The Journal of Immunology recent issues

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2008-11-18

[PILLARS OF IMMUNOLOGY] Bringing the Thymus to the Bench

Anderson, G., Jenkinson, E. J. — 2008-11-18

[PILLARS OF IMMUNOLOGY] Pillars Article: Generation of T-cell Function in Organ Culture of Foetal Mouse Thymus I. Mitogen Responsiveness

2008-11-18

[CUTTING EDGE] Cutting Edge: CD4+ T Cell-Derived IL-2 Is Essential for Help-Dependent Primary CD8+ T Cell Responses

Wilson, E. B., Livingstone, A. M. — 2008-11-18

CD4⁺ T cell help is essential for primary CD8⁺ T cell responses to noninflammatory Ags. IL-2 is one of the principal cytokines made by naive CD4⁺ T cells, and we show in this study that it is an essential component of help. Adoptively transferred naive CD4⁺ TCR-transgenic OT-II cells supported endogenous primary CD8⁺ T cell responses, but IL-2-deficient OT-II cells were unable to provide help, although they responded to Ag in vivo and up-regulated CD40 ligand in vitro. Wild -type OT-II cells helped endogenous CD8⁺ T cell responses in IL-2-deficient mice, but not in IL-2R-deficient mice. Thus, CD4⁺ T cell-derived IL-2 is essential for CD8⁺ T cell responses to noninflammatory, cell-associated Ags. We suggest that it is also a critical component of help for CD8⁺ T cell responses to pathogens, because protective memory also requires CD8⁺ T cell stimulation by IL-2 during priming.

[CUTTING EDGE] Cutting Edge: The Idd3 Genetic Interval Determines Regulatory T Cell Function through CD11b+CD11c- APC

Anderson, A. C., Chandwaskar, R., Lee, D. H., Kuchroo, V. K. — 2008-11-18

The Idd3 genetic interval confers protection against multiple autoimmune diseases, including type 1 diabetes and experimental autoimmune encephalomyelitis. The favored candidate gene in this interval is Il2, which is polymorphic between susceptible and resistant strains of mice. IL-2 regulates the growth/death of effector T cells as well as the generation/maintenance of regulatory T cells (Tregs), and recent studies have shown that NOD.Idd3 Tregs are more suppressive than their NOD counterparts. We have further dissected the mechanisms underlying the differential suppression by NOD and NOD.Idd3 Tregs and find that it is determined by CD11b⁺CD11c^– APCs. Thus, contrary to what might be expected, our data suggest that the differential suppressive activity of NOD and NOD.Idd3 Tregs is not due to an effect of the Idd3 genetic interval on T cells but rather is due to differences in the APC compartment.

[CUTTING EDGE] Cutting Edge: Viral Infection Breaks NK Cell Tolerance to "Missing Self"

Sun, J. C., Lanier, L. L. — 2008-11-18

NK cells attack cells lacking MHC class I, yet MHC class I-deficient mice have normal numbers of NK cells with intact, albeit diminished, functions. Moreover, wild-type NK cells are tolerant of MHC class I-deficient cells in mixed bone marrow chimeras. In this study, we investigated how the absence of MHC class I affects NK cells. NK cells from β₂-microglobulin-deficient (B2m^–/–) and wild-type mice exhibit similar phenotypic and functional characteristics. Both B2m^–/– and wild-type Ly49H⁺ NK cells proliferated robustly and produced IFN- after infection with mouse CMV. NK cells in mixed wild-type:B2m^–/– chimeric mice were initially tolerant of MHC class I-deficient host cells. However, this tolerance was gradually lost over time and after mouse CMV infection was rapidly broken, with a pronounced rejection of host B2m^–/– hematopoietic cells. Thus, although NK cells can be held in check against "missing self," acute inflammation driven by infection can rapidly break established self-tolerance.

[CUTTING EDGE] Cutting Edge: Selective Blockade of LIGHT-Lymphotoxin {beta} Receptor Signaling Protects Mice from Experimental Cerebral Malaria Caused by Plasmodium berghei ANKA

2008-11-18

Studies in experimental cerebral malaria (ECM) in mice have identified T cells and TNF family members as critical mediators of pathology. In this study we report a role for LIGHT-lymphotoxin β Receptor (LTβR) signaling in the development of ECM and control of parasite growth. Specific blockade of LIGHT-LTβR, but not LIGHT-herpesvirus entry mediator interactions, abrogated the accumulation of parasites and the recruitment of pathogenic CD8⁺ T cells and monocytes to the brain during infection without affecting early activation of CD4⁺ T cells, CD8⁺ T cells, or NK cells. Importantly, blockade of LIGHT-LTβR signaling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process Ag during infection, as well as reduced systemic cytokine levels when control mice displayed severe ECM symptoms. In summary, we have discovered a novel pathogenic role for LIGHT and LTβR in ECM, identifying this TNF family receptor-ligand interaction as an important immune regulator during experimental malaria.

[CUTTING EDGE] Cutting Edge: Members of the Staphylococcus aureus Extracellular Fibrinogen-Binding Protein Family Inhibit the Interaction of C3d with Complement Receptor 2

2008-11-18

Staphylococcus aureus expresses a highly diversified arsenal of immune evasion proteins, many of which target the complement system. The extracellular fibrinogen-binding protein (Efb) and the Efb homologous protein (Ehp) have previously been demonstrated to bind to C3 and inhibit complement activation and amplification. In this study we present the first evidence that Efb and Ehp are also capable of inhibiting the interaction of C3d with complement receptor 2 (CR2), which plays an important role in B cell activation and maturation. The C-terminal domain of Efb efficiently blocked this interaction both in surface plasmon resonance-based competition studies and cellular assays and prevented the CR2-mediated stimulation of B cells. Furthermore, analyses of the available structural data were consistent with a molecular mechanism that reflects both steric and electrostatic effects on the C3d-CR2 interaction. Our study therefore suggests that S. aureus may disrupt both the innate and adaptive immune responses with a single protein module.

