Transfusion-related immunomodulation: gamma irradiation alters the effects of leukoreduction on alloimmunization.

BACKGROUND: Adverse events following blood transfusion include allosensitization and generalized immunosuppression, collectively referred to as transfusion-related immune modulation. We evaluated the immunological effects of red blood cell (RBC) and platelet transfusions on alloantibody responses and on immunoregulatory cells in nonimmunosuppressed patients undergoing cardiovascular surgery. STUDY DESIGN AND METHODS: Patients were randomized to receive standard unmodified (STD), leukoreduced (LR), or leukoreduced and γ-irradiated (LRγ) RBCs. Patients received only apheresis platelets that were in-process LR and were γ-irradiated for the third arm. Nontransfused patients served as controls for the effects of surgery itself on immunologic changes. Antibodies to HLA were assessed with use of solid-phase assays. The effects of transfusion on adaptive and innate immunity were evaluated by assessing T regulatory cells (Tregs) and invariant natural killer T (iNKT) cells. RESULTS: LR of blood products reduced the development of human leukocyte antigen (HLA) alloantibodies, but only in patients without preexisting HLA antibodies. However, if LR blood products were γ-irradiated, HLA antibody production was not reduced. Compared to nontransfused patients, recipients of STD or LR transfusions showed a significant increase in CD4+CD25hi T cells expressing FoxP3 or CTLA4 and also of iNKT cells producing interleukin-4. In contrast, recipients of LRγ blood products showed markedly lower increases in all three cellular assays. CONCLUSION: LR decreased HLA alloantibody production in naïve recipients, but did not reduce the immunosuppressive effects of transfusion. LRγ reduced immunosuppression and was not associated with decreased HLA alloantibody production.

Leukofiltration plus pathogen reduction prevents alloimmune platelet refractoriness in a dog transfusion model.

Human lymphocyte antigen alloimmunization to filter leukoreduced (F-LR) platelets occurs in about 18% of immunosuppressed thrombocytopenic hematology/oncology patients and represents a significant challenge for effective chemotherapy. In a dog platelet transfusion model, we have evaluated other methods of preventing alloimmune platelet refractoriness and demonstrated that successful methods in our dog model are transferable to man. In the present study, donor/recipient pairs were dog lymphocyte antigen DR-B incompatible (88% of the pairs), and recipient dogs received up to 8 weekly treated transfusions from a single donor (a highly immunogenic stimulus), or until platelet refractoriness. Continued acceptance of F-LR platelets occurred in 6 of 13 recipients (46%), but neither γ-irradiation (γ-I; 0 of 5) nor Mirasol pathogen reduction (MPR; 1 of 7) treatment of donor platelets prevented alloimmune platelet refractoriness. Combining γ-I with F-LR was associated with only 2 of 10 (20%) recipients accepting the transfused platelets. Surprisingly, F-LR platelets that then underwent MPR were accepted by 21 of 22 (95%) recipients (P < .001 vs F-LR + γ-I recipients). Furthermore, 7 of 21 (33%) of these accepting recipients demonstrated specific tolerance to 8 more weekly donor transfusions that had not been treated. In addition, platelet concentrates prepared from F-LR + MPR whole blood were also nonimmunogenic; that is, 10 of 10 (100%) recipients accepted donor platelets. Overall, 31 of 32 (97%) recipients accepted F-LR + MPR platelets; none developed antibodies to donor lymphocytes. These data are the highest rate of acceptance for platelet transfusions reported in either animals or man. This approach to platelet transfusion may be particularly important when supporting patients with intact immune systems, such as in myelodysplastic syndromes.

Ultraviolet B irradiation in the prevention of alloimmunization in a dog platelet transfusion model.

BACKGROUND: Alloimmune platelet (PLT) refractoriness remains a significant problem for chronically transfused patients with thrombocytopenia. STUDY DESIGN AND METHODS: In a dog PLT transfusion model, we evaluated ultraviolet B irradiation (UV-B) of donor PLTs-either alone or in combination with centrifuge leukoreduction (C-LR) or filtration leukoreduction (F-LR)-to prevent refractoriness to donor PLTs and to induce tolerance to standard (STD) PLTs from the same donor or to tertiary donors. RESULTS: Recipient acceptance rates for C-LR donor PLT transfusions were 14%, F-LR were 33%, and UV-B irradiated were 45% with no significant differences among the treatments given to the donor's PLTs. Adding UV-B irradiation to C-LR or F-LR PLTs increased acceptance rates to 50 and 68% (p = 0.02 and p = 0.05), respectively, comparing single treatments to the combined treatments. After a recipient had accepted any type of UV-B-treated donor PLTs, specific tolerance to subsequent transfusions of the same donor's STD PLTs averaged 65%. Nonspecific tolerance to third-party donor's STD PLTs averaged 36% if they had accepted their initial donor's treated PLTs but was only 4% (p < 0.001) if they had rejected these PLTs. CONCLUSION: Combining UV-B irradiation with a method of leukoreduction produces additive effects on prevention of alloimmune PLT refractoriness.

