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Background: Bronchopulmonary dysplasia (BPD) is a common neonatal lung disease that is underpinned by pulmonary inflammation. We recently discovered the dominance of type 2-polarised immunity in BPD. Method(s): Cord and peripheral blood were collected from: i) infants born at 24 to 29 gestational weeks at birth, day 1, weeks 1 and 2, and 36 weeks gestational age; ii) healthy term infants at birth and 4-16 weeks of age; iii) healthy adults. Following overnight stimulation with PMA + ionomycin or vehicle, polarisation of CD8+ and CD4-CD8-double-negative (DN)-T cells (including type 1/2/17) was enumerated by flow cytometry. Results were analysed against BPD status. Result(s): 51 preterm infants, 20 term infants and five adults were enrolled, and 254 unique samples collected. Type 2 polarisation predominated in preterm and term infants up to 16 weeks of age. The 41 preterm infants with BPD had significantly more type 2-polarised cells than infants without BPD on day 1 and week 1 for both CD8+ and DN T cells (4-6% vs 0.5-1.5% for different time points of CD8+ cells; 14% vs 1-3% for DN-T cells). There was no difference in type 1-and type 17-polarised cells based on BPD status, except in week 2 when BPD infants also had more type 1-polarised CD8+ cells than No-BPD infants. Conclusion(s): BPD is strongly associated with type 2 polarisation not only in CD4+ T cells as presented previously, but also in CD8+ and DN-T cells. These findings strengthen the concept that therapies targeting the type 2 pathway may offer benefit to BPD patients.

Background: Bronchopulmonary dysplasia (BPD) is a common neonatal lung disease that is underpinned by pulmonary inflammation. We recently discovered the dominance of type 2-polarised immunity in BPD. Method(s): Cord and peripheral blood were collected from: i) infants born at 24 to 29 gestational weeks at birth, day 1, weeks 1 and 2, and 36 weeks gestational age; ii) healthy term infants at birth and 4-16 weeks of age; iii) healthy adults. Following overnight stimulation with PMA + ionomycin or vehicle, polarisation of CD8+ and CD4-CD8-double-negative (DN)-T cells (including type 1/2/17) was enumerated by flow cytometry. Results were analysed against BPD status. Result(s): 51 preterm infants, 20 term infants and five adults were enrolled, and 254 unique samples collected. Type 2 polarisation predominated in preterm and term infants up to 16 weeks of age. The 41 preterm infants with BPD had significantly more type 2-polarised cells than infants without BPD on day 1 and week 1 for both CD8+ and DN T cells (4-6% vs 0.5-1.5% for different time points of CD8+ cells; 14% vs 1-3% for DN-T cells). There was no difference in type 1-and type 17-polarised cells based on BPD status, except in week 2 when BPD infants also had more type 1-polarised CD8+ cells than No-BPD infants. Conclusion(s): BPD is strongly associated with type 2 polarisation not only in CD4+ T cells as presented previously, but also in CD8+ and DN-T cells. These findings strengthen the concept that therapies targeting the type 2 pathway may offer benefit to BPD patients.

Background: Indolent T-lymphoblastic proliferations (iT-LBP) are reactive expansions of TdT+ T-cells outside of thymic tissue which may be erroneously misdiagnosed as T-lymphoblastic lymphoma (T-LBL). This is in part due to the complex morphologic, immunophenotypic and clinical overlap that can occur between these entities. A singular definitive method for addressing the diagnostic quandary of iT-LBP versus T-LBL is lacking. However, in recent years, LMO2 has been described as a positive marker of T-LBL but consistently negative in reactive immature thymocytes. Design(s): We searched our pathology archives for cases of indolent T-lymphoblastic proliferations and identified 7 with adequate material for further immunohistochemical studies. 3 new cases that have not been described in the literature to date were also identified. We performed immunohistochemical staining for LMO2 on these cases with comparison to LMO2 staining patterns on 12 cases of T-LBL. Result(s): All cases of iT-LBP were negative for LMO2 (0/7) whereas all cases of T-LBL showed expression of LMO2 (12/12) (p-value < 0.001; Fisher exact test). We additionally describe 3 new cases of iTLBP which show typical features of iT-LBP in expression of CD4 and CD8, and TdT, absence of CD34, as well as non-clonal TCR studies and clinical indolence without mediastinal adenopathy. One case was seen in a patient with lymphoid architecture showing Castleman-like features. Conclusion(s): LMO2 is a diagnostically useful marker for differentiating between iT-LBP and T-LBL. As previously described, iTLBP typically express CD4 and CD8 and lack CD34. The absence of mediastinal enlargement is also a characteristic feature of this entity which has not been focused on previously.

Background: Indolent T-lymphoblastic proliferations (iT-LBP) are reactive expansions of TdT+ T-cells outside of thymic tissue which may be erroneously misdiagnosed as T-lymphoblastic lymphoma (T-LBL). This is in part due to the complex morphologic, immunophenotypic and clinical overlap that can occur between these entities. A singular definitive method for addressing the diagnostic quandary of iT-LBP versus T-LBL is lacking. However, in recent years, LMO2 has been described as a positive marker of T-LBL but consistently negative in reactive immature thymocytes. Design(s): We searched our pathology archives for cases of indolent T-lymphoblastic proliferations and identified 7 with adequate material for further immunohistochemical studies. 3 new cases that have not been described in the literature to date were also identified. We performed immunohistochemical staining for LMO2 on these cases with comparison to LMO2 staining patterns on 12 cases of T-LBL. Result(s): All cases of iT-LBP were negative for LMO2 (0/7) whereas all cases of T-LBL showed expression of LMO2 (12/12) (p-value < 0.001; Fisher exact test). We additionally describe 3 new cases of iTLBP which show typical features of iT-LBP in expression of CD4 and CD8, and TdT, absence of CD34, as well as non-clonal TCR studies and clinical indolence without mediastinal adenopathy. One case was seen in a patient with lymphoid architecture showing Castleman-like features. Conclusion(s): LMO2 is a diagnostically useful marker for differentiating between iT-LBP and T-LBL. As previously described, iTLBP typically express CD4 and CD8 and lack CD34. The absence of mediastinal enlargement is also a characteristic feature of this entity which has not been focused on previously.