[CUTTING EDGE] Cutting Edge: Migration of Langerhans Dendritic Cells Is Impaired in Autoimmune Dermatitis

Eriksson, A. U., Singh, R. R. — 2008-11-18

Tissue-resident dendritic cells, such as Langerhans cells (LC), normally carry Ags from tissues to lymph nodes to induce immunity to tissue Ags. In this study, we report that LC are reduced in the skin-draining lymph nodes of MRL-Fas^lpr/lpr and MRL-Fas^+/+ mice that develop T cell-mediated autoimmune skin inflammation as compared with MHC-matched healthy strains. This deficiency of LC in skin-draining lymph nodes is due to a profound impairment of LC migration, resulting in the accumulation of activated LC in the skin. Such a defect in LC migration develops before the onset of skin lesions and correlates with the onset and severity of dermatitis. The reduced, rather than increased, migration of LC from skin to skin-draining lymph nodes represents a novel functional abnormality of LC in autoimmune dermatitis.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Requirement of IL-17RA in Con A Induced Hepatitis and Negative Regulation of IL-17 Production in Mouse T Cells

2008-11-18

Th17 cells, a subset of T cells involved in autoimmunity and host defense against extracellular Gram-negative infection, express both IL-17A and IL-17F. Both IL-17A and IL-17F can signal via the IL-17RA; however, IL-17F does so at a 1- to 2-log higher concentration than IL-17A. In this study, we show that the IL-17F homodimer via IL-17RA is a negative regulator of IL-17 production in T cells and suggest a mechanism whereby IL-17RA on T cells serves as an autocrine/paracrine regulator of IL-17 synthesis in T cells.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Increased Osteopontin Expression in Dendritic Cells Amplifies IL-17 Production by CD4+ T Cells in Experimental Autoimmune Encephalomyelitis and in Multiple Sclerosis

Murugaiyan, G., Mittal, A., Weiner, H. L. — 2008-11-18

Osteopontin (Opn) is a broadly expressed pleiotropic cytokine, and has been shown to play an important role in various autoimmune diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). It is reported that Opn exacerbates EAE by skewing T cell differentiation toward IFN--producing Th1 cells. Opn expression in dendritic cells (DCs) and its role in IL-17 induction from T cells during EAE or MS are unknown. We found that during EAE, Opn expression is elevated in DCs both in the periphery and in the CNS. There was increased expression of Opn receptor on T cells, and Opn induced IL-17 production by CD4⁺ T cells via the β₃ integrin receptor and Opn inhibited IL-10 production via the CD44 receptor. Furthermore, anti-Opn treatment reduced clinical severity of EAE by reducing IL-17 production. Anti-Opn was also effective in reducing clinical severity of EAE when given after the appearance of clinical symptoms. Analogous to EAE, in subjects with MS, we found increased expression of Opn in DCs and increased expression of the Opn receptors CD44, β₃, and _v on T cells. Furthermore, Opn-stimulated CD4⁺ T cells from MS patients produced significantly higher amounts of IL-17. Our results demonstrate a role for DC-produced Opn both in EAE and MS that is linked to the production of IL-17.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] NK Cells Induce Apoptosis in Tubular Epithelial Cells and Contribute to Renal Ischemia-Reperfusion Injury

2008-11-18

Renal ischemia-reperfusion injury (IRI) can result in acute renal failure with mortality rates of 50% in severe cases. NK cells are important participants in early-stage innate immune responses. However, their role in renal tubular epithelial cell (TEC) injury in IRI is currently unknown. Our data indicate that NK cells can kill syngeneic TEC in vitro. Apoptotic death of TEC in vitro is associated with TEC expression of the NK cell ligand Rae-1, as well as NKG2D on NK cells. In vivo following IRI, there was increased expression of Rae-1 on TEC. FACS analyses of kidney cell preparations indicated a quantitative increase in NKG2D-bearing NK cells within the kidney following IRI. NK cell depletion in wild-type C57BL/6 mice was protective, while adoptive transfer of NK cells worsened injury in NK, T, and B cell-null Rag2^–/–_c^–/– mice with IRI. NK cell-mediated kidney injury was perforin (PFN)-dependent as PFN^–/– NK cells had minimal capacity to kill TEC in vitro compared with NK cells from wild-type, FasL-deficient (gld), or IFN-^–/– mice. Taken together, these results demonstrate for the first time that NK cells can directly kill TEC and that NK cells contribute substantially to kidney IRI. NK cell killing may represent an important underrecognized mechanism of kidney injury in diverse forms of inflammation, including transplantation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Inhibition of the Alloimmune Response Through the Generation of Regulatory T Cells by a MHC Class II-Derived Peptide

2008-11-18

We have previously shown that HLA-DQA1, a peptide derived from a highly conserved region of MHC class II, prevents alloreactive T cell priming and effector function in vivo, although underlying mechanisms are obscure. In this study, we demonstrate that 28% of mice treated with HLA-DQA1 combined with low-dose rapamycin achieved permanent engraftment of fully MHC-disparate islet allografts and significantly prolonged survival in the remaining animals (log rank, p < 0.001). Immunohistologic examination of the grafts from HLA-DQA1/rapamycin-treated animals revealed up-regulated expression of TGF-ß and FoxP3. In vivo administration of blocking anti-TGF-ß or depleting anti-CD25 mAb augmented T cell alloimmunity and prevented the long-term engraft induced by HLA-DQA1. In vitro experiments further showed that HLA-DQA1 induced differentiation of CD4⁺ T cells into CD4⁺CD25⁺FoxP3⁺ regulatory T cells. Together, these data provide the first demonstration that HLA-DQA1, a MHC class II-derived peptide, can prolong allograft survival via a TGF-β and regulatory T cell-dependent mechanisms.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Lin-Sca1+Kit- Bone Marrow Cells Contain Early Lymphoid-Committed Precursors That Are Distinct from Common Lymphoid Progenitors

