Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen.

BACKGROUND: In an earlier study we developed a unique strategy allowing us to specifically eliminate antigen-specific murine B cells via their distinct B cell receptors using a new class of fusion proteins. In the present work we elaborated our idea to demonstrate the feasibility of specifically addressing and eliminating human memory B cells. RESULTS: The present study reveals efficient adaptation of the general approach to selectively target and eradicate human memory B cells. In order to demonstrate the feasibility we engineered a fusion protein following the principle of recombinant immunotoxins by combining a model antigen (tetanus toxoid fragment C, TTC) for B cell receptor targeting and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA') to induce apoptosis after cellular uptake. The TTC-ETA' fusion protein not only selectively bound to a TTC-reactive murine B cell hybridoma cell line in vitro but also to freshly isolated human memory B cells from immunized donors ex vivo. Specific toxicity was confirmed on an antigen-specific population of human CD27(+) memory B cells. CONCLUSIONS: This protein engineering strategy can be used as a generalized platform approach for the construction of therapeutic fusion proteins with disease-relevant antigens as B cell receptor-binding domains, offering a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.

Δγ1134.5 herpes simplex viruses encoding human cytomegalovirus IRS1 or TRS1 induce interferon regulatory factor 3 phosphorylation and an interferon-stimulated gene response.

The chimeric herpes simplex viruses (HSV) are Δγ134.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ134.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ134.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ134.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).

Murine Cytomegalovirus-induced Complement-fixing Antibodies Deposit in Murine Renal Allografts During Acute Rejection.

BACKGROUND: Human cytomegalovirus (CMV) infection is associated with renal allograft dysfunction and loss, particularly in combination with acute rejection. Emerging literature suggests that non-HLA antibodies may contribute to antibody-mediated rejection, but pathogen-induced antibodies have not been investigated in this context. This study examines the presence of CMV-induced antibodies in murine CMV (MCMV)-infected renal allografts during acute rejection. METHODS: Intragraft immunoglobulin G (IgG) and complement C3 immunostaining were compared among allogeneic MCMV D-/R-, D+/R-, and D+/R+ renal transplants. Intragraft antibody deposition was examined in B cell-deficient recipients treated with MCMV immune sera. Antibody binding and complement-dependent cytotoxicity (CDC) of D-/R- and D+/R+ sera against infected renal tubular epithelial cells (TECs) were measured in vitro. IgG immunostaining was performed in D+/R+ allografts and native kidneys and in D+/R- allografts treated with ganciclovir to inhibit viral replication. RESULTS: D+/R- and D+/R+ transplants had more abundant IgG and C3 deposition compared with D-/R- recipients. Greater IgG deposition was associated with more severe allograft injury in B cell-deficient recipients treated with MCMV immune sera compared with nonimmune sera. D+/R+ sera induced greater CDC of infected TECs compared with D-/R- sera. Native kidneys had lower IgG deposition compared with allografts, despite similar organ viral loads. Ganciclovir-treated allografts had reduced IgG deposition compared with untreated allografts. CONCLUSIONS: In this murine model, complement-fixing antibodies can deposit into MCMV-infected renal allografts, are associated with allograft damage, and can induce CDC of MCMV-infected renal TECs. The allogeneic response and viral replication may also contribute to intragraft antibody deposition.

Chimeric HCMV/HSV-1 and Δγ134.5 oncolytic herpes simplex virus elicit immune mediated antigliomal effect and antitumor memory.

Malignant gliomas are the most common primary brain tumor and are characterized by rapid and highly invasive growth. Because of their poor prognosis, new therapeutic strategies are needed. Oncolytic virotherapy (OV) is a promising strategy for treating cancer that incorporates both direct viral replication mediated and immune mediated mechanisms to kill tumor cells. C134 is a next generation Δγ134.5 oHSV-1 with improved intratumoral viral replication. It remains safe in the CNS environment by inducing early IFN signaling which restricts its replication in non-malignant cells. We sought to identify how C134 performed in an immunocompetent tumor model that restricts its replication advantage over first generation viruses. To achieve this we identified tumors that have intact IFN signaling responses that restrict C134 and first generation virus replication similarly. Our results show that both viruses elicit a T cell mediated anti-tumor effect and improved animal survival but that subtle difference exist between the viruses effect on median survival despite equivalent in vivo viral replication. To further investigate this we examined the anti-tumor activity in immunodeficient mice and in syngeneic models with re-challenge. These studies show that the T cell response is integral to C134 replication independent anti-tumor response and that OV therapy elicits a durable and circulating anti-tumor memory. The studies also show that repeated intratumoral administration can extend both OV anti-tumor effects and induce durable anti-tumor memory that is superior to tumor antigen exposure alone.

