, as demonstrated using low numbers of adoptively transferred cells, and the responding OT-I cells display marked site-dependent functional heterogeneity with respect to IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) production and granzyme B expression. OT-I cells responding to VV-OVAP develop the capacity to produce IFN-gamma in response to antigen as they proliferate and differentiate. In marked contrast, naive OT-I cells rapidly produce TNF-alpha upon antigen recognition, and this capacity declines as the cells proliferate in response to the virus, suggesting that this potent inflammatory cytokine may be important primarily during initiation of the response. At the peak of clonal expansion, a large fraction (30-60%) of the OT-I cells responding to the virus express high IL-7Ralpha levels, and the majority of these cells is subsequently lost. While high IL-7Ralpha expression may be necessary for a CD8 T cell to transition to memory, it is clearly not sufficient. Thus, OT-I cells responding to VV infection exhibit a high degree of heterogeneity within the responding population that differs depending on their anatomical location, despite the specificity and affinity of the TCR being identical on all of the cells.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance

We have developed a model of autoimmunity to investigate autoantibody-mediated cross-presentation of self antigen. RIP-mOVA mice, expressing OVA in pancreatic beta cells, develop severe autoimmune diabetes when given OT-I cells (OVA-specific CD8(+) T cells) and anti-OVA IgG but not when given T cells alone. Anti-OVA IgG is not directly injurious to the islets but rather enhances cross-presentation of apoptotic islet antigen to the OT-I cells, leading to their differentiation into potent effector cells. Antibody-driven effector T cell activation is dependent on the presence of activating Fc receptors for IgG (FcgammaRs) and cross-priming DCs. As a consequence, diabetes incidence and severity was reduced in mice lacking activating FcgammaRs. An intact complement pathway was also required for disease development, as C3 deficiency was also partially protective. C3-deficient animals exhibited augmented T cell priming overall, indicating a proinflammatory role for complement activation after the T cell priming phase. Thus, we show that autoreactive antibody can potently enhance the activation of effector T cells in response to cross-presented self antigen, thereby contributing to T cell-mediated autoimmunity.

Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection

Neutrophils, monocytes, and dendritic cells (DC) are phenotypically and functionally related phagocytes whose presence in infected tissues is critical to host survival. Their overlapping expression pattern of surface molecules, the differentiation capacity of monocytes, and the presence of monocyte subsets underscores the complexity of understanding the role of these cells during infection. In this study we use five- to seven-color flow cytometry to assess the phenotype and function of monocytes recruited to Peyer's patches (PP) and mesenteric lymph nodes (MLN) after oral Salmonella infection of mice. The data show that CD68(high)Gr-1(int) (intermediate) monocytes, along with CD68(int)Gr-1(high) neutrophils, rapidly accumulate in PP and MLN. The monocytes have increased MHC-II and costimulatory molecule expression and, in contrast to neutrophils and DC, produce inducible NO synthase. Although neutrophils and monocytes from infected mice produce TNF-alpha and IL-1beta upon ex vivo culture, DC do not. In addition, although recruited monocytes internalize Salmonella in vitro and in vivo they did not induce the proliferation of OT-II CD4(+) T cells after coincubation with Salmonella expressing OVA despite their ability to activate OT-II cells when pulsed with the OVA(323-339) peptide. We also show that recruited monocytes enter the PP of infected mice independently of the mucosal address in cell adhesion molecule-1 (MAdCAM-1). Finally, recruited but not resident monocytes increase in the blood of orally infected mice, and MHC-II up-regulation, but not TNF-alpha or iNOS production, occur already in the blood. These studies are the first to describe the accumulation and function of monocyte subsets in the blood and GALT during oral Salmonella infection.

Role of Hepatic Kupffer Cells in Inducing T cell Tolerance (128.12)

Abstract The liver is known to favor the induction of immunological tolerance rather than immunity. Although Kupffer cells (KC) have been indicated to play a role in liver tolerance to allografts and soluble antigens, the mechanisms involved remain unclear. We hypothesized that KC could induce T cell tolerance by acting as ¡°poor¡± antigen presenting cells, as well as an active suppressor of T cell activation. The phenotypes of KC were characterized by flow cytometry. The abilities of KC to activate T cells and to suppress T cell activation induced by spleen dendritic cells (DC) were examined by in vitro proliferation assays using OTII cells. We found that, comparing with DC, KC expressed significantly lower levels of MHCII, B7.1, B7.2, and CD40. This is consistent with our observation that KC were not as potent as DC in eliciting OVA-specific T cell proliferation. More importantly, we found that KC could inhibit DC-induced OVA-specific T cell response. Further investigation of the mechanism involved in such suppression revealed that KC could induce apoptosis of the activated T cells. In summary, our data suggest that KC are ¡°poor¡± stimulators of T cells, and that they can cause tolerance through induction of T cell apoptosis. These findings indicate that KC play a critical role in regulating immune reactions within the liver and contributing to liver-mediated systemic immune tolerance.