Kumar, R., Fossati, V., Israel, M., Snoeck, H.-W. — 2008-11-18

The significance of a population in mouse bone marrow of lineage-negative (Lin^–), Sca1-positive, c-kit-negative (LSK^–) cells, which is reported to be devoid of long-term repopulation capacity or myeloid potential, is unknown. In this study, we show that the LSK^– population is composed of several subsets defined by the expression of flt3, CD25, and IL-7R. The first subset was CD25^– and more than 90% expressed either flt3, IL-7R, or both. The CD25^–LSK^– population had T cell, B cell, and NK cell potential in vivo, and most of this activity was localized in the flt3⁺ subset, irrespective of the expression of IL-7R. Although lymphoid potential of flt3⁺LSK^– cells in vivo was 3-fold lower than that of lin^–Sca1^lowkit^lowIL7R⁺ common lymphoid progenitors (CLPs), their cloning efficiency in vitro was 10-fold lower than that of CLPs. Furthermore, although the myeloid potential of flt3⁺LSK^– cells was 10-fold lower than that of CLPs in the absence of M-CSF, the relative myeloid potential of both populations was similar in its presence. These observations suggest differential growth factor requirements of both populations. The second subset of LSK^– cells was homogeneously CD25⁺flt3^–IL7R⁺ and could be generated from both CD25^–LSK^– cells and from CLPs, but did not engraft in immunodeficient Rag1^–/– or Rag1^–/–_c^–/– hosts. This population, of which the significance is unclear, was increased in Rag1^–/– mice and in old mice. Thus, the LSK^– population is phenotypically and functionally heterogeneous and contains early lymphoid-committed precursors. Our findings imply that the early stages of lymphoid commitment are more complex than was thus far assumed.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Resolution of Unique Sca-1highc-Kit- Lymphoid-Biased Progenitors in Adult Bone Marrow

Harman, B. C., Northrup, D. L., Allman, D. — 2008-11-18

We have identified a distinctive lymphoid-restricted progenitor population in adult mouse bone marrow based on a unique c-Kit^–Sca-1^highFlt3⁺ AA4⁺ surface phenotype. These cells are highly lymphoid biased and rapidly generate B and T cells after adoptive transfer. However, whereas previously described lymphoid progenitors such as common lymphoid progenitors express TdT and relatively high levels of RAG2, and are enriched for cells with an active V(D)J recombinase, Flt3⁺ AA4⁺ cells within the c-Kit^–Sca-1^high bone marrow fraction are TdT^–, are RAG2^low, and do not display evidence for ongoing or past recombinase activity. Furthermore, unlike common lymphoid progenitors that readily generate B cells upon stimulation with IL-7, c-Kit^–Sca-1^highFlt3⁺ precursors do not express abundant levels of the IL-7R, and require costimulation with Flt3 ligand and IL-7 to generate B cells in vitro. Moreover, these findings suggest that hematopoietic stem cells in adults generate an array of lymphoid-biased progenitor populations characterized by distinct gene expression and cytokine response profiles.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Retinal Pigment Epithelium-Derived CTLA-2{alpha} Induces TGF{beta}-Producing T Regulatory Cells

2008-11-18

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4⁺ T cells into Treg cells by producing and secreting CTLA-2, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2 converted CD4⁺ T cells into Treg cells in vitro, and CTLA-2 small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2 induced CD4⁺CD25⁺Foxp3⁺ Treg cells that produced TGFβ in vitro. Moreover, CTLA-2 produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4⁺ T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2 effect on Treg cell induction occurred through TGFβ signaling, because CTLA-2 promoted activation of TGFβ in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Deregulation of c-Myc Confers Distinct Survival Requirements for Memory B Cells, Plasma Cells, and Their Progenitors

Khuda, S. E., Loo, W. M., Janz, S., Van Ness, B., Erickson, L. D. — 2008-11-18

Deregulation of the c-Myc oncogene is tightly associated with human and murine plasma cell (PC) neoplasms. Through the analysis of Ag-specific B cell responses in mice where Myc is targeted to the Igh C locus, we show here that c-Myc dramatically impairs the primary and secondary Ab response. This impairment is differentiation stage specific, since germinal center B cell formation, affinity maturation, and class switch recombination were intact. Examination of PC viability revealed that c-Myc triggered apoptosis only upon final maturation when Ab is secreted and is resistant to the survival factor BAFF (B cell-activating factor belonging to the TNF family). In contrast, PC precursors (PC_pre) that ultimately give rise to mature PCs survived normally and vigorously expanded with BAFF signaling. We further show that c-Myc also facilitates the apoptosis of memory B cells. Thus, C-Myc controls both cellular arms of long-lived B cell immunity than previously anticipated. Only when deregulation of c-Myc was combined with enforced Bcl-x_L expression were mature PCs able to survive in response to BAFF. These data indicate that the survival requirements for tumor-susceptible PC_pre and PCs are distinct and that tumor progression likely develops as PC_pre transition to functional PCs when apoptotic pathways such as members of the Bcl-2 family are disabled.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Targeting the Neonatal Fc Receptor for Antigen Delivery Using Engineered Fc Fragments

Mi, W., Wanjie, S., Lo, S.-T., Gan, Z., Pickl-Herk, B., Ober, R. J., Ward, E. S. — 2008-11-18

The development of approaches for Ag delivery to the appropriate subcellular compartments of APCs and the optimization of Ag persistence are both of central relevance for the induction of protective immunity or tolerance. The expression of the neonatal Fc receptor, FcRn, in APCs and its localization to the endosomal system suggest that it might serve as a target for Ag delivery using engineered Fc fragment-epitope fusions. The impact of FcRn binding characteristics of an Fc fragment on in vivo persistence allows this property to also be modulated. We have therefore generated recombinant Fc (mouse IgG1-derived) fusions containing the N-terminal epitope of myelin basic protein that is associated with experimental autoimmune encephalomyelitis in H-2^u mice. The Fc fragments have distinct binding properties for FcRn that result in differences in intracellular trafficking and in vivo half-lives, allowing the impact of these characteristics on CD4⁺ T cell responses to be evaluated. To dissect the relative roles of FcRn and the "classical" FcRs in Ag delivery, analogous aglycosylated Fc-MBP fusions have been generated. We show that engineered Fc fragments with increased affinities for FcRn at pH 6.0–7.4 are more effective in delivering Ag to FcRn-expressing APCs in vitro relative to their lower affinity counterparts. However, higher affinity of the FcRn-Fc interaction at near neutral pH results in decreased in vivo persistence. The trade-off between improved FcRn targeting efficiency and lower half-life becomes apparent during analyses of T cell proliferative responses in mice, particularly when Fc-MBP fusions with both FcRn and FcR binding activity are used.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Uncoupling of Induced Protein Processing from Maturation in Dendritic Cells Exposed to a Highly Antigenic Preparation from a Helminth Parasite