Background CPI-444, an A2AR antagonist, inhibits suppression of T cell function by adenosine and is active in multiple preclinical models. CPI-444 is being investigated as a single agent and in combination with atezolizumab in an ongoing Phase 1/1b clinical trial in patients with advanced cancers (NCT02655822). Biomarker investigations were conducted to explore immune modulation in serial tumor biopsies and peripheral blood as well associations between adenosine pathway genes and clinical activity. Methods Tumor biopsies and blood samples were analyzed in 80 patients with renal cell carcinoma (RCC: n=35), non-small cell lung cancer (NSCLC: n=26), triple negative breast cancer (TNBC: n=17), microsatellite instable colorectal cancer (MSI-CRC n=2) treated with CPI-444 100mg BID as single agent ('SA' n=18) or in combination with atezolizumab ('combo' n=62). Paired tumor biopsies were analyzed: gene expression profiles (Nanostring), CD8, PD-L1 and CD73 (IHC). T cell repertoires were examined by sequencing of the T cell receptor beta chain gene in PBMCs and tumors. Results Of 80 evaluable patients, 54 had progressed on prior anti-PD-(L) 1 therapy ('IO-refractory'), 26 were IO-naive. Most patients were PD-L1 negative in archival tumor (80% TNBC, 91% RCC, 69% NSCLC). Six objective responses were observed: 2 partial responses (PR) in RCC (1 SA and 1 combo); 2 PRs in NSCLC (both combo); 1 PR MSICRC (combo) and 1 PR in TNBC (combo). Notably, 3 PRs were observed in IO-refractory patients (1 RCC, 2 NSCLC) and 2 PRs were PD-L1 negative (1 RCC, 1 NSCLC). SA and combo treatment significantly increased CD8+ cell infiltration (mean 2-fold; 95%CI 1.18-2.91), T cell activation and interferongamma pathway gene expression in biopsies as well as T cell clone expansion in both tumor biopsies and periphery. Shared clonotypes expanded in matched post-dose PBMCs and tumor suggesting trafficking of T cells following CPI-444 treatment. This included IOrefractory and PD-L1-negative patients. Pat

Background CPI-444, an A2AR antagonist, inhibits suppression of T cell function by adenosine and is active in multiple preclinical models. CPI-444 is being investigated as a single agent and in combination with atezolizumab in an ongoing Phase 1/1b clinical trial in patients with advanced cancers (NCT02655822). Biomarker investigations were conducted to explore immune modulation in serial tumor biopsies and peripheral blood as well associations between adenosine pathway genes and clinical activity. Methods Tumor biopsies and blood samples were analyzed in 80 patients with renal cell carcinoma (RCC: n=35), non-small cell lung cancer (NSCLC: n=26), triple negative breast cancer (TNBC: n=17), microsatellite instable colorectal cancer (MSI-CRC n=2) treated with CPI-444 100mg BID as single agent ('SA' n=18) or in combination with atezolizumab ('combo' n=62). Paired tumor biopsies were analyzed: gene expression profiles (Nanostring), CD8, PD-L1 and CD73 (IHC). T cell repertoires were examined by sequencing of the T cell receptor beta chain gene in PBMCs and tumors. Results Of 80 evaluable patients, 54 had progressed on prior anti-PD-(L) 1 therapy ('IO-refractory'), 26 were IO-naive. Most patients were PD-L1 negative in archival tumor (80% TNBC, 91% RCC, 69% NSCLC). Six objective responses were observed: 2 partial responses (PR) in RCC (1 SA and 1 combo); 2 PRs in NSCLC (both combo); 1 PR MSICRC (combo) and 1 PR in TNBC (combo). Notably, 3 PRs were observed in IO-refractory patients (1 RCC, 2 NSCLC) and 2 PRs were PD-L1 negative (1 RCC, 1 NSCLC). SA and combo treatment significantly increased CD8+ cell infiltration (mean 2-fold; 95%CI 1.18-2.91), T cell activation and interferongamma pathway gene expression in biopsies as well as T cell clone expansion in both tumor biopsies and periphery. Shared clonotypes expanded in matched post-dose PBMCs and tumor suggesting trafficking of T cells following CPI-444 treatment. This included IOrefractory and PD-L1-negative patients. Pat

Background Pembrolizumab, a PD-1 immune checkpoint inhibitor, and dinaciclib, a cyclin-dependent kinase inhibitor, have demonstrated antitumor activity as monotherapies in various tumor types, including hematologic malignancies. In a preclinical study using a solidtumor syngeneic model (MC38), enhanced antitumor activity was observed with the combination of pembrolizumab and dinaciclib, with no significant toxicities. KEYNOTE-155 is a phase Ib study designed to evaluate the safety and efficacy of the combination of pembrolizumab and dinaciclib in patients with relapsed or refractory chronic lymphocytic leukemia (CLL), multiple myeloma (MM), or diffuse large B cell lymphoma (DLBCL). Methods Key eligibility criteria include >=18 years, ECOG performance status 0-1, and confirmed diagnosis of one of the following: CLL as defined by 2008 iwCLL criteria with >=1 prior therapy; active MM with measurable disease and >=2 prior therapies including an IMiD and proteasome inhibitor; or DLBCL with progression following >=2 prior therapies including autologous stem cell transplantation (ASCT; or ineligible for ASCT). An initial cohort of 12 patients (>=3 with each disease type) will be enrolled in the dose-evaluation phase (two 21-day cycles) to determine dose-limiting toxicities (DLTs). In cycle 1, patients will receive pembrolizumab 200 mg on day 1, dinaciclib 7 mg/m2 on day 1, and dinaciclib 10 mg/m2 on day 8. In cycle 2 and beyond, patients will receive pembrolizumab 200 mg on day 1 and dinaciclib 14 mg/m2 on days 1 and 8. If <=4 patients experience DLTs in the first 2 cycles, expansion cohorts (~30 patients each) will be opened for signal detection. If >=5 DLTs occur in the first 2 cycles, a lower dose of dinaciclib will be studied in up to 24 patients. Treatment will continue until disease progression, unacceptable toxicity, or 35 cycles. Response will be assessed every 3 cycles for CLL and DLBCL, and at the start of each cycle for MM. Primary end points are safety and tole

Aim: The aims of the study are to define the presence of glomerular neutrophil extracellular traps (NETs) in a murine model of experimental myeloperoxidase anti neutrophil cytoplasmic antibody associated glomerulonephritis (MPO-ANCA GN) and determine whether human recombinant deoxyribonuclease 1 (DNase 1) can inhibit NET formation and limit inflammation in this disease model. Background(s): Glomerular neutrophil traps containing extracellular MPO are a prominent feature of the renal injury seen in biopsies from patients with MPO-ANCA GN. DNase 1 in addition to its other anti-inflammatory properties can inhibit NET formation invitro and is used as a safe and effective therapy in cystic fibrosis. Method(s): Experimental MPO-ANCA GN was induced using a standard protocol. DNase 1 (10mg/kg, n=8) or saline control was administered intravenously twice daily to inhibit net formation 10 days after establishing MPO autoimmunity. Result(s): Glomerular NETs defined as colocalization of NET components: citrullinated histone 3 (H3Cit), protein arginine deiminase 4 (PAD4), MPO and chromatin via confocal microscopy were prominent within kidneys of mice receiving saline control (38+/- 5.6%glomeruli had NETs), compared to the treatment group receiving DNase1 (2.5 +/- 1.5% of glomeruli, P<0.0001), with an accompanied reduction in glomerular MPO (P<0.05). Histological injury (segmental necrosis) was present in the saline group (38.5+/- 1.5% affected glomeruli) and significantly reduced in the treated group (13.2+/- 1.9%, P<0.05). A significant reduction in glomerular CD4 and CD8 T cells, macrophages and neutrophils was evident in the DNase 1 treated group (all P<0.05). Functional kidney injury assessed by proteinuria was significantly reduced (24 hour albuminuria 1650 +/- 515.7 mug in controls vs 368.7 +/- 166 mug, P<0.05). Conclusion(s): Net formation is prominent in experimental MPO- ANCA GN and contributes to the inflammatory component of the disease. Administration of DNAse1 is eff