Bcl-3 is required for adjuvant mediated T cell survival and sustained inhibition of GSK3ß (87.38)

Abstract Increased transcription of bcl3 correlates with adjuvant-mediated survival of activated T cells. To determine whether Bcl3 is required for adjuvant-dependent survival of activated T cells, OT-II.Bcl3 knockout (KO) mice were challenged with ova peptide (Ova) in the presence or absence of LPS and T cell survival was assessed after 20 hr culture. Ova-treated CD4 T cells from the Bcl3 KO mice activated with or without LPS failed to survive whereas wild type (WT) OTII cells survived when activated with LPS. Using low-number adoptive transfer of WT or KO OTIIs, we show that Bcl3 is required for effective adjuvant-dependent increase in clonal expansion in response to antigen. At peak expansion, 10-fold fewer KO OTIIs were recovered after LPS treatment, compared to the WT OTIIs. Similar defects were seen in adjuvant treated OT-I.Bcl3 KO cells. GSK3ß is a target molecule whose activity is affected by adjuvants during T cell expansion. Bcl3 KO T cells do not maintain the inhibitory p(ser9) on GSK3ß as the WT OTII cells although the initial increase after T cell activation is not affected. This indicates increased phosphatase activity in Bcl3 KO T cells. Studies are underway to address unchecked phosphatase activity as a mechanism of death in the KO cells. These data establish the importance of Bcl-3 in adjuvant- mediated survival of in vivo activated T cells. This work was supported by USPHS grants AI51377 and AI059023 and the Commonwealth of Kentucky Research Challenge Trust Fund.

SHPS-1 positively regulates T cell priming and cytokine production by dendritic cells (DCs). (89.15)

Abstract [Background] SHPS-1, a transmembrane protein that binds protein tyrosine phosphatases in its cytoplasmic region, is predominantly expressed on DCs, but the role of SHPS-1 signals during T cell priming by DCs is largely unknown. Here, we investigated the physiological roles of SHPS-1 in the functions of DCs by the use of SHPS-1 mutant mice, which lack most of the cytoplasmic region of this protein. [Methods] DCs were prepared from spleens by Auto MACS. The expression of SHPS-1 or costimulatory molecules on DCs were examined by FCM. IL-12-induced IFN-γ production by DCs was determined by ELISA. The ability of DCs for antigen presentation was evaluated by the incubation of OT-II cells with OVA-loaded DCs. [Results] FCM analysis showed that SHPS-1 was predominantily expressed on CD8α− DCs. The expression level of SHPS-1 was reduced in DCs from SHPS-1 mutant mice, but the expression levels of costimulatory molecules on DCs from SHPS-1 mutant mice were almost comparable to that on WT DCs. On the contrary, IL-12-induced production of IFN-γ was reduced in DCs from SHPS-1 mutant mice. The ability of DCs to stimulate proliferation of OVA-specific CD4+ T cells was also impaired in DCs from SHPS-1 mutant mice. [Conclusion] SHPS-1 positively regulates DC functions including CD4+ T cell priming and cytokine production.

Programming for a CD8 T Cell Memory Response Requires IL-12 and/or Type I IFN (95.23)

Abstract Naïve CD8 T cells proliferate in response to Ag and costimulation, but require signals from IL-12 or IFNα/β to make a productive response. Whether these are the only cytokines with this activity, and their relative contributions, are only beginning to be defined. Response to LCMV infection by CD8 cells lacking the IFNα/β receptor (IFNIR−/−) is reduced > 99%, but response to vaccinia virus (VV) remains largely intact (J.Exp.Med. 202, 637; J. Immunol. 176, 4225). We have studied responses of OT-I T cells to OVA-expressing VV and Listeria monocytogenes (LM), using cells that lack IFNIR, IL-12 receptor (IL-12R−/−), or both (DblKO). Clonal expansion of receptor-deficient OT-I cells to LM-OVA or VV-OVA is comparable to that of wild type (wt) cells, but effector functions are compromised, and their numbers decline more rapidly. IL-12R−/− cells exhibit greater defects than do IFN-IR−/− cells, and DblKO cells are the most compromised. Compared to the wt memory population following VV-OVA infection (> day 30), memory cell numbers are reduced by 27% for IFN-IR−/− cells, 96% for IL-12R−/− cells, and >99% for DblKO cells. For LM-OVA, the reduction in memory is 76% for IFN-IR−/− cells, 97% for IL-12R−/− cells, and >99% for DblKO cells. Thus, both IL-12 and IFNα/β contribute to responses to VV and LM, with IL-12 making the greater contribution, and programming for CD8 T cell memory requires signals from one or the other of these cytokines.