Marshall, F. A., Pearce, E. J. — 2008-11-18

TLR ligands induce dendritic cell (DC) maturation. During this process, cells initiate proteolytic degradation of internalized protein Ags into peptides that complex with MHC class II (MHC II) and simultaneously increase expression of costimulatory molecules and of cytokines such as IL-6, IL-12, and IL-23. In these ways, TLR-activated DCs are able to activate naive Th cells and initiate Th1 and Th17 responses, and TLR ligands thus serve as adjuvants for these types of responses. In contrast, products from helminth parasites generally do not activate DCs and act as adjuvants for Th2 response induction. We have explored the underlying basis for this form of adjuvanticity. We show that exposure of DCs to soluble Ags from the eggs of the helminth parasite Schistosoma mansoni (schistosome egg Ag (SEA)) leads to the induction of proteolysis of internalized Ag. This occurs in the absence of significant induction of costimulatory molecule expression or production of proinflammatory cytokines. SEA-induced Ag processing occurs independently of MyD88 or Toll/IL-1 receptor domain containing adaptor inducing IFN-β (Trif), but is significantly attenuated by inhibition of p38, but not ERK, signaling. In DCs exposed to SEA, ligation of CD40 provides a necessary second signal that stimulates costimulatory molecule expression, allowing DCs to mature into capable APCs. Collectively, the data demonstrate the existence of a MyD88/Trif-independent, p38-dependent pathway of Ag processing in DCs, which is uncoupled from conventional DC maturation and is associated with induction of Th2-type immune responses.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Transplantation of Bone Marrow Transduced to Express Self-Antigen Establishes Deletional Tolerance and Permanently Remits Autoimmune Disease

2008-11-18

Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-, and GM-CSF in response to MOG_35–55 peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4⁺ MOG_35–55-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] A New Approach to the Blocking of Alloreactive T Cell-Mediated Graft-versus-Host Disease by In Vivo Administration of Anti-CXCR3 Neutralizing Antibody

2008-11-18

Chemokines and chemokine receptors play critical roles in directing the migration of alloreactive donor T cells into graft-vs-host disease (GVHD) target organs. However, blockade of GVHD by antagonist Ab against chemokine receptors remains an elusive goal. Using a mouse model of human GVHD, we demonstrate that in vivo administration of anti-CXCR3 Ab for 21 days (long-term), but not for 7 days (short-term), inhibits alloreactive CD8⁺ T cell-mediated GVHD. During a graft-vs-host reaction, infused donor CD8⁺ T cells generate two subsets of potent inducers of GVHD: CXCR3⁺CD8⁺ and CXCR3^–CD8⁺ T cells. Compared with CXCR3⁺CD8⁺ T cells, CXCR3^–CD8⁺ T cells produce less granzyme B, Fas ligand, IFN-, and TNF-. Interestingly, stimulation with either dendritic cells or IL-2 induces a dynamic conversion between CXCR3⁺CD8⁺ and CXCR3^–CD8⁺ T cells. Short-term anti-CXCR3 Ab treatment inhibits only CXCR3⁺CD8⁺ T cell-mediated GVHD, but not the disease induced by CXCR3^–CD8⁺ T cells. Prolonged in vivo administration of anti-CXCR3 Ab significantly reduces the infiltration of alloreactive CD8⁺ T cells into GVHD target organs and inhibits GVHD mediated by either CXCR3⁺CD8⁺ or CXCR3^–CD8⁺ T cells. Thus, we have established a novel and effective approach with the potential to give rise to new clinical methods for preventing and treating GVHD after allogeneic hematopoietic stem cell transplantation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Drak2 Regulates the Survival of Activated T Cells and Is Required for Organ-Specific Autoimmune Disease

McGargill, M. A., Choy, C., Wen, B. G., Hedrick, S. M. — 2008-11-18

Drak2 is a serine/threonine kinase expressed in T and B cells. The absence of Drak2 renders T cells hypersensitive to suboptimal stimulation, yet Drak2^–/– mice are enigmatically resistant to experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We show in this study that Drak2^–/– mice were also completely resistant to type 1 diabetes when bred to the NOD strain of mice that spontaneously develop autoimmune diabetes. However, there was not a generalized suppression of the immune system, because Drak2^–/– mice remained susceptible to other models of autoimmunity. Adoptive transfer experiments revealed that resistance to disease was intrinsic to the T cells and was due to a loss of T cell survival under conditions of chronic autoimmune stimulation. Importantly, the absence of Drak2 did not alter the survival of naive T cells, memory T cells, or T cells responding to an acute viral infection. These experiments reveal a distinction between the immune response to persistent self-encoded molecules and transiently present infectious agents. We present a model whereby T cell survival depends on a balance of TCR and costimulatory signals to explain how the absence of Drak2 affects autoimmune disease without generalized suppression of the immune system.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Enhanced T Cell Apoptosis within Drak2-Deficient Mice Promotes Resistance to Autoimmunity

Ramos, S. J., Hernandez, J. B., Gatzka, M., Walsh, C. M. — 2008-11-18

Clonal expansion of T cells is vital to adaptive immunity, yet this process must be tightly controlled to prevent autoimmune disease. The serine/threonine kinase death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK2) is a negative regulator of TCR signaling and sets the threshold for the activation of naive and memory T cells and selected thymocytes. Despite enhanced T cell activation, Drak2^–/– mice are resistant to experimental autoimmune encephalomyelitis, an autoimmune demyelinating disease that resembles multiple sclerosis. However, the basis for this autoimmune resistance is currently unknown. In this study, we show that, in the absence of DRAK2 signaling, T cells require greater tonic signaling for maintenance during clonal expansion. Following stimulation, Drak2^–/– T cells were more sensitive to an intrinsic form of apoptosis that was prevented by CD28 ligation, homeostatic cytokines, or enforced Bcl-x_L expression. T cell-specific Bcl-x_L expression also restored the susceptibility of Drak2^–/– mice to experimental autoimmune encephalomyelitis and enhanced thymic positive selection. These findings demonstrate that DRAK2 is selectively important for T cell survival and highlight the potential that DRAK2 blockade may lead to permanent autoimmune T cell destruction via intrinsic apoptosis pathways.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The Erk2 MAPK Regulates CD8 T Cell Proliferation and Survival