The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic.

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer.

A phase 1/2 clinical trial evaluating dosing, safety, immunogenicity, and overall survival on metastatic colorectal cancer (mCRC) patients after immunotherapy with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine was performed. We report our extended observations on long-term overall survival and further immune analyses on a subset of treated patients including assessment of cytolytic T cell responses, T regulatory (Treg) to T effector (Teff) cell ratios, flow cytometry on peripheral blood mononuclear cells (PBMCs), and determination of HLA-A2 status. An overall survival of 20 % (median survival 11 months) was observed during long-term follow-up, and no long-term adverse effects were reported. Cytolytic T cell responses increased after immunizations, and cell-mediated immune (CMI) responses were induced whether or not patients were HLA-A2 positive or Ad5 immune. PBMC samples from a small subset of patients were available for follow-up immune analyses. It was observed that the levels of carcinoembryonic antigen (CEA)-specific CMI activity decreased from their peak values during follow-up in five patients analyzed. Preliminary results revealed that activated CD4+ and CD8+ T cells were detected in a post-immunization sample exhibiting high CMI activity. Treg to Teff cell ratios were assessed, and samples from three of five patients exhibited a decrease in Treg to Teff cell ratio during the treatment protocol. Based upon the favorable safety and immunogenicity data obtained, we plan to perform an extensive immunologic and survival analysis on mCRC patients to be enrolled in a randomized/controlled clinical trial that investigates Ad5 [E1-, E2b-]-CEA(6D) as a single agent with booster immunizations.

Expression of plasma membrane receptor genes during megakaryocyte development.

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, ß receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-ß, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-ß, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function.

Suppression of the immune response to FVIII in hemophilia A mice by transgene modified tolerogenic dendritic cells.

Current methods for eradicating clinically significant inhibitory antibodies to human factor VIII (hFVIII) in patients with hemophilia A rely on repeated delivery of high doses of factor concentrates for a minimum of many months. We hypothesize that tolerance can be induced more efficiently and reliably through hFVIII antigen presentation by tolerogenic dendritic cells (tDCs). In this study, we generated tDCs from hemophilia A mice and modified them with a foamy virus vector expressing a bioengineered hFVIII transgene. Naive and preimmunized mice infused with hFVIII expressing tDCs showed suppression of the T cell and inhibitor responses to recombinant hFVIII (rhFVIII). Treatment with hFVIII expressing tDCs was also associated with a higher percentage of splenocytes demonstrating a regulatory T cell phenotype in immunized mice. Furthermore, CD4(+) T cells harvested from recipients of hFVIII expression vector-modified tDCs were able to mediate antigen-specific immune suppression in naive secondary recipients. We also demonstrated a trend for improved suppression of inhibitor formation by coexpressing interleukin-10 (IL-10) and hFVIII from a bicistronic vector. These preclinical results demonstrate the potential for employing vector modified ex vivo generated tDCs to treat high titer inhibitors in patients with hemophilia A.

The SLAM family member CD48 (Slamf2) protects lupus-prone mice from autoimmune nephritis.

Polymorphisms in the SLAM family of leukocyte cell surface regulatory molecules have been associated with lupus-like phenotypes in both humans and mice. The murine Slamf gene cluster lies within the lupus-associated Sle1b region of mouse chromosome 1. Non-autoreactive C57BL/6 (B6) mice that have had this region replaced by syntenic segments from other mouse strains (i.e. 129, NZB and NZW) are B6 congenic strains that spontaneously produce non-nephritogenic lupus-like autoantibodies. We have recently reported that genetic ablation of the SLAM family member CD48 (Slamf2) drives full-blown autoimmune disease with severe proliferative glomerulonephritis (CD48GN) in B6 mice carrying 129 sequences of the Sle1b region (B6.129CD48(-/-)). We also discovered that BALB/c mice with the same 129-derived CD48-null allele (BALB.129CD48(-/-)) have neither nephritis nor anti-DNA autoantibodies, indicating that strain specific background genes modulate the effects of CD48 deficiency. Here we further examine this novel model of lupus nephritis in which CD48 deficiency transforms benign autoreactivity into fatal nephritis. CD48GN is characterized by glomerular hypertrophy with mesangial expansion, proliferation and leukocytic infiltration. Immune complexes deposit in mesangium and in sub-endothelial, sub-epithelial and intramembranous sites along the glomerular basement membrane. Afflicted mice have low-grade proteinuria, intermittent hematuria and their progressive renal injury manifests with elevated urine NGAL levels and with uremia. In contrast to the lupus-like B6.129CD48(-/-) animals, neither BALB.129CD48(-/-) mice nor B6 × BALB/c F1.129CD48(-/-) progeny have autoimmune traits, indicating that B6-specific background genes modulate the effect of CD48 on lupus nephritis in a recessive manner.