CD8aa-specific CD8ab Effector Memory T Cell Differentiation in the Gut (42.6)

Abstract CD8 effector memory T cells at the intestinal mucosa are important for rapid containment of the invading pathogens. CD8aa, a homodimer of CD8a, has been found on gut intraepithelial lymphocytes (IELs). It is also transiently expressed on some peripheral CD8ab primary effector T cells that differentiate to memory T cells. In vitro stimulated sorted CD8aa+ and CD8aa− OT-I cells migrate with equal capacity to the gut and spleen of recipient mice. One month later, oral re-challenge with Listeria Monocytogenes-OVA (LM-OVA) leads to an immediate secondary expansion of CD8aa+, but not CD8aa−, donor cells among the IELs, which suggests that only CD8aa+ precursors can differentiate into mucosal memory T cells and respond immediately to an oral pathogen challenge. Later, both CD8aa+ and CD8aa− donor cell-derived secondary responses are detected in spleen and gut. However, 45 days after the secondary response, only CD8aa+donor cells generate memory IELs. It indicates that CD8aa+ primary effector cells are programmed during the initial priming to survive and to become mucosal effector memory T cells. CD8aa expression is transient on primary effector cells and regained after relocation into the gut. The specific role of CD8aa in the survival and differentiation of intestinal CD8 effector memory T cells was investigated.

KERATINOCYTES INDUCE GRAFT-VERSUS-HOST DISEASE BY PRESENTATION OF EPIDERMAL SELF-ANTIGEN TO CD8 T CELLS.

The precise contribution(s) of skin dendritic cells (DCs) (epidermal Langerhans cells (LCs) and dermal dendritic cells (dDCs)) to graft-versus-host-disease (GvHD) in the skin has not been well delineated. We therefore developed an intradermal (id) injection model whereby CD8+ T (OT-I) cells that express ovalbumin (OVA)-peptide-specific T-cell receptors (Vα2/Vβ5) are delivered directly to the dermis of transgenic (Tg) mice expressing OVA targeted to the epidermis under a keratin 14 (K14) promoter. To determine the relative contribution of LCs to the GvHD reaction, we have generated K14-OVA x Langerin-diphtheria-toxin-receptor (Langerin-DTR) Tg mice to conditionally ablate LCs in the epidermis. Additionally, to identify the role of dDCs in the reaction we generated K14-OVA Tg chimeras using β2-microglobulin-deficient (b2m) congenic donor bone marrow cells. These mice were chimerized with donor b2m-derived MHC II high cells in the spleen (> 98%), LN (> 98%), and dermis (> 90%), whereas the majority of epidermal LCs remained recipient derived (> 95%). The dDCs in these mice do not have the capacity to present OVA to OT-I cells, whereas the LCs maintain their antigen-presenting functions, as previously reported. Following id injection of OT-I cells, the K14-OVA Tg mice developed localized skin GvHD on day 7 regardless of the level of OVA expression in the epidermis. Local injection of OT-I cells into the b2m→K14-OVA chimeric mice and LC-depleted K14-OVA x Langerin-DTR double Tg mice resulted in skin GvHD by day 7, comparable to normal K14-OVA Tg mice. Unexpectedly, OT-I cell injection into diptheria toxin (DT)-treated b2m→K14-OVA x Langerin-DTR Tg mice also resulted in skin GvHD. Thus, in vivo, even in the absence of both LCs in the epidermis and dDCs that can present OVA peptide in association with MHC I molecules, GvHD ensued. Furthermore, in vitro, OVA-expressing epidermal cells depleted of LCs (> 98% depletion) induced OT-I cell priming but did not induce a detectable allogeneic epidermal lymphocyte reaction. These results indicate that keratinocytes can function as accessory cells to induce the priming of naive autoreactive T cells in the skin.