D'Souza, W. N., Chang, C.-F., Fischer, A. M., Li, M., Hedrick, S. M. — 2008-11-18

The magnitude of T cell responses is determined by proliferation and survival decisions made by the responding cells. We now demonstrate that the Erk MAPK pathway plays a critical role in these cell fate decisions within CD8 T cells. While Erk1 is dispensable for all aspects of CD8 T cell activation, Erk2 is required for the proliferation of CD8 T cells activated in the absence of costimulation. Surprisingly, Erk2 is not required for proliferation following the addition of a costimulatory signal in vitro, or upon viral infection in vivo, but regulates the size of the responding population by enhancing cell survival. An important component of this Erk2-derived signal is the transcriptional regulation of Bcl-2 family members Bcl-x_L and Bim, and impaired Erk2-deficient CD8 T cell survival can be rescued by genetic ablation of Bim. These studies ascribe multifaceted functions specific to Erk2 in CD8 T cell activation, proliferation, and survival.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The Immunosuppressor Mycophenolic Acid Kills Activated Lymphocytes by Inducing a Nonclassical Actin-Dependent Necrotic Signal

2008-11-18

Mycophenolate mofetil (MMF) is an immunosuppressive agent used in transplantation. Over the last decade, MMF has also emerged as an alternative therapeutic regimen for autoimmune diseases, mainly for patients refractory to other therapies. The active compound of MMF, mycophenolic acid (MPA), depletes the intracellular pool of guanosine tri-phosphate through inosine monophosphate dehydrogenase blockade. The molecular mechanism involved in the elimination of T and B lymphocytes upon inhibition of inosine monophosphate dehydrogenase remains elusive. In this study, we showed that in contrast to the immunosuppressors azathioprine, cyclosporin A, and tacrolimus, MPA killed lymphocytes through the activation of a caspase-independent necrotic signal. Furthermore, the MPA-mediated necrotic signal relied on the transmission of a novel intracellular signal involving Rho-GTPase Cdc42 activity and actin polymerization. In addition to its medical interest, this study sheds light on a novel and atypical molecular mechanism leading to necrotic cell death.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Signals and Sequences That Control CD28 Localization to the Central Region of the Immunological Synapse

Sanchez-Lockhart, M., Graf, B., Miller, J. — 2008-11-18

During T cell interaction with APC, CD28 is recruited to the central region (cSMAC) of the immunological synapse. CD28-mediated signaling through PI3K results in the recruitment of protein kinase C- (PKC) to the cSMAC, activation of NF-B, and up-regulation of IL-2 transcription. However, the mechanism that mediates CD28 localization to the cSMAC and the functional consequences of CD28 localization to the cSMAC are not understood. In this report, we show that CD28 recruitment and persistence at the immunological synapse requires TCR signals and CD80 engagement. Addition of mAb to either MHC class II or CD80 results in the rapid displacement of CD28 from the immunological synapse. Ligand binding is not sufficient for CD28 localization to the immunological synapse, as truncation of the cytosolic tail of CD28 disrupts synapse localization without effecting the ability of CD28 to bind CD80. Furthermore, a single point mutation in the CD28 cytosolic tail (tyrosine 188) interferes with the ability of CD28 to preferentially accumulate at the cSMAC. PKC distribution at the immunological synapse mirrors the distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKC even when CD28 is not localized to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-B, suggesting that CD28-mediated localization of PKC to the cSMAC is important for efficient signal transduction. These data reinforce the importance of the interplay of signals between TCR and CD28 and suggest that CD28 signaling through PCK may be mediated through localization to the cSMAC region of the immunological synapse.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Abnormal Regulatory and Effector T Cell Function Predispose to Autoimmunity following Xenogeneic Thymic Transplantation

2008-11-18

Porcine thymus grafts support robust murine and human thymopoiesis, generating a diverse T cell repertoire that is deleted of donor and host-reactive cells, achieving specific xenograft tolerance. Positive selection is mediated exclusively by the xenogeneic thymic MHC. Although thymectomized, T cell-depleted normal mice usually remain healthy following xenogeneic thymic transplantation, thymus-grafted congenitally athymic mice frequently develop multiorgan autoimmunity. We investigated the etiology of this syndrome by adoptively transferring lymphocyte populations from fetal pig thymus-grafted BALB/c nude mice to secondary BALB/c nude recipients. Fetal pig thymus-grafted nude mice generated normal numbers of CD25⁺Foxp3⁺CD4 T cells, but these cells lacked the capacity to block autoimmunity. Moreover, thymocytes and peripheral CD4⁺CD25^– cells from fetal pig thymus-grafted nude mice, but not those from normal mice, induced autoimmunity in nude recipients. Injection of thymic epithelial cells from normal BALB/c mice into fetal pig thymus grafts reduced autoimmunity and enhanced regulatory function of splenocytes. Our data implicate abnormalities in postthymic maturation, expansion, and/or survival of T cells positively selected by a xenogeneic MHC, as well as incomplete intrathymic deletion of thymocytes recognizing host tissue-specific Ags, in autoimmune pathogenesis. Regulatory cell function is enhanced and negative selection of host-specific thymocytes may potentially also be improved by coimplantation of recipient thymic epithelial cells in the thymus xenograft.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Galectin-9 Increases Tim-3+ Dendritic Cells and CD8+ T Cells and Enhances Antitumor Immunity via Galectin-9-Tim-3 Interactions