Background Pembrolizumab, a PD-1 immune checkpoint inhibitor, and dinaciclib, a cyclin-dependent kinase inhibitor, have demonstrated antitumor activity as monotherapies in various tumor types, including hematologic malignancies. In a preclinical study using a solidtumor syngeneic model (MC38), enhanced antitumor activity was observed with the combination of pembrolizumab and dinaciclib, with no significant toxicities. KEYNOTE-155 is a phase Ib study designed to evaluate the safety and efficacy of the combination of pembrolizumab and dinaciclib in patients with relapsed or refractory chronic lymphocytic leukemia (CLL), multiple myeloma (MM), or diffuse large B cell lymphoma (DLBCL). Methods Key eligibility criteria include >=18 years, ECOG performance status 0-1, and confirmed diagnosis of one of the following: CLL as defined by 2008 iwCLL criteria with >=1 prior therapy; active MM with measurable disease and >=2 prior therapies including an IMiD and proteasome inhibitor; or DLBCL with progression following >=2 prior therapies including autologous stem cell transplantation (ASCT; or ineligible for ASCT). An initial cohort of 12 patients (>=3 with each disease type) will be enrolled in the dose-evaluation phase (two 21-day cycles) to determine dose-limiting toxicities (DLTs). In cycle 1, patients will receive pembrolizumab 200 mg on day 1, dinaciclib 7 mg/m2 on day 1, and dinaciclib 10 mg/m2 on day 8. In cycle 2 and beyond, patients will receive pembrolizumab 200 mg on day 1 and dinaciclib 14 mg/m2 on days 1 and 8. If <=4 patients experience DLTs in the first 2 cycles, expansion cohorts (~30 patients each) will be opened for signal detection. If >=5 DLTs occur in the first 2 cycles, a lower dose of dinaciclib will be studied in up to 24 patients. Treatment will continue until disease progression, unacceptable toxicity, or 35 cycles. Response will be assessed every 3 cycles for CLL and DLBCL, and at the start of each cycle for MM. Primary end points are safety and tole

Aim: The aims of the study are to define the presence of glomerular neutrophil extracellular traps (NETs) in a murine model of experimental myeloperoxidase anti neutrophil cytoplasmic antibody associated glomerulonephritis (MPO-ANCA GN) and determine whether human recombinant deoxyribonuclease 1 (DNase 1) can inhibit NET formation and limit inflammation in this disease model. Background(s): Glomerular neutrophil traps containing extracellular MPO are a prominent feature of the renal injury seen in biopsies from patients with MPO-ANCA GN. DNase 1 in addition to its other anti-inflammatory properties can inhibit NET formation invitro and is used as a safe and effective therapy in cystic fibrosis. Method(s): Experimental MPO-ANCA GN was induced using a standard protocol. DNase 1 (10mg/kg, n=8) or saline control was administered intravenously twice daily to inhibit net formation 10 days after establishing MPO autoimmunity. Result(s): Glomerular NETs defined as colocalization of NET components: citrullinated histone 3 (H3Cit), protein arginine deiminase 4 (PAD4), MPO and chromatin via confocal microscopy were prominent within kidneys of mice receiving saline control (38+/- 5.6%glomeruli had NETs), compared to the treatment group receiving DNase1 (2.5 +/- 1.5% of glomeruli, P<0.0001), with an accompanied reduction in glomerular MPO (P<0.05). Histological injury (segmental necrosis) was present in the saline group (38.5+/- 1.5% affected glomeruli) and significantly reduced in the treated group (13.2+/- 1.9%, P<0.05). A significant reduction in glomerular CD4 and CD8 T cells, macrophages and neutrophils was evident in the DNase 1 treated group (all P<0.05). Functional kidney injury assessed by proteinuria was significantly reduced (24 hour albuminuria 1650 +/- 515.7 mug in controls vs 368.7 +/- 166 mug, P<0.05). Conclusion(s): Net formation is prominent in experimental MPO- ANCA GN and contributes to the inflammatory component of the disease. Administration of DNAse1 is eff

Myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) associated glomerulonephritis (MPO-ANCA GN) is an important cause of renal failure. With current standard treatment 30% of patients die or become dialysis dependent within 3 years. Studies in murine models have shown that the disease is neutrophil dependent. We have shown recently in patients that glomerular neutrophil extracellular traps (NETS) are a prominent feature. Human recombinant deoxyribonuclease 1 (DNase 1) is already used safely in children with Cystic Fibrosis. DNase 1 in addition to its other antiinflammatory properties can inhibit net formation invitro. We used DNase 1 intravenously twice daily to inhibit net formation in a murine model of experimental MPO-ANCA glomerulonephritis. DNase 1 treatment significantly decreased the percentage of glomeruli with NETs as defined by co-localisation of NET components: citrullinated histone 3 (H3Cit), protein arginine deiminase 4 (PAD4), MPO and chromatin (2.5% +/- SEM 1.5, versus 38% +/- SEM 5.6 in saline treated, P< 0.0001) and significantly reduced the amount of extracellular MPO (P< 0.05). Functional injury as assessed by proteinuria was significantly reduced (albuminuria/creatinine ratio reduced from 0.3+/- 0.09 in controls versus 0.07+/-0.03) as was histological injury (segmental glomerular necrosis, 38.5% +/- 4.2% in controls versus 13.2% +/- 1.9%). Glomerular recruitment of CD4 T cells, CD8 T cells, macrophages and neutrophils, were reduced with DNase 1 treatment (all, P< 0.05).This data suggests that NET formation is a critical component of inflammatory injury in this disease and that DNase 1 is effective in inhibiting NET formation, and reducing inflammation.

Aims: Aggressive epidermotropic cutaneous CD8+ lymphoma is currently afforded provisional status in the WHO classification of lymphomas. An EORTC Workshop was convened to describe in detail the features of this putative neoplasm and evaluate its nosological status with respect to other cutaneous CD8+ lymphomas. Methods and results: Sixty-one CD8+ cases were analysed at the workshop; clinical details, often with photographs, histological sections, immunohistochemical results, treatment and patient outcome were discussed and recorded. Eighteen cases had distinct features and conformed to the diagnosis of aggressive epidermotropic cutaneous CD8+ lymphoma. The patients typically present with widespread plaques and tumours, often ulcerated and haemorrhagic, and histologically have striking pagetoid epidermotrophism. A CD8+/CD45RA+/CD45RO-/CD2-/CD5-/CD56- phenotype, with one or more cytotoxic markers, was found in seven of 18 patients, with a very similar phenotype in the remainder. The tumours seldom involve lymph nodes, but mucosal and central nervous system involvement are not uncommon. The prognosis is poor, with a median survival of 12 months. Examples of CD8+ mycosis fungoides, lymphomatoid papulosis and Woringer-Kolopp disease presented the typical features well documented in the CD4+ forms of those diseases. Conclusions: Aggressive epidermotropic cutaneous CD8+ lymphoma is a distinct lymphoma that warrants inclusion as a distinct entity in future revisions of lymphoma classifications.