Cutting Edge: Programed Death (PD) Ligand-1/PD-1 Interaction Is Required for CD8+ T Cell Tolerance to Tissue Antigens

Constitutive presentation of tissue Ags by dendritic cells results in tolerance of autoreactive CD8+ T cells; however, the underlying molecular mechanisms are not well understood. In this study we show that programmed death (PD)-1, an inhibitory receptor of the CD28 family, is required for tolerance induction of autoreactive CD8+ T cells. An antagonistic Ab against PD-1 provoked destructive autoimmune diabetes in RIP-mOVA mice expressing chicken OVA in the pancreatic islet cells, which received naive OVA-specific CD8+ OT-I cells. This effect was mediated by the PD ligand (PD-L) PD-L1 but not by PD-L2. An increased number of effector OT-I cells recovered from the pancreatic lymph nodes of anti-PD-L1-treated mice showed down-regulation of PD-1. Furthermore, the blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect OT-I cell division but enhanced its granzyme B, IFN-gamma, and IL-2 production. Thus, during the presentation of tissue Ags to CD8+ T cells, PD-1/PD-L1 interaction crucially controls the effector differentiation of autoreactive T cells to maintain self-tolerance.

Cytotoxic T-Cell Response Following Mouse Liver Transplantation Is Independent of the Initial Site of T-Cell Priming

Background Liver transplantation in the mouse results in systemic induction of tolerance. The underlying mechanisms may also account for the persistence of chronic liver infections. It has therefore been hypothesized that antigen (Ag) presentation within the liver by nonprofessional antigen-presenting cells (APC) leads to incomplete T-cell activation, ultimately resulting in tolerance induction. We tested this hypothesis in an orthotopic mouse liver transplantation model. Methods Mouse liver transplantation was used to manipulate antigen presentation in major histocompatibility complex (MHC)-disparate donor and recipient strains. The effect of restricted Ag presentation was studied using CD8 + T-cell receptor transgenic OT-I cells. Transgenic OT-I cells were activated by injection of their cognate peptide antigen SIINFEKL, which could be presented by the MHC class I of only one of the mouse strains. Depending on the strain combination, Ag presentation was restricted to either the transplanted liver itself, the recipient (excluding the transplanted liver), or systemically throughout the recipient. Extrahepatic Ag presentation by passenger leukocytes was eliminated by using donors of chimeric bone marrow. Results OT-I cells encountering antigen only in the transplanted liver were activated, underwent extensive proliferation, and developed effector functions, based on IFN-γ production and in vivo cytotoxicity assays. This T-cell activation and differentiation within the liver was comparable to animals with systemic Ag presentation and to animals with absent hepatic-parenchymal Ag presentation. Conclusions The restricted presentation of antigen in the liver showed no immunosuppressive effect on activation of CD8 + T cells. In contrast, the liver may be an excellent priming site for naive CD8 + T cells.

Transplanted Donor CD8 TN Convert to TM in Severely Lymphopenic HCT Recipients and Are Distinguishable from Bona Fide Donor CD8 TM.