Auto-antibody production and glomerulonephritis in congenic Slamf1-/- and Slamf2-/- [B6.129] but not in Slamf1-/- and Slamf2-/- [BALB/c.129] mice.

Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.

Targeting NKT cells and PD-L1 pathway results in augmented anti-tumor responses in a melanoma model.

Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8(+) OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8(+) cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8(+) T cells were adoptively transferred into PD-L1(-/-) recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1(-/-) mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8(+) T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.

Suppression of FVIII inhibitor formation in hemophilic mice by delivery of transgene modified apoptotic fibroblasts.

The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20-40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4+ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4+CD25+ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.

Absence of alpha 4 but not beta 2 integrins restrains development of chronic allergic asthma using mouse genetic models.

OBJECTIVE: Chronic asthma is characterized by ongoing recruitment of inflammatory cells and airway hyperresponsiveness leading to structural airway remodeling. Although alpha 4 beta 1 and beta2 integrins regulate leukocyte migration in inflammatory diseases and play decisive roles in acute asthma, their role has not been explored under the chronic asthma setting. To extend our earlier studies with alpha 4(Delta/Delta) and beta2(-/-) mice, which showed that both alpha 4 and beta2 integrins have nonredundant regulatory roles in acute ovalbumin (OVA)-induced asthma, we explored to what extent these molecular pathways control development of structural airway remodeling in chronic asthma. MATERIALS AND METHODS: Control, alpha 4(Delta/Delta), and beta2(-/-) mouse groups, sensitized by intraperitoneal OVA as allergen, received intratracheal OVA periodically over days 8 to 55 to induce a chronic asthma phenotype. Post-OVA assessment of inflammation and pulmonary function (airway hyperresponsiveness), together with airway modeling measured by goblet cell metaplasia, collagen content of lung, and transforming growth factor beta1 expression in lung homogenates, were evaluated. RESULTS: In contrast to control and beta2(-/-) mice, alpha 4(Delta/Delta) mice failed to develop and maintain the composite chronic asthma phenotype evaluated as mentioned and subepithelial collagen content was comparable to baseline. These data indicate that beta2 integrins, although required for inflammatory migration in acute asthma, are dispensable for structural remodeling in chronic asthma. CONCLUSION: alpha 4 integrins appear to have a regulatory role in directing transforming growth factor beta-induced collagen deposition and structural alterations in lung architecture likely through interactions of Th2 cells, eosinophils, or mast cells with endothelium, resident airway cells, and/or extracellular matrix.

Ly6C(low) monocytes differentiate into dendritic cells and cross-tolerize T cells through PDL-1.

Monocyte-derived dendritic cells are active participants during the immune response against infection, but whether they play a role in maintaining self-tolerance under steady-state conditions is not known. Here we investigated the differentiation of monocytes, their ability to ingest apoptotic cells, and their potential functionality in vivo. We observed that Ly6C (Gr-1)(low) mature monocytes up-regulate their MHC II level in the spleen, express high levels of PDL-1 (programmed death ligand 1), and are more efficient than Ly6C(high) immature monocytes in the ingestion of apoptotic cells in vivo. Sorted circulating Ly6C(low) monocytes were able to cross-present both apoptotic cell-associated OVA and soluble OVA protein. Monocytes containing apoptotic cells can further differentiate into CD11c(+)CD8alpha(-)MHC II(+) splenic dendritic cells that maintained high expression of PDL-1. Since wild-type but not PDL-1-deficient peripheral blood monocytes containing apoptotic cell-associated OVA suppressed the response to OVA immunization, PDL-1 expression was required for monocyte-mediated T cell tolerance. These observations demonstrate that Ly6C(low) mature monocytes can promote tolerance to self Ag contained in apoptotic cells through a PDL-1-dependent mechanism.