Introduction: Chronic liver disease affects >600 million people worldwide and is characterised by inflammation driving progressive fibrosis cumulatively resulting in cirrhosis. The consequences of cirrhosis include liver failure and hepatocellular carcinoma (HCC). Measures to diminish established liver injury targeting either inflammation or fibrosis progression are urgently required. Leukocyte infiltration is the hallmark of liver inflammation and is seen in virtually every form of liver injury. The inflammatory infiltration occurs in a pattern of leukocyte aggregates, and we have shown that these aggregates determine the extent of liver injury. Inflammatory mediators from the leukocyte infiltrate drive hepatic stellate cell (HSC) activation and progression of liver fibrosis.We also discovered that the glycoprotein CD147 is increased in human liver disease, and that it is essential for intrahepatic immune aggregate formation and injury. Furthermore we have found that CD147 regulates the induction of Matrix Metalloproteinases (MMPs) in hepatocytes, thereby contributing significantly to the extracellular matrix (ECM) turnover during injury. Method(s): Mouse models of progressive liver injury were induced in C57BL/6 and BALB/c mice by either thioacetamide (TAA) for 4, 8 or 20 weeks or intraperitoneal injection of carbon tetrachloride (CCl4) twice weekly for 72 hours or 4 weeks. In-vitro interventions consisted of alpha-CD147 mAb (RL73.2) or an isotype control. Additionally we have utilized CD147-/- animals. Liver leukocytes were analysed by eight-colour flow cytometry analysis for CD3, CD4, CD8alpha, CD19, NK1.1, F4/80 and CD147 cell surface expression. Cyclophilin (CyP) A and B gene expression in liver and liver leukocytes were analysed by qRT-PCR. Chemotaxis towards CyPA was assessed including a chemokinesis control. Gene and protein expressions of fibrotic markers were analysed in mouse liver tissues but also human cirrhotic liver explants by qRT-PCR, immunoblottin

Introduction: Chronic liver disease affects >600 million people worldwide and is characterised by inflammation driving progressive fibrosis cumulatively resulting in cirrhosis. The consequences of cirrhosis include liver failure and hepatocellular carcinoma (HCC). Measures to diminish established liver injury targeting either inflammation or fibrosis progression are urgently required. Leukocyte infiltration is the hallmark of liver inflammation and is seen in virtually every form of liver injury. The inflammatory infiltration occurs in a pattern of leukocyte aggregates, and we have shown that these aggregates determine the extent of liver injury. Inflammatory mediators from the leukocyte infiltrate drive hepatic stellate cell (HSC) activation and progression of liver fibrosis.We also discovered that the glycoprotein CD147 is increased in human liver disease, and that it is essential for intrahepatic immune aggregate formation and injury. Furthermore we have found that CD147 regulates the induction of Matrix Metalloproteinases (MMPs) in hepatocytes, thereby contributing significantly to the extracellular matrix (ECM) turnover during injury. Method(s): Mouse models of progressive liver injury were induced in C57BL/6 and BALB/c mice by either thioacetamide (TAA) for 4, 8 or 20 weeks or intraperitoneal injection of carbon tetrachloride (CCl4) twice weekly for 72 hours or 4 weeks. In-vitro interventions consisted of alpha-CD147 mAb (RL73.2) or an isotype control. Additionally we have utilized CD147-/- animals. Liver leukocytes were analysed by eight-colour flow cytometry analysis for CD3, CD4, CD8alpha, CD19, NK1.1, F4/80 and CD147 cell surface expression. Cyclophilin (CyP) A and B gene expression in liver and liver leukocytes were analysed by qRT-PCR. Chemotaxis towards CyPA was assessed including a chemokinesis control. Gene and protein expressions of fibrotic markers were analysed in mouse liver tissues but also human cirrhotic liver explants by qRT-PCR, immunoblottin

Background. Macrophages constitute 38% to 60% of infiltrating cells during acute renal allograft rejection. Their contribution to tissue damage during acute rejection was examined by depleting macrophages in a rat model. Methods. Lewis rats underwent bilateral nephrectomy and then received a Dark Agouti renal allograft and liposomal-clodronate, control phosphate-buffered saline liposomes, or saline intravenously (n=7 per group) on days 1 and 3 postsurgery. Grafts were harvested on day 5. Results. Liposomal-clodronate treatment resulted in a 70% reduction in blood ED1+ monocytes and 60% reduction in intragraft ED1+ macrophages (both P < 0.01). Half of all remaining interstitial ED1+ cells were undergoing apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling +/ED1+), and thus functional depletion of more than 75% of macrophages was achieved. Histologic and functional parameters of acute rejection were attenuated: interstitial infiltrate, tubulitis, and glomerulitis (P < 0.01); tubular cell apoptosis (P < 0.001); tubular cell proliferation (P < 0.001); and serum creatinine (P < 0.01). Production of inducible nitric oxide synthase by infiltrating cells and urinary nitric oxide excretion was reduced by 90% (P < 0.001). In contrast, no reduction in the number of other leukocytes was seen (CD3+, CD4+, CD8 +, and natural killer cells). Activation of lymphocytes (CD25 +) and production of lymphocyte effector molecules (granzyme B) were unaltered. Conclusion. This study demonstrates that macrophages contribute to tissue damage during acute rejection.

Mycophenolate mofetil (MMF) is a recently introduced immunosuppressive drug. Its active form is mycophenolic acid (MPA). MPA specifically inhibits de novo purine synthesis, which is vital for T and B lymphocyte proliferation. We measured lymphocyte subset numbers and mitogen induced proliferation in kidney transplant recipients on different combinations of MMF, cyclosporin A (C), azathioprine (A) and prednisolone (P) (C+A n=70; C+A+P n=15; C+MMF n=45; C+MMF+P n=37) and normals (n=73). Patients on MMF had severely reduced phytohaemagglutinin A (PHA) induced proliferation compared to normals (Nml 2766+/-926 CPM/1000 lymphocytes [mean+/-S.D.]; C+MMF 282+/-406; C+MMF+P 195+/-496); non-MMF patients did not differ from normal. Similar inhibition of Poke Weed Mitogen and Staphylococcal enterotoxin B induced proliferation was observed. Cell cycle studies established that MMF patients had a significantly higher proportion of lymphocytes in the G0/G1 phase following PHA stimulation than the non-MMF patients. All transplant groups had significantly lower B cell numbers than the normal controls but no differences in CD4 and CD8 T cell numbers. All but the C+MMF group had significantly lower CD16+NK cell numbers than normal, while only the non-MMF groups had significantly lower CD56+NK cell numbers. The proliferation assay used was an ex vivo diluted whole blood technique. Removal of residual MPA by washing the plasma out prior to mitogen stimulation led to a significant increase in proliferation in six out of seven cases. In summary we have found that MMF treatment has a strikingly inhibitory effect on patient ex vivo lymphocyte mitogenic function. © 2004 Elsevier B.V. All rights reserved.