During the past several years, it has been appreciated that lymphoid cells expand under conditions of lymphopenia. T cell replete hematopoietic cell transplants (HCT) can therefore potentially provide both immediate and long-term immune function. Recent studies have reported that naïve CD8 cells (TN) undergo a phenotypic and functional conversion to memory CD8 cells (TN→TM) during homeostatic expansion. We proposed that transplanted antigen-specific CD8 TN can convert to memory-like cells post-HCT and subsequently provide immune function equivalent to that by infused memory CD8 cells (TM). A syngeneic murine HCT model was therefore utilized to investigate the homeostatic expansion, phenotype, and effector function of TM and TN populations following transplant. The CD8 TM population was generated in vitro from OT-I-Rag1−/− spleen cells cultured with rmIL-2 and OVA peptide followed by rmIL-15. Resultant OT-I TM (>99%) exhibited a central memory (CD44+/Ly6C+) phenotype. The OT-I TN population (CD44lo/Ly6Clo) was positively selected from OT-I mice using Miltenyi CD8 beads (>99% OT-I). Either 1.5×106 TM or TN, with 2×106 T cell-depleted B6 BM, were transplanted into 9.0-Gy conditioned syngeneic recipients. Both CFSE-labeled TM and TN proliferated almost immediately post-transplant. By day 7, TN had undergone slightly more divisions than TM. Between days 8 and 10, TN exhibited greater levels of BrudU incorporation compared to TM. Homeostatic expansion by CD8 TM lasted approximately 2 weeks whereas expansion of CD8 TN continued for another week and ultimately reached greater overall numbers. Cell surface analysis of transplanted populations demonstrated that CD8 TM retained their memory phenotype >7 months post-HCT. In contrast, the initially naive phenotype of transplanted TN began to resemble a memory phenotype by 14 days post-HCT and by 6 weeks exhibited such a phenotype. At this time, the transplanted TN→TM generated ex vivo lytic activity which was indistinguishable from transplanted TM. Interestingly, when challenged in vivo with peptide-pulsed DC >6 weeks post-HCT, OT-I cells in HCT recipients of TM and TN displayed differences in their ability to undergo antigen-driven expansion. The transplanted TM expanded following challenge, reaching maximal levels 8 days later, then contracted to a set-point higher than pre-challenge levels. In contrast, the transplanted TN→TM showed considerably less expansion post-challenge. These findings demonstrate that the conditions for the homeostatic expansion of donor TN and TM are present in recipients immediately following ablative conditioning and transplant. Moreover, expansion conditions appear to favor TN which reached higher homeostatic numbers. Although transplanted TN converted to a memory phenotype and demonstrated ex vivo lytic activity equivalent to transplanted TM, both memory populations did not undergo equivalent expansion following in vivo antigen exposure. These findings challenge the notion that antigen-independent TN→TM populations are functionally indistinguishable from bona fide TM cells. We propose that on a per-cell basis, transplanted TN→TM may function indistinguishably from transplanted TM. However, their initial period of rapid proliferation immediately following infusion may have diminished the ability of TN→TM to subsequently expand following in vivo antigen challenge.

An important role of Tyk2 in APC function of dendritic cells for priming CD8+ T cells producing IFN‐γ

Tyrosine kinase 2 (Tyk2) contributes to the signals triggered by IL-12 for IFN-gamma production by NK cells and T cells. We found in this study that Tyk2-deficient (-/-) mice showed increased susceptibility at the early stage after an i.p. infection with Listeria monocytogenes, accompanied by impaired IFN-gamma production. The numbers of both MHC class Ib (H2-M3)- or MHC class Ia (Kb)-restricted CD8+ T cells producing IFN-gamma and exhibiting cytotoxicity were significantly decreased in Tyk2-/- mice after infection with L. monocytogenes. Using an adoptive transfer system of OT-I cells expressing OVA(257-264)/Kb-specific TCR into Tyk2-/- mice followed by challenge with recombinant L. monocytogenes expressing OVA, we found that the defective Tyk2 signaling in the host environment was at least partially responsible for the impaired CD8+ T cytotoxic-1 (Tc1) cell responses in Tyk2-/- mice following the infection. Adoptive transfer with MHC class Ib- or MHC class Ia-binding peptide-pulsed BM-derived DC from Tyk2-/- mice induced lower levels of the Ag-specific CD8+ Tc1 cells producing IFN-gamma. These results suggest that Tyk2 signaling is also important for DC function in the induction of MHC class Ia- and class Ib-restricted CD8+ Tc1 cells following L. monocytogenes infection.

Novel Model of Antigen-Specific Induction of Bile Duct Injury

Biliary-directed inflammation is an important cause of acute and chronic liver disease. We developed and characterized a transgenic mouse model of immune-mediated hepatobiliary injury. Ovalbumin (OVA)-BIL mice were developed using 3.0 kilobase of the rat apical sodium-dependent bile acid transporter promoter to drive aberrant expression of a membrane form of ovalbumin (OVA) on biliary epithelium. Liver inflammation resulted from adoptive transfer of OVA-specific T cells. Liver immune cells were characterized to determine the mechanism of the response by assessing activation, proliferation, and intracellular cytokine expression. OVA-BIL transgenic mice were tolerant to OVA, without evidence of liver disease. Adoptive transfer of OVA-specific CD4+ and CD8+ T cells into naïve OVA-BIL mice led to biliary-centered necroinflammatory damage in a dose-dependent manner. This inflammation absolutely required CD8+ T cells and was augmented by CD4+ T cells. Adoptively transferred OVA CD8+ cells homed to and proliferated in the liver but not the spleen. These activated, adoptively transferred cytotoxic T lymphocytes produced elevated levels of tumor necrosis factor alpha and interferon gamma. T-cell recognition of antigen aberrantly expressed on bile duct epithelium induced an acute necroinflammatory response specific to the liver, with activation, proliferation, and cytokine production predominantly by the OVA-specific cytotoxic T cells. Thus, OVA BIL represents an antigen-specific animal model of inflammatory bile duct injury.