2008-11-18

A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of T cell-mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN--producing Tim-3⁺ CD8⁺ T cells with enhanced granzyme B and perforin expression, although it induced CD4⁺ T cell apoptosis. It simultaneously increased the number of Tim-3⁺CD86⁺ mature dendritic cells (DCs) in vivo and in vitro. Coculture of CD8⁺ T cells with DCs from Gal-9-treated mice increased the number of IFN- producing cells and IFN- production. Depletion of Tim-3⁺ DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN--producing CD8⁺ T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8⁺ T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8⁺ T cells.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Targeting Poly(I:C) to the TLR3-Independent Pathway Boosts Effector CD8 T Cell Differentiation through IFN-{alpha}/{beta}

Ngoi, S. M., Tovey, M. G., Vella, A. T. — 2008-11-18

Poly(I:C) is an adjuvant used for antitumor treatment and vaccines because of its prominent effects on CD8 T cells and NK cells. Poly(I:C) binds TLR3 and this interaction is thought to be central for driving cell-mediated immune responses. We investigated the importance of TLR3 in poly(I:C)-mediated endogenous CD8 T cell responses using the pathogenic T cell stimulant Staphylococcus aureus enterotoxin A. While the responsive CD8 T cells expanded comparably in both wild-type and TLR3^–/– mice, differentiation of effector CD8 T cells was enhanced by poly(I:C) in the TLR3^–/– mice. A higher percentage of Ag-specific CD8 T cells became IFN- and TNF- producers in the absence of TLR3 signaling. Consistent with this boosted response was the observation that TLR3-deficient cells synthesized less IL-10 compared with TLR3-sufficient cells in response to poly(I:C). Ultimately, however, the fundamental mechanism of CD8 effector T cell differentiation through the TLR3-independent pathway was shown to be completely IFN-/β-dependent. Administration of IFN-/β-neutralizing Abs abolished the poly(I:C) effects in TLR3^–/– mice. These findings reveal specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly(I:C) is authenticated as a therapeutic adjuvant used in vaccines.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Dynamic Modulation of CCR7 Expression and Function on Naive T Lymphocytes In Vivo

Britschgi, M. R., Link, A., Lissandrin, T. K. A., Luther, S. A. — 2008-11-18

The chemokine receptor CCR7 is critical for the recirculation of naive T cells. It is required for T cell entry into secondary lymphoid organs (SLO) and for T cell motility and retention within these organs. How CCR7 activity is regulated during these processes in vivo is poorly understood. Here we show strong modulation of CCR7 surface expression and occupancy by the two CCR7 ligands, both in vitro and in vivo. In contrast to blood, T cells in SLO had most surface CCR7 occupied with CCL19, presumably leading to continuous signaling and cell motility. Both ligands triggered CCR7 internalization in vivo as shown in Ccl19^–/– and plt/plt mice. Importantly, CCR7 occupancy and down-regulation led to strongly impaired chemotactic responses, an effect reversible by CCR7 resensitization. Therefore, during their recirculation, T cells cycle between states of free CCR7 with high ligand sensitivity in blood and occupied CCR7 associated with continual signaling and reduced ligand sensitivity within SLO. We propose that these two states of CCR7 are important to allow the various functions CCR7 plays in T cell recirculation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Retinoic Acids Are Potent Inhibitors of Spontaneous Human Eosinophil Apoptosis

2008-11-18

Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10^–6M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The Role of Thymic Stromal Lymphopoietin in CD8+ T Cell Homeostasis

Rochman, Y., Leonard, W. J. — 2008-11-18

Thymic stromal lymphopoietin (TSLP) is a cytokine produced by stromal cells, epithelial cells, and basophils that acts on dendritic cells, mast cells, and CD4⁺ T cells. The receptor for TSLP contains a TSLP-specific receptor chain (TSLPR) and the IL-7R -chain. Although IL-7 critically controls the expansion and survival of naive and memory CD8⁺ T cells, an action for TSLP on CD8⁺ T cells has not been reported. We now demonstrate that CD8⁺ T cells express TSLPR and that TSLP activates both STAT5 and Akt and induces Bcl-2 in these cells. Correspondingly, TSLP increases CD8⁺ T cell survival in vitro as well as in wild-type and T-depleted mice in vivo, without altering the homeostatic proliferation of these cells. Moreover, TSLP can maintain CD8⁺ T cells even in the absence of IL-7. Thus, our data reveal that TSLP contributes to CD8⁺ T cell homeostasis in both normal and lymphopenic conditions.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The Phosphoinositide 3-Kinase-Dependent Activation of Btk Is Required for Optimal Eicosanoid Production and Generation of Reactive Oxygen Species in Antigen-Stimulated Mast Cells

2008-11-18

Activated mast cells are a major source of the eicosanoids PGD₂ and leukotriene C₄ (LTC₄), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A₂, thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC₄ and PGD₂, respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcRI-dependent PGD₂, LTC₄, and ROS production in bone marrow-derived mast cells of Btk^–/– mice. The FcRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A₂ phosphorylation but rather appears to regulate the generation of ROS, LTC₄, and PGD₂ by contributing to the necessary Ca²⁺ signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] An In Vitro System to Model the Establishment and Reactivation of HIV-1 Latency

Marini, A., Harper, J. M., Romerio, F. — 2008-11-18

HIV-1 establishes latency primarily by infecting activated CD4⁺ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. The lack of a valid cellular model to study HIV-1 latency has hindered advances in the understanding of its biology. In this study, we attempted to model HIV-1 latency using human primary CD4⁺ T cells infected in vitro with HIV-1 after activation with Ag-loaded dendritic cells and then brought back to quiescence through a resting phase in the presence of IL-7. During the resting phase, expression of cellular activation markers disappeared and cell proliferation and viral replication ceased, but resumed following restimulation of rested cells with Ag or mAbs directed to CD3/CD28. In addition, higher cell death rates were observed in HIV-1-infected than uninfected cultures during secondary but not primary stimulation. Thus, this system may allow us to study the biology of HIV-1 latency, as well as the mechanisms of CD4⁺ T cell death following HIV-1 reactivation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Enhancement in Specific CD8+ T Cell Recognition of EphA2+ Tumors In Vitro and In Vivo after Treatment with Ligand Agonists