Background. Macrophages constitute 38% to 60% of infiltrating cells during acute renal allograft rejection. Their contribution to tissue damage during acute rejection was examined by depleting macrophages in a rat model. Methods. Lewis rats underwent bilateral nephrectomy and then received a Dark Agouti renal allograft and liposomal-clodronate, control phosphate-buffered saline liposomes, or saline intravenously (n=7 per group) on days 1 and 3 postsurgery. Grafts were harvested on day 5. Results. Liposomal-clodronate treatment resulted in a 70% reduction in blood ED1+ monocytes and 60% reduction in intragraft ED1+ macrophages (both P < 0.01). Half of all remaining interstitial ED1+ cells were undergoing apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling +/ED1+), and thus functional depletion of more than 75% of macrophages was achieved. Histologic and functional parameters of acute rejection were attenuated: interstitial infiltrate, tubulitis, and glomerulitis (P < 0.01); tubular cell apoptosis (P < 0.001); tubular cell proliferation (P < 0.001); and serum creatinine (P < 0.01). Production of inducible nitric oxide synthase by infiltrating cells and urinary nitric oxide excretion was reduced by 90% (P < 0.001). In contrast, no reduction in the number of other leukocytes was seen (CD3+, CD4+, CD8 +, and natural killer cells). Activation of lymphocytes (CD25 +) and production of lymphocyte effector molecules (granzyme B) were unaltered. Conclusion. This study demonstrates that macrophages contribute to tissue damage during acute rejection.

Mycophenolate mofetil (MMF) is a recently introduced immunosuppressive drug. Its active form is mycophenolic acid (MPA). MPA specifically inhibits de novo purine synthesis, which is vital for T and B lymphocyte proliferation. We measured lymphocyte subset numbers and mitogen induced proliferation in kidney transplant recipients on different combinations of MMF, cyclosporin A (C), azathioprine (A) and prednisolone (P) (C+A n=70; C+A+P n=15; C+MMF n=45; C+MMF+P n=37) and normals (n=73). Patients on MMF had severely reduced phytohaemagglutinin A (PHA) induced proliferation compared to normals (Nml 2766+/-926 CPM/1000 lymphocytes [mean+/-S.D.]; C+MMF 282+/-406; C+MMF+P 195+/-496); non-MMF patients did not differ from normal. Similar inhibition of Poke Weed Mitogen and Staphylococcal enterotoxin B induced proliferation was observed. Cell cycle studies established that MMF patients had a significantly higher proportion of lymphocytes in the G0/G1 phase following PHA stimulation than the non-MMF patients. All transplant groups had significantly lower B cell numbers than the normal controls but no differences in CD4 and CD8 T cell numbers. All but the C+MMF group had significantly lower CD16+NK cell numbers than normal, while only the non-MMF groups had significantly lower CD56+NK cell numbers. The proliferation assay used was an ex vivo diluted whole blood technique. Removal of residual MPA by washing the plasma out prior to mitogen stimulation led to a significant increase in proliferation in six out of seven cases. In summary we have found that MMF treatment has a strikingly inhibitory effect on patient ex vivo lymphocyte mitogenic function. © 2004 Elsevier B.V. All rights reserved.

COPD is characterized by infl ammation that persists despite smoking cessation. This infl ammation is driven to a large extent by T lymphocytes. The antigens that stimulate the T cells have not been defi ned although autoimmunity and infection have been suggested as potential candidates. Aim To measure the response of lung T cells to stimulation by the bacterium NTHi. Methods Subjects were those with and without COPD who were having a lobectomy. Lung tissue was stimulated with live NTHi and the next day cells were permeabilized and antibodies for surface markers and intracellular cytokines added. Cells were analysed by FACS. Results 8 control and 13 COPD subjects have been recruited so far. Large numbers of antigen specifi c CD4 and CD8 T cells were detected in both groups. Levels were higher in COPD subjects. Results are shown in the table as mean % and range. (Table Presented) Conclusion There are large numbers of lung T cells specific for NTHi in COPD.

Aim: To define the role of T cell immunoglobulin mucin 1 (Tim-1) in experimental renal injury. Background(s): Tim-1 modulates T cell responses, but is also known as Kim-1 and expressed by damaged proximal tubules. Method(s): Tim-1 was neutralized by anti-Tim-1 monoclonal antibodies (RMT1- 10, 250 mug, alternate days, controls received non-immune rat IgG) that do not affect Kim-1 binding to apoptotic/necrotic cells (Kim-1's major intrarenal effect), in two murine models 1) anti-glomerular basement membrane (GBM) glomerulonephritis, where glomerular injury is Th1 and Th17 cell-mediated; 2) cisplatin nephrotoxicity, where T cells are 'innate' effectors mediating acute tubulointerstitial injury. Result(s): In anti-GBM glomerulonephritis (day 14, non-sensitized mice) anti- Tim-1 antibodies attenuated crescents, proliferative injury and leukocyte accumulation, but interstitial foxp3+ regulatory T cells and interleukin-10 mRNA were increased. Intrarenal neutrophil, macrophage and T cell chemokine mRNA were decreased, except for CCL20 (important in recruiting Tregs). Th1 and Th17 cellular responses (within the immune system and intrarenally) were selectively reduced. Day 6 injury was unaffected demonstrating that Tim-1 promotes a mature autologous response. In cisplatin nephrotoxicity, anti-Tim-1 antibodies reduced early (day 1) systemic CD4+ and CD8+ T cell activation, apoptosis, and cytokine production, with less histologic damage, improved renal function and fewer leukocytes infiltrating the kidney (day 3). Renal NF-kappaB activation, apoptosis, and proinflammatory cytokine/chemokine mRNA expression were reduced. To determine whether the actions of anti-Tim-1 antibodies could be due to effects on tubular cells, anti-Tim-1 antibodies were given to cisplatin-treated Rag1-/- mice, with no effect, demonstrating that the benefits of Tim-1 inhibition come from its T cell effects. Conclusion(s): Via its T cell effects, Tim-1 mediates both glomerular injury induced by adaptive i

COPD is characterized by infl ammation that persists despite smoking cessation. This infl ammation is driven to a large extent by T lymphocytes. The antigens that stimulate the T cells have not been defi ned although autoimmunity and infection have been suggested as potential candidates. Aim To measure the response of lung T cells to stimulation by the bacterium NTHi. Methods Subjects were those with and without COPD who were having a lobectomy. Lung tissue was stimulated with live NTHi and the next day cells were permeabilized and antibodies for surface markers and intracellular cytokines added. Cells were analysed by FACS. Results 8 control and 13 COPD subjects have been recruited so far. Large numbers of antigen specifi c CD4 and CD8 T cells were detected in both groups. Levels were higher in COPD subjects. Results are shown in the table as mean % and range. (Table Presented) Conclusion There are large numbers of lung T cells specific for NTHi in COPD.