Abrogation of Functional Selectin-Ligand Expression Reduces Migration of Pathogenic CD8+ T Cells into Heart

CD8+ T cells are involved in autoimmune and infectious myocarditis and cardiac allograft rejection. The role of selectins in cardiac recruitment of CD8+ T cells is not understood. In this study, the contribution of T cell selectin ligands to effector CD8+ T cell recruitment into the heart was examined using a model of myocarditis, which depends on transfer of OVA peptide-specific CD8+ T cells (OT-I) into mice (CMy-mOva) that express OVA in the heart. alpha-(1,3)-Fucosyltransferase (FucT)-VII-deficient OT-I cells displayed over a 95% reduction in their ability to interact with P-selectin under flow conditions in vitro, compared with wild-type OT-I cells. Interaction of FucT-VII-deficient OT-I cells with E-selectin was reduced approximately 50%. FucT-VII-deficient OT-I cells were also less efficiently recruited into a dermal site of Ag and adjuvant injection. Significantly, FucT-VII-deficient OT-I cells were also impaired in their ability to migrate into CMy-mOva hearts, compared with wild-type OT-I cells. Transfer of FucT-VII-deficient T cells caused less severe early myocarditis and myocyte damage than transfer of wild-type T cells. Combined FucT-IV/VII-deficient OT-I cells displayed a more profound reduction in E-selectin interactions in vitro compared with FucT-VII-deficient T cells, and the FucT-IV/VII-deficient T cells also showed less early recruitment and pathogenicity in the CMy-mOva myocarditis model. These results identify a prominent role for selectin ligands in contributing to effector CD8+ T cell recruitment into the myocardium and indicate that selectin-dependent T cell recruitment is relevant to other tissues besides the skin.

The Mannose Receptor Mediates Uptake of Soluble but Not of Cell-Associated Antigen for Cross-Presentation

The mannose receptor (MR) has been implicated in the recognition and clearance of microorganisms and serum glycoproteins. Its endocytic function has been studied extensively using macrophages, although it is expressed by a variety of cell types, including dendritic cells (DC). In this study, we investigated its role in Ag presentation by DC using MR-/- mice. Uptake of the model Ag, soluble OVA, by bone marrow-derived DC and in vitro activation of OVA-specific CD8 T cells (OT-I cells) strictly depended on the MR. In vivo, MR deficiency impaired endocytosis of soluble OVA by DC and concomitant OT-I cell activation. No alterations in the DC subtype composition in MR-/- mice were accountable. Uptake of cell-associated OVA was unaffected by MR deficiency, resulting in unchanged activation of OT-I cells. These findings demonstrate that DC use the MR for endocytosis of a particular Ag type intended for cross-presentation.

Gut-associated lymphoid tissue–primed CD4+ T cells display CCR9-dependent and -independent homing to the small intestine

CD4(+) T-cell entry to the intestinal mucosa is central to the generation of mucosal immunity as well as chronic intestinal inflammation, yet the mechanisms regulating this process remain poorly defined. Here we show that murine small intestinal CD4(+) lamina propria lymphocytes express a heterogeneous but restricted array of chemokine receptors including CCR5, CCR6, CCR9, CXCR3, and CXCR6. CD4(+) T-cell receptor transgenic OT-II cells activated in mesenteric lymph nodes acquired a distinct chemokine receptor profile, including expression of CCR6, CCR9, and CXCR3 that was only partially reproduced in vitro after priming with mesenteric lymph node dendritic cells. A subset of these effector CD4(+) T cells, expressing CD69 and alpha(4)beta(7), entered the intestinal lamina propria and the majority of these cells expressed CCR9. CCR9(-/-) OT-II cells were disadvantaged in their ability to localize to the intestinal lamina propria; however, they were readily detected at this site and expressed alpha(4)beta(7), but little CCR2, CCR5, CCR6, CCR8, CCR10, CXCR3, or CXCR6. Thus, whereas CD4(+) T cells activated in gut-associated lymphoid tissue express a restricted chemokine receptor profile, including CCR9, targeting both CCR9-dependent and CCR9-independent entry mechanisms is likely to be important to maximally inhibit accumulation of these cells within the small intestinal mucosa.