2008-11-18

The EphA2 receptor tyrosine kinase is an attractive therapeutic target that is commonly overexpressed on solid tumors, with the degree of overexpression associated with disease progression, metastatic potential, and poor prognosis. Agonistic mAbs or ligand (ephrinA1)-Fc fusion protein are capable of inducing EphA2 internalization and degradation, thereby (at least transiently) eliminating the influence of this oncoprotein. We and others have also shown that EphA2 contains multiple peptide epitopes that can be recognized by effector CD4⁺ and CD8⁺ T cells isolated from tumor-bearing patients. Herein, we show that "agonist" reagents that trigger the proteasome-dependent degradation of tumor cell EphA2 result in the improved presentation of peptides derived from (both the extracellular and intracellular domains of) EphA2 in MHC class I complexes expressed on the tumor cell membrane for at least 48 h, as manifested by increased recognition by EphA2-specific CD8⁺ T cells in vitro. We also observed that while delivery of ephrinA1-Fc fusion protein or agonist mAb into EphA2⁺ tumor lesions promotes EphA2 degradation in situ, this single administration of agent does not dramatically alter tumor progression in a humanized SCID model. However, when combined with the adoptive transfer of normally nontherapeutic (human) anti-EphA2 CD8⁺ CTL, this dual-agent regimen results in complete tumor eradication. These results suggest that strategies targeting the conditional proteasome-mediated destruction of tumor cell EphA2 may enable EphA2-specific CD8⁺ T cells (of modest functional avidity) to realize improved therapeutic potential.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Self-Antigen Prevents CD8 T Cell Effector Differentiation by CD134 and CD137 Dual Costimulation

2008-11-18

We compared how CD4 vs CD8 cells attain the capacity to express the effector cytokine IFN- under both immunogenic and tolerogenic conditions. Although the Ifng gene locus was epigenetically repressed in naive Ag-inexperienced CD4 cells, it had already undergone partial remodeling toward a transcriptionally competent configuration in naive CD8 cells. After TCR stimulation, CD8 cells fully remodeled the Ifng locus and gained the capacity to express high levels of IFN- more rapidly than CD4 cells. Enforced dual costimulation through OX40 and 4-1BB redirected CD8 cells encountering soluble exogenous peptide to expand and differentiate into IFN- and TNF- double-producing effectors rather than becoming tolerant. Despite this and the stronger tendency of CD8 compared with CD4 cells to differentiate into IFN--expressing effectors, when parenchymal self-Ag was the source of tolerizing Ag, enforced dual costimulation selectively boosted expansion but did not push effector differentiation in CD8 cells while both expansion and effector differentiation were dramatically boosted in CD4 cells. Notably, enforced dual costimulation was able to push effector differentiation in CD8 cells encountering cognate parenchymal self-Ag when CD4 cells were simultaneously engaged. Thus, the ability of enforced OX40 plus 4-1BB dual costimulation to redirect CD8 cells to undergo effector differentiation was unexpectedly influenced by the source of tolerizing Ag and help was selectively required to facilitate CD8 cell effector differentiation when the tolerizing Ag derived from self.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Photoaffinity Antigens for Human {gamma}{delta} T Cells

2008-11-18

V2V2 T cells comprise the major subset of peripheral blood T cells in humans and expand during infections by recognizing small nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate, an endogenous isoprenoid intermediate. Recognition of these nonpeptide Ags is mediated by the V2V2 T cell Ag receptor. Several findings suggest that prenyl pyrophosphates are presented by an Ag-presenting molecule: contact between T cells and APC is required, the Ags do not bind the V2V2 TCR directly, and Ag recognition is abrogated by TCR mutations in CDRs distant from the putative Ag recognition site. Identification of the putative Ag-presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate Ags with the presenting molecule. In this study, we show that photoaffinity analogues of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C₅-OPP), can crosslink to the surface of tumor cell lines and be presented as Ags to T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β₂-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C₅-OPP. Finally, pulsing of BZ-(C)-C₅-OPP is inhibited by isopentenyl pyrophosphate and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide Ags are presented by a novel-Ag-presenting molecule that is widely distributed and nonpolymorphic, but not classical MHC class I, MHC class II, or CD1.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Coexpression of TGF-{beta}1 and IL-10 Enables Regulatory T Cells to Completely Suppress Airway Hyperreactivity

2008-11-18

In allergic airway disease, Treg may play an important role in the modulation of airway hyperreactivity (AHR) and inflammation. We therefore investigated the therapeutic potential of Treg in an Ag-dependent murine asthma model. We here describe that AHR can be completely suppressed by adoptive transfer of Treg overexpressing active TGF-β1. Using mice with impaired TGF-β signaling in T cells, we could demonstrate that TGF-β signaling in recipient effector T cells or transferred Treg themselves is not required for the protective effects on AHR. However, the expression of IL-10 by Treg was found to be essential for the suppression of AHR, since Treg overexpressing active TGF-β1 but deficient in IL-10 lacked protective effects. Airway inflammation could not be significantly suppressed by wild-type or transgenic Treg. In conclusion, modulation of cytokine expression by Treg may have therapeutic potential for the treatment of AHR in asthma. The mechanisms of the effects of Treg on airway inflammation require further clarification.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Rapid Deletion of Antigen-Specific CD4+ T Cells following Infection Represents a Strategy of Immune Evasion and Persistence for Anaplasma marginale

Han, S., Norimine, J., Palmer, G. H., Mwangi, W., Lahmers, K. K., Brown, W. C. — 2008-11-18