Aim: To define the role of T cell immunoglobulin mucin 1 (Tim-1) in experimental renal injury. Background(s): Tim-1 modulates T cell responses, but is also known as Kim-1 and expressed by damaged proximal tubules. Method(s): Tim-1 was neutralized by anti-Tim-1 monoclonal antibodies (RMT1- 10, 250 mug, alternate days, controls received non-immune rat IgG) that do not affect Kim-1 binding to apoptotic/necrotic cells (Kim-1's major intrarenal effect), in two murine models 1) anti-glomerular basement membrane (GBM) glomerulonephritis, where glomerular injury is Th1 and Th17 cell-mediated; 2) cisplatin nephrotoxicity, where T cells are 'innate' effectors mediating acute tubulointerstitial injury. Result(s): In anti-GBM glomerulonephritis (day 14, non-sensitized mice) anti- Tim-1 antibodies attenuated crescents, proliferative injury and leukocyte accumulation, but interstitial foxp3+ regulatory T cells and interleukin-10 mRNA were increased. Intrarenal neutrophil, macrophage and T cell chemokine mRNA were decreased, except for CCL20 (important in recruiting Tregs). Th1 and Th17 cellular responses (within the immune system and intrarenally) were selectively reduced. Day 6 injury was unaffected demonstrating that Tim-1 promotes a mature autologous response. In cisplatin nephrotoxicity, anti-Tim-1 antibodies reduced early (day 1) systemic CD4+ and CD8+ T cell activation, apoptosis, and cytokine production, with less histologic damage, improved renal function and fewer leukocytes infiltrating the kidney (day 3). Renal NF-kappaB activation, apoptosis, and proinflammatory cytokine/chemokine mRNA expression were reduced. To determine whether the actions of anti-Tim-1 antibodies could be due to effects on tubular cells, anti-Tim-1 antibodies were given to cisplatin-treated Rag1-/- mice, with no effect, demonstrating that the benefits of Tim-1 inhibition come from its T cell effects. Conclusion(s): Via its T cell effects, Tim-1 mediates both glomerular injury induced by adaptive i

Aims/hypothesis: Diabetic nephropathy is an inflammatory disease with prominent leucocyte infiltration of the kidneys. While the importance of macrophages in diabetic renal injury has been clearly demonstrated, the role of lymphocytes is still unknown. We therefore examined the development of diabetic renal injury in lymphocyte-deficient mice. Method(s): Streptozotocin was used to induce diabetes in Rag1-/- mice, which lack mature T and B lymphocytes, and in wild-type (Rag1+/+) controls. The development of renal injury was examined over 20 weeks of diabetes. Result(s): Both groups developed equivalent diabetes, however only Rag1+/+ mice had kidney infiltration with CD4, CD8, CD22 and forkhead box P3-positive cells, as well as glomerular immunoglobulin deposition. At 20 weeks, Rag1+/+ mice exhibited renal hypertrophy, increased mesangial and interstitial matrix, kidney macrophage accumulation, tubular injury, progressive albuminuria and a decline in renal function. In comparison, diabetic Rag1-/- mice showed similar histological damage, matrix expansion, macrophage accrual and loss of renal function, but were protected from increasing albuminuria. This protection was associated with protection against loss of podocytes and glomerular podocin production, and with reduced glomerular macrophage activation. Conclusions/interpretation: These results show that lymphocytes contribute to the development of diabetic albuminuria, which may partly arise from increasing glomerular macrophage activation and podocyte damage. In contrast, lymphocytes do not appear to promote tubular injury, increased matrix deposition or decline in renal function in a mouse model of type 1 diabetes. Our findings suggest that innate immunity rather than adaptive immune responses are the major inflammatory contributor to the progression of diabetic renal injury. © 2010 Springer-Verlag.

Introduction: Natural Killer (NK) cells play a key role in the interaction between the innate and adaptive immune systems. Toll like receptors(TLR) are key mediators of innate immunity, and we have previously demonstrated TLR mediated dysfunction of monocytes and Dendritic cells(DC) in chronic hepatitis B infection(CHB). NK cells express a number of TLRs, however their functional significance remains unclear. The aim of this study was to determine whether a TLR mediated dysfunction of NK cells contributes to the immune hyporesponsiveness seen in CHB Methods: Peripheral blood was obtained from patients in immunoactive, immunotolerant (IT) and inactive stages of chronic HBV infection and from healthy volunteers. PBMCs were isolated using Ficoll Density Centrifugation and then cultured with the following TLR ligands: poly I:C (TLR3), purified LPS (TLR4), R848 (TLR 7/8) and CpG 2216(TLR9). After 24 hours, the cells were stained with flurochrome conjugated antibodies against CD3, CD56, CD4, CD8, and CD45RA to identify NK, NKT, and naive and memory CD4 and CD8 T cells. Anti-IFN-gamma Abs were used to detect intracellular cytokine staining(ICS). Flow cytometry was used to determine the frequency of lymphocyte subsets and levels of ICS (expressed as percentage of cell subtype producing cytokine) Results: Samples from 22 CHB patients (representative of the different clinical phases) and 8 controls were analysed. The frequency of circulating NK cells ranged from 8.45% to 42% of the PBMC population excluding granulocytes, with the lowest frequency observed in the HBeAg negative immunoactive group (mean 20.5+/-2.3) though no significant differences were noted either between the groups of CHB patients or compared to controls. The frequency of NKT cells did also not differ significantly between groups, with a range of 1.82 to 9.5%. NK cell IFN-gamma production was demonstrated in response to all 4 TLR ligands in all patient groups. IFN-gamma production was significantly lower in

Aims/hypothesis: Diabetic nephropathy is an inflammatory disease with prominent leucocyte infiltration of the kidneys. While the importance of macrophages in diabetic renal injury has been clearly demonstrated, the role of lymphocytes is still unknown. We therefore examined the development of diabetic renal injury in lymphocyte-deficient mice. Method(s): Streptozotocin was used to induce diabetes in Rag1-/- mice, which lack mature T and B lymphocytes, and in wild-type (Rag1+/+) controls. The development of renal injury was examined over 20 weeks of diabetes. Result(s): Both groups developed equivalent diabetes, however only Rag1+/+ mice had kidney infiltration with CD4, CD8, CD22 and forkhead box P3-positive cells, as well as glomerular immunoglobulin deposition. At 20 weeks, Rag1+/+ mice exhibited renal hypertrophy, increased mesangial and interstitial matrix, kidney macrophage accumulation, tubular injury, progressive albuminuria and a decline in renal function. In comparison, diabetic Rag1-/- mice showed similar histological damage, matrix expansion, macrophage accrual and loss of renal function, but were protected from increasing albuminuria. This protection was associated with protection against loss of podocytes and glomerular podocin production, and with reduced glomerular macrophage activation. Conclusions/interpretation: These results show that lymphocytes contribute to the development of diabetic albuminuria, which may partly arise from increasing glomerular macrophage activation and podocyte damage. In contrast, lymphocytes do not appear to promote tubular injury, increased matrix deposition or decline in renal function in a mouse model of type 1 diabetes. Our findings suggest that innate immunity rather than adaptive immune responses are the major inflammatory contributor to the progression of diabetic renal injury. © 2010 Springer-Verlag.