PD-L1/PD-1 signal deficiency promotes allogeneic immune responses and accelerates heart allograft rejection.

BACKGROUND: PD-L1, a ligand for programmed death 1 (PD-1), delivers a negative costimulatory signal to T cells and plays a critical role in the regulation of peripheral tolerance. METHODS: We used PD-L1(-/-) mice to evaluate the role of the PD-L1 signal on allogeneic immune responses in vivo and the underlying mechanisms. Heart transplantation was performed from PD-L1(-/-) donors or recipients in major histocompatibility complex fully mismatched mouse combinations. The immunologic function of allograft recipients was evaluated ex vivo by enzyme-linked immunospot, mixed lymphocytes reaction, cytotoxic T lymphocyte, and flow cytometry. RESULTS: Our results demonstrated that PD-L1(-/-) T cells proliferated vigorously under alloantigen stimulation, and also that the antigen-presenting cells (APCs) from PD-L1(-/-) mice exhibited a stronger allostimulatory activity compared with that in wild-type mice. Heart allografts were rejected at an accelerated rate in both PD-L1(-/-) donors and recipients. This was associated with significantly augmented donor specific T-cell proliferation and antidonor cytotoxic T lymphocyte activities, and enhanced Th1- or Th2-type immune responses of heart allograft recipients. CONCLUSIONS: Absence of PD-L1 input triggers a stimulatory signal to effector T cells and APCs, accelerating heart allograft rejection. Engagement of the PD-L1 signal on T cells or APCs may be necessary to induce transplant tolerance.

Distinct changes in adult lymphopoiesis in Rag2-/- mice fully reconstituted by alpha4-deficient adult bone marrow cells.

OBJECTIVE: alpha4 Integrins are major players in lymphoid cell trafficking and immune responses. However, their importance in lymphoid reconstitution and function, studied by antibody blockade or in genetic models of chimeric animals with alpha4(KO) embryonic stem (ES) cells, competitive repopulation experiments with fetal liver(KO) cells, or in beta1/beta7 doubly-deficient mice has yielded disparate conclusions. MATERIALS AND METHODS: To study the role of alpha4 integrin (alpha4beta1, alpha4beta7) during adult life, we transplanted lethally irradiated Rag2(-/-) mice with alpha4(Delta/Delta) or alpha4(f/f) adult bone marrow (BM) cells and evaluated recipients at several points after transplantation. RESULTS: Lymphomyeloid repopulation (8 months later) was entirely donor-derived in all recipients, and novel insights regarding lymphoid reconstitution and function were revealed. Thymic repopulation was impaired in all alpha4(Delta/Delta) recipients, likely because of homing defects of BM-derived progenitors, although a role of alpha4 integrin in intrathymic expansion/maturation of T cells cannot be excluded; reconstitution of gut lymphoid tissue was also greatly diminished because of homing defects of alpha4(Delta/Delta) cells; impaired immunoglobulin (Ig) M and IgE, but normal IgG responses were seen, suggesting compromised initial B-/T-cell interactions, whereas interferon-gamma production from ovalbumin-stimulated cells was increased, possibly reflecting a bias against Th2 stimulation. CONCLUSION: These data complement previous observations by defending the role of alpha4 integrin in thymic and gut lymphoid tissue homing, and by strengthening evidence of attenuated B-cell responses in alpha4-deficient mice.

Tissue expression of PD-L1 mediates peripheral T cell tolerance.

Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2(-/-) mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-gamma production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2(-/-) non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell-mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1-PD-L1 interactions in mediating tissue tolerance.

CD48 controls T-cell and antigen-presenting cell functions in experimental colitis.