Acquired T cell immunity is central for protection against infection. However, the immunological consequences of exposing memory T cells to high Ag loads during acute and persistent infection with systemic pathogens are poorly understood. We investigated this by using infection with Anaplasma marginale, a ruminant pathogen that replicates to levels of 10⁹ bacteria per ml of blood during acute infection and maintains mean bacteremia levels of 10⁶ per ml during long-term persistent infection. We established that immunization-induced Ag-specific peripheral blood CD4⁺ T cell responses were rapidly and permanently lost following infection. To determine whether these T cells were anergic, sequestered in the spleen, or physically deleted from peripheral blood, CD4⁺ T lymphocytes from the peripheral blood specific for the major surface protein (MSP) 1a T cell epitope were enumerated by DRB3*1101 tetramer staining and FACS analysis throughout the course of immunization and challenge. Immunization induced significant epitope-specific T lymphocyte responses that rapidly declined near peak bacteremia to background levels. Concomitantly, the mean frequency of tetramer⁺CD4⁺ cells decreased rapidly from 0.025% before challenge to a preimmunization level of 0.0003% of CD4⁺ T cells. Low frequencies of tetramer⁺CD4⁺ T cells in spleen, liver, and inguinal lymph nodes sampled 9–12 wk postchallenge were consistent with undetectable or unsustainable Ag-specific responses and the lack of T cell sequestration. Thus, infection of cattle with A. marginale leads to the rapid loss of Ag-specific T cells and immunologic memory, which may be a strategy for this pathogen to modulate the immune response and persist.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] T Cell-Independent Spontaneous Loss of Tolerance by Anti-Double-Stranded DNA B Cells in C57BL/6 Mice

Tsao, P. Y., Jiao, J., Ji, M. Q., Cohen, P. L., Eisenberg, R. A. — 2008-11-18

Systemic lupus erythematosus is characterized by loss of tolerance to DNA and other nuclear Ags. To understand the role of T cells in the breaking of tolerance, an anti-DNA site-specific transgenic model of spontaneous lupus, B6.56R, was studied. T cells were eliminated by crossing B6.56R with CD4^–/^– or TCRβ^–/–^–/– mice, and the effects on anti-dsDNA serum levels, numbers of anti-dsDNA Ab-secreting cells, and isotypes of anti-dsDNA were analyzed. In addition, the development and activation of B cells in these mice were examined. Surprisingly, the presence of T cells made little difference in the development and character of the serum anti-dsDNA Ab in B6.56R mice. At 1 mo of age, anti-dsDNA Abs were somewhat lower in mice deficient in β and T cells. Levels of Abs later were not affected by T cells, nor was autoantibody class switching. B cell activation was somewhat diminished in T cell-deficient mice. Thus, in the B6 background, the presence of an anti-dsDNA transgene led the production of autoantibodies with a specificity and isotype characteristic of murine systemic lupus erythematosus with little influence from T cells. TLR9 also did not appear to play a role. Although we do not yet understand the mechanism of this failure of immunoregulation, these results suggest that similar processes may influence autoimmunity associated with clinical disease.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Egr2 Is Required for Bcl-2 Induction during Positive Selection

2008-11-18

The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2^–/– thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] TCR Down-Regulation Controls Virus-Specific CD8+ T Cell Responses

2008-11-18

The CD3 di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3 di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8⁺ T cells is impaired in mice with a mutated CD3 di-leucine-based motif. The CD3 mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8⁺ T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3-mediated TCR down-regulation in virus-specific CD8⁺ T cell responses.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] TNF Receptor-Associated Factor 5 Is Required for Optimal T Cell Expansion and Survival in Response to Infection

Kraus, Z. J., Haring, J. S., Bishop, G. A. — 2008-11-18

Receptors belonging to the TNF-receptor (TNF-R) superfamily include important costimulatory molecules, many of which specifically affect T cell activation. TNF receptor-associated factors (TRAFs) are recruited to many TNF-R superfamily members and are important modulators of the proximal signaling events that occur at the time of receptor engagement and activation. TRAF5 has been shown to be a positive regulator of a number of these receptors that are involved in T cell costimulation. However, the potential importance of TRAF5 in cellular immune responses to infection or in T cell expansion and memory have not been studied. We report in this study that TRAF5 was required for optimal CD8⁺ T cell responses following infection with Listeria monocytogenes expressing OVA (LM-OVA). TRAF5 was necessary for optimal T cell expansion following primary infection with LM-OVA, and its absence resulted in fewer memory CD8⁺ T cells following LM-OVA infection, together with higher bacterial loads in the liver. The effect of TRAF5 on CD8⁺ T cell expansion was T cell intrinsic and not due to effects of TRAF5 deficiency on APCs. Although their proliferative ability remained intact, CD8⁺ T cells from TRAF5^–/– mice were more sensitive to apoptosis and were unresponsive to the prosurvival effects of the TNF-R superfamily costimulator CD27. Collectively, these studies identify TRAF5 as an important positive signaling element that enhances T cell expansion and pathogen containment by providing a survival advantage to responding Ag-specific CD8⁺ T cells during infection.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses

Getachew, Y., Stout-Delgado, H., Miller, B. C., Thiele, D. L. — 2008-11-18

It is well established that granzymes A and B play a role in CTL killing of target cells by the perforin-dependent granule exocytosis pathway. The functions of multiple additional granzymes expressed in CTL are less well defined. In the present studies, CTL generated from mice deficient in dipeptidyl peptidase 1 (DPP1) were used to investigate the contribution of granzyme C to CTL killing of allogeneic target cells. DPP1 is required for activation of granzymes A and B by proteolytic removal of their N-terminal dipeptide prodomains while a significant portion of granzyme C is processed normally in the absence of DPP1. Cytotoxicity of DPP1^–/– CTL generated in early (5-day) MLC in vitro and in peritoneal exudate cells 5 days after initial allogeneic sensitization in vivo was significantly impaired compared with wild-type CTL. Following 3 days of restimulation with fresh allogeneic stimulators however, cytotoxicity of these DPP1^–/– effector cells was comparable to that of wild-type CTL. Killing mediated by DPP1^–/– CTL following restimulation was rapid, perforin dependent, Fas independent and associated with early mitochondrial injury, phosphatidyl serine externalization, and DNA degradation, implicating a granzyme-dependent apoptotic pathway. The increased cytotoxicity of DPP1^–/– CTL following restimulation coincided with increased expression of granzyme C. Moreover, small interfering RNA inhibition of granzyme C expression during restimulation significantly decreased cytotoxicity of DPP1^–/– but not wild-type CTL. These results indicate that during late primary alloimmune responses, granzyme C can support CTL-mediated killing by the granule exocytosis pathway in the absence of functional granzymes A or B.