Introduction: Natural Killer (NK) cells play a key role in the interaction between the innate and adaptive immune systems. Toll like receptors(TLR) are key mediators of innate immunity, and we have previously demonstrated TLR mediated dysfunction of monocytes and Dendritic cells(DC) in chronic hepatitis B infection(CHB). NK cells express a number of TLRs, however their functional significance remains unclear. The aim of this study was to determine whether a TLR mediated dysfunction of NK cells contributes to the immune hyporesponsiveness seen in CHB Methods: Peripheral blood was obtained from patients in immunoactive, immunotolerant (IT) and inactive stages of chronic HBV infection and from healthy volunteers. PBMCs were isolated using Ficoll Density Centrifugation and then cultured with the following TLR ligands: poly I:C (TLR3), purified LPS (TLR4), R848 (TLR 7/8) and CpG 2216(TLR9). After 24 hours, the cells were stained with flurochrome conjugated antibodies against CD3, CD56, CD4, CD8, and CD45RA to identify NK, NKT, and naive and memory CD4 and CD8 T cells. Anti-IFN-gamma Abs were used to detect intracellular cytokine staining(ICS). Flow cytometry was used to determine the frequency of lymphocyte subsets and levels of ICS (expressed as percentage of cell subtype producing cytokine) Results: Samples from 22 CHB patients (representative of the different clinical phases) and 8 controls were analysed. The frequency of circulating NK cells ranged from 8.45% to 42% of the PBMC population excluding granulocytes, with the lowest frequency observed in the HBeAg negative immunoactive group (mean 20.5+/-2.3) though no significant differences were noted either between the groups of CHB patients or compared to controls. The frequency of NKT cells did also not differ significantly between groups, with a range of 1.82 to 9.5%. NK cell IFN-gamma production was demonstrated in response to all 4 TLR ligands in all patient groups. IFN-gamma production was significantly lower in

CD4+ T cells with immune regulatory function can be either FOXP3+ or FOXP3-. We have previously shown that priming of naturally occurring TCR-peptide-reactive CD4+FOXP3- Treg specifically controls VÎ28.2+CD4+ T cells mediating EAE. However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVÎ28.2-chain, is not known. In this study we show that APC derived from splenocytes of naà ̄ve mice are able to stimulate cloned CD4+ Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations, DC were the most efficient in stimulating the Treg. Stimulation of CD4+ Treg was dependent upon processing and presentation of TCR peptides from ingested VÎ28.2TCR+CD4 + T cells. Additionally, DC pulsed with TCR peptide or apoptotic VÎ28.2+ T cells were able to prime Treg in vivo and mediate protection from disease in a CD8-dependent fashion. These data highlight a novel mechanism for the priming of CD4+ Treg by CD8α+ DC and suggest a pathway that can be exploited to prime antigen-specific regulation of T-cell-mediated inflammatory disease. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells including neutrophils, macrophages,T lymphocytes, and dendritic cells (DCs). The relative contributions of these various inflammatory cells to airway injury and remodeling are not well documented. In particular, the potential role of DCs as mediators of inflammation in the smoker's airways and COPD patients is poorly understood. In the current study we analyzed the effects of cigarette smoke extract on mouse bone marrow derived DC and the production of chemokines and cytokines were studied. In addition, we assessed CSE-induced changes in cDC function in the mixed lymphocyte reaction (MLR) examining CD4+ and CD8+ T cell proliferation. Cigarette smoke extract induces the release of the chemokines CCL3 and CXCL2 (but not cytokines), via the generation of reactive oxygen species (ROS). In a mixed-leukocyte reaction assay, cigarette smoke-primed DCs potentiate CD8+T cell proliferation via CCL3. In contrast, proliferation of CD4+T cells is suppressed via an unknown mechanism. The cigarette smoke-induced release of CCL3 and CXCL2 by DCs may contribute to the influx of CD8+T cells and neutrophils into the airways, respectively.

Nasopharyngeal carcinoma is a neoplasm with a high incidence in Southeast Asia, and it is strongly associated with Epstein-Barr virus (EBV) activation involving the expression of a weakly immunogenic protein, namely, latent membrane protein (LMP)-2. Previous immunological studies already identified the human leukocyte antigen (HLA)-A11 restricted peptide epitope (SSCSSCPLSK) in the LMP-2 antigen. In this work, we prepared gold nanoparticle (AuNP)-peptide conjugate 1 by treating the nanoparticles with the N-cysteinated LMP-2 epitope. The AuNP-peptide conjugates have been characterized by TEM (15-24 nm in diameter) and UV-vis spectroscopy (surface plasmon resonance absorption band at lambda(max) = 520 nm). In the presence of a CALNN capping peptide, the AuNP-peptide conjugates are stable in solution without aggregation at room temperature for at least 48 In. By ELIspot studies, AuNP-peptide conjugate 1 was found to elicit a significantly stronger INF-gamma response [number of spot forming cells (SPC) = 727 +/- 198] from peripheral blood mononuclear cells of healthy HLA-A11 donors when compared to that induced by the unconjugated LMP-2 peptides (SFC = 73 +/- 28). Further studies showed that dendritic cells treated with conjugate I can effect CD8+ T-cell activation leading to epitope-specific cytotoxic T lymphocyte killing responses in vitro.

Nasopharyngeal carcinoma is a neoplasm with a high incidence in Southeast Asia, and it is strongly associated with Epstein-Barr virus (EBV) activation involving the expression of a weakly immunogenic protein, namely, latent membrane protein (LMP)-2. Previous immunological studies already identified the human leukocyte antigen (HLA)-A11 restricted peptide epitope (SSCSSCPLSK) in the LMP-2 antigen. In this work, we prepared gold nanoparticle (AuNP)-peptide conjugate 1 by treating the nanoparticles with the N-cysteinated LMP-2 epitope. The AuNP-peptide conjugates have been characterized by TEM (15-24 nm in diameter) and UV-vis spectroscopy (surface plasmon resonance absorption band at lambda(max) = 520 nm). In the presence of a CALNN capping peptide, the AuNP-peptide conjugates are stable in solution without aggregation at room temperature for at least 48 In. By ELIspot studies, AuNP-peptide conjugate 1 was found to elicit a significantly stronger INF-gamma response [number of spot forming cells (SPC) = 727 +/- 198] from peripheral blood mononuclear cells of healthy HLA-A11 donors when compared to that induced by the unconjugated LMP-2 peptides (SFC = 73 +/- 28). Further studies showed that dendritic cells treated with conjugate I can effect CD8+ T-cell activation leading to epitope-specific cytotoxic T lymphocyte killing responses in vitro.