BACKGROUND & AIMS: The cell-surface receptor CD48 is a lipid-anchored protein expressed on all antigen-presenting cells and T cells. CD2 and 2B4 are known ligands for CD48, which themselves are expressed on the surface of hematopoietic cells. Here we examine the effect of CD48 in the development of chronic experimental colitis and how CD48 affects adaptive and innate immune functions. METHODS: The role of CD48 in experimental colitis was first assessed by transferring CD4(+)CD45RB(hi) cells isolated from either wild-type or CD48(-/-) mice into either Rag-2(-/-) or CD48(-/-) x Rag-2(-/-) mice. Development of chronic colitis in these adoptively transferred mice was assessed by disease activity index, histology, and production of interferon-gamma in mesenteric lymph nodes. Relevant functions of CD48(-/-)CD4(+) T cells and CD48(-/-) macrophages were examined using in vitro assays. In a second set of experiments, the efficacy of anti-CD48 in prevention or treatment of chronic colitis was determined. RESULTS: CD48(-/-)CD4(+) cells induced colitis when transferred into Rag-2(-/-) mice, but not when introduced into CD48(-/-) x Rag-2(-/-) recipients. However, both recipient mouse strains developed colitis upon adoptive transfer of wild-type CD4(+) cells. Consistent with a CD4(+) T-cell defect was the observation that in vitro proliferation of CD48(-/-)CD4(+) T cells was impaired upon stimulation with CD48(-/-) macrophages. In vitro evidence for a modest macrophage functional defect was apparent because CD48(-/-) macrophages produced less tumor necrosis factor alpha and interleukin 12 than wild-type cells upon stimulation with lipopolysaccharide. Peritoneal macrophages also showed a defect in clearance of gram-negative bacteria in vitro. Treatment of the CD4(+)CD45RB(hi)-->Rag-2(-/-) mice or the wild-type BM-->tg26 mice with anti-CD48 (HM48-1) ameliorated development of colitis, even after its induction. CONCLUSIONS: Both CD48-dependent activation of macrophages and CD48-controlled activation of T cells contribute to maintaining the inflammatory response. Consequently, T cell-induced experimental colitis is ameliorated only when CD48 is absent from both T cells and antigen-presenting cells. Because anti-CD48 interferes with these processes, anti-human CD48 antibody treatment may represent a novel therapy for inflammatory bowel disease patients.

Requirement of homotypic NK-cell interactions through 2B4(CD244)/CD48 in the generation of NK effector functions.

2B4 belongs to the CD2 subset of the IgG family of receptors. Members in this family have been shown to function as coreceptors via homophilic or heterophilic interactions. Both 2B4 and CD2 bind to CD48, another member of this family. Because all 3 molecules are expressed on natural killer (NK) cells, it raises a possibility that the binding of 2B4 and CD2 to CD48 among NK cells may have functional consequences. Using specific monoclonal antibodies and gene-deficient NK cells, we found that 2B4/CD48, but not CD2/CD48, interaction is essential for IL-2-driven expansion and activation of murine NK cells. In the absence of 2B4/CD48 interaction, NK cytotoxicity and IFN-gamma secretion on tumor target exposure is severely impaired. Impaired activation of NK cells in 2B4-deficient mice was also demonstrated by poor NK-mediated clearance of syngeneic tumor cells in these mice. Functional impairment of NK cells in the absence of 2B4/CD48 interactions was accompanied by defective calcium signaling, suggesting that the early signaling pathway of NK receptors is inhibited. Finally, homotypic interactions among NK cells through 2B4/CD48 was visualized by specific localization of GFP-tagged 2B4 onto NK-NK conjugation sites. Thus, these data identify a novel mechanism whereby NK effector function is regulated via homotypic 2B4/CD48 interactions.

PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis.

To compare the roles of programmed death 1 ligand 1 (PD-L1) and PD-L2 in regulating immunity to infection, we investigated responses of mice lacking PD-L1 or PD-L2 to infection with Leishmania mexicana. PD-L1(-/-) and PD-L2(-/-) mice exhibited distinct disease outcomes following infection with L. mexicana. In comparison to susceptible WT mice, PD-L1(-/-) mice showed resistance to L. mexicana, as demonstrated by reduced growth of cutaneous lesions and parasite burden. In contrast, PD-L2(-/-) mice developed exacerbated disease with increased parasite burden. Host resistance to L. mexicana is partly associated with the development of a Th1 response and down-regulation of the Th2 response. Both PD-L1(-/-) and PD-L2(-/-) mice produced levels of IFN-gamma similar to WT mice. However, the development of IL-4-producing cells was reduced in PD-L1(-/-) mice, demonstrating a role for PD-L1 in regulating Th cell differentiation. This inadequate Th2 response may explain the increased resistance of PD-L1(-/-) mice. Although no alterations in Th1/Th2 skewing were observed in PD-L2(-/-) mice, PD-L2(-/-) mice exhibited a marked increase in L. mexicana-specific antibody production. Increased Leishmania-specific IgG production may suppress the healing response through FcgammaR ligation on macrophages. Taken together, our results demonstrate that PD-L1 and PD-L2 have distinct roles in regulating the immune response to L. mexicana.

Programmed death-1 (PD-1):PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes.

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.