Genital infection with human papillomavirus (HPV) is usually transient, as the immune system is capable of eliminating the virus. When immunity "fails" and the infection persists, vulvar intraepithelial neoplasia (VIN) may develop. In this study, we examined the distribution of inflammatory cells in 51 patients with HPV-associated usual-type VIN and in 19 healthy controls. Frozen vulvar tissue samples were tested for the presence of HPV-DNA, and immunohistochemical staining for

Antigen stimulation of naive T cells in conjunction with strong costimulatory signals elicits the generation of effector and memory populations. Such terminal differentiation transforms naive T cells capable of differentiating along several terminal pathways in response to pertinent environmental cues into cells that have lost developmental plasticity and exhibit heightened responsiveness. Because these cells exhibit little or no need for the strong costimulatory signals required for full activation of naive T cells, it is generally considered memory and effector T cells are released from the capacity to be inactivated. Here, we show that steadystate dendritic cells constitutively presenting an endogenously expressed antigen inactivate fully differentiated memory and effector CD8+ T cells in vivo through deletion and inactivation. These findings indicate that fully differentiated effector and memory T cells exhibit a previously unappreciated level of plasticity and provide insight into how memory and effector T-cell populations may be regulated. © 2008 by The American Society of Hematology.

Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here, it is reported that CD46 transgenic mice (MYII-strain) express CD46 in all major organs and that it functions as a receptor for rAd35 vectors. Similar to monkeys and humans, MYII mice highly express CD46 in their lungs and kidneys and demonstrate low expression in muscle. Upon intravenous administration, rAd35 vector genomes as well as expression are detected in lungs of MYII mice, in contrast to wild-type littermates. Expression was predominantly detected in lung epithelial cells. Upon intramuscular administration, the initial level of luciferase expression is higher in MYII mice as compared with wild-type littermates, in spite of the fact that CD46 expression is low in muscle of MYII mice. The higher level of expression in muscle of MYII mice results in prolonged gene expression as assessed by CCD camera imaging for luciferase activity. Finally, a significant dose-sparing effect in MYII mice as compared with wild-type littermates on anti-SIVgag CD8+ T-cell induction following intramuscular vaccination with an rA35.SIVgag vaccine was observed. This dose-sparing effect was also observed when reinfusing dendritic cells derived from MYII mice after exposure to rAd35.SIVgag vaccine as compared with rAd35.SIVgag exposed dendritic cells from wild-type littermates. It was concluded that MYII mice represent an interesting preclinical model to evaluate potency and safety of rAd35 vectors. © 2006 SGM. Chemicals / CAS: Antigens, CD46

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

The atypical Nef protein (NefF12) from human immunodeficiency virus type 1 strain F12 (HIV-1F12) interferes with virion production and infectivity via a mysterious mechanism. The correlation of these effects with the unusual perinuclear subcellular localization of NefF12 suggested that the wild-type Nef protein could bind to assembly intermediates in late stages of viral replication. To test this hypothesis, Nef from HIV-1NL4-3 was fused to an endoplasmic reticulum (ER) retention signal (NefKKXX). This mutant NefKKXX protein recapitulated fully the effects of NefF12 on Gag processing and virion production, either alone or as a CD8 fusion protein. Importantly, the mutant NefKKXX protein also localized to the intermediate compartment, between the ER and the trans-Golgi network. Furthermore, Nef bound the GagPol polyprotein in vitro and in vivo. This binding mapped to the C-terminal flexible loop in Nef and the transframe p6* protein in GagPol. The significance of this interaction was demonstrated by a genetic assay in which the release of a mutant HIV-1 provirus lacking the PTAP motif in the late domain that no longer binds Tsg101 was rescued by a Nef.Tsg101 chimera. Importantly, this rescue as well as incorporation of Nef into HIV-1 virions correlated with the ability of Nef to interact with GagPol. Our data demonstrate that the retention of Nef in the intermediate compartment interferes with viral replication and suggest a new role for Nef in the production of HIV-1.

In this study, a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia with an early onset and high incidence was established and characterized. All tumors analyzed were found to express the alpha,beta T-cell receptor, and the majority of them had a mature, CD3+CD4+CD8- immunophenotype. In a few cases, tumors with a more immature CD3+CD4+CD8+ phenotype were isolated. Expanded phenotyping using a broad panel of lymphocyte differentiation markers confirmed the mature T-cell phenotype of the tumors. Histologic and cell cycle analysis of the tumors revealed an aggressive lymphoblastic malignancy with a very high proliferation rate and widespread engagement of bone marrow and lymphoid as well as nonlymphoid organs. Thus, the TLL mouse strain represents a unique model for the analysis of the oncogenesis and progression of mature T-cell tumors and for the development of therapeutic measures to combat such tumors.

Positive selection has been proposed to be involved in protection from diabetes. We examined positive selection by fluorescence-activated cell sorter analyses in thymocytes of protected and susceptible E-transgenic and non-transgenic NOD mice. Three Vb families showed positive selection in E-transgenic mice. Vb6+CD4+ and Vb10+CD4+ thymocytes were found at higher frequencies in both protected NOD-Ea and susceptible NOD-DY mice. The increased frequencies of Vb13+CD8+ thymocytes were found in protected NOD-Ea mice only, and not in susceptible NOD-DY transgenic mice. These three Vb families were further examined in bone-marrow chimeras between NOD-Ea and non-transgenic NOD mice, where we could examine the contribution of E-expressing bone-marrow-derived cells in positive selection. We find that NOD-Ea→NOD-Ea chimeras have an increased positive selection of Vb13+CD8+ cells and that positive selection is more efficient when both thymic epithelium and bone-marrow-derived cells express the E molecule. This was also seen for Vb6+CD4+ cells. However, for Vb6, bone-marrow-derived cells alone were also capable of positive selection. Positive selection of Vb10+CD4+ cells was restricted to E-expressing thymic epithelium only. For Vb13+CD8+ cells, we found that positive selection is most efficient with E-expression on both thymic epithelium and bone-marrow-derived cells, although positive selection also occurs with E-positive epithelium only. For Vb6+CD4+ cells, the dominating selecting cells are bone-marrow-derived cells, and Vb10+CD4+ cells seem to be selected exclusively by the thymic epithelium. Thus, the conditions for positive selection seem to vary considerably between different Vb families.

The non-obese diabetic (NOD) mouse is an animal model for insulin- dependent diabetes that has many similarities to the human disease. NOD mice transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. An Ea transgene mutated in the promoter region, (ΔY) lacks E expression on most B cells, thymic medullary epithelium and primary antigen-presenting cells, and confers no protection whatsoever. We have used these transgenic NOD mice, together with non-transgenic NOD mice, to study the correlation of E expression and production of interleukin-4 (IL-4) and interferon-γ (IFN-γ). We show that protected E-transgenic NOD mice have elevated levels of IL-4 compared with non-transgenic mice, both in the thymus and in the periphery. However, susceptible ΔY-transgenic mice have elevated thymic IL-4 levels, but express almost as little IL-4 as non-transgenic NOD mice in the periphery. This drop in peripheral IL-4 production seen in ΔY-transgenic mice thus correlates with the decreased E expression in the periphery of ΔY-transgenic NOD mice. In contrast, there were no differences in IFN-γ production between the three NOD lines. We suggest that Ea-transgenic NOD mice have E-selected regulatory T cells producing IL-4, which are subsequently activated by E-expressing primary antigen-presenting cells in the periphery. This activation would then be instrumental for the E-mediated protection from disease in NOD mice. Such a process would explain the total absence of protection in ΔY-transgenic NOD mice, despite their widespread E expression.