Differential effects of cyclophosphamide and mycophenolate mofetil on cellular and serological parameters in patients with systemic lupus erythematosus.

INTRODUCTION: Disease activity and therapy show an impact on cellular and serological parameters in patients with systemic lupus erythematosus (SLE). This study was performed to compare the influence of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) therapy on these parameters in patients with flaring, organ-threatening disease. METHODS: SLE patients currently receiving CYC (n = 20), MMF (n = 25) or no immunosuppressive drugs (n = 22) were compared using a cross-sectional design. Median disease activity and daily corticosteroid dose were similar in these treatment groups. Concurrent medication, organ manifestations, and disease activity were recorded, and cellular and serological parameters were determined by routine diagnostic tests or flow cytometric analysis. In addition follow-up data were obtained from different sets of patients (CYC n = 24; MMF n = 23). RESULTS: Although both drugs showed a significant effect on disease activity and circulating B cell subsets, only MMF reduced circulating plasmablasts and plasma cells as well as circulating free light chains within three months of induction therapy. Neither MMF nor CYC were able to reduce circulating memory B cells. MMF lowered IgA levels more markedly than CYC. We did not observe a significant difference in the reduction of IgG levels or anti-dsDNA antibodies comparing patients receiving MMF or CYC. In contrast to MMF, induction therapy with CYC was associated with a significant increase of circulating CD8+ effector T cells and plasmacytoid dendritic cells (PDCs) after three months. CONCLUSIONS: The results indicate differences between MMF and CYC with regard to the mechanism of action. MMF, but not CYC, treatment leads to a fast and enduring reduction of surrogate markers of B cell activation, such as circulating plasmablasts, plasma cells and free light chains but a comparable rate of hypogammaglobulinemia.

Ganciclovir prophylaxis improves late murine cytomegalovirus-induced renal allograft damage.

BACKGROUND: Human cytomegalovirus (CMV) infection is associated with inferior survival in renal transplant patients, and ganciclovir (GCV) prophylaxis is associated with improved survival. In a murine CMV (MCMV) renal transplantation model, ganciclovir prophylaxis improved innate infiltrates and allograft damage during the period of prophylaxis. In this study, late effects were examined after the discontinuation of prophylaxis. METHODS: MCMV D+/R- and D-/R- allogeneic transplants were performed with cyclosporine immunosuppression. One D+/R- cohort received ganciclovir prophylaxis for 14 days after transplantation followed by 28 days without ganciclovir. At 42 days after transplantation, grafts were analyzed for histologic tissue damage and immune infiltrates. Another D+/R- cohort was treated with anti-NK1.1 antibodies for 14 days after transplantation and compared with animals without natural killer (NK) cell depletion. RESULTS: At day 42, MCMV-infected transplants had higher damage scores (15.6±0.6) compared with uninfected transplants (8.3±0.9; P<0.01), which improved in ganciclovir-treated allografts (9.5±1.4). MCMV-infected grafts contained greater frequencies of NK cell and myeloid infiltrates compared with uninfected grafts (P<0.05), which decreased in the ganciclovir-treated grafts. NK cell depletion improved allograft histology of MCMV-infected grafts. CONCLUSIONS: MCMV infection exacerbates late renal allograft damage and is associated with NK and myeloid cell infiltrates. Ganciclovir prophylaxis reduces allograft injury and NK cell and myeloid infiltrates even after the cessation of prophylaxis. NK cell depletion in MCMV-infected transplants also improves histology. These results suggest that ganciclovir prophylaxis may have a long-term beneficial effect on CMV-infected renal allografts and suggest a potential role for NK cells in the pathogenesis of CMV-associated allograft injury.

Ganciclovir transiently attenuates murine cytomegalovirus-associated renal allograft inflammation.

BACKGROUND: Prophylactic ganciclovir (GCV) is used in high-risk renal transplant patients to prevent acute cytomegalovirus (CMV) disease, but its impact on inflammation within the allograft itself remains undefined. METHODS: To study the effect of GCV prophylaxis on allograft inflammation, murine CMV (MCMV)-infected allografts were analyzed in a murine donor positive/recipient negative allogeneic renal transplantation model by flow cytometry and immunofluorescent staining. RESULTS: By flow cytometry, CD45+ leukocyte infiltrates were more abundant in MCMV-infected allografts at 14 days posttransplant compared with uninfected grafts (P<0.01) and decreased in the presence of GCV (P<0.05). CD11c+ dendritic cells, Gr-1+ myeloid cells, CD204+ macrophages, and CD49b+ natural killer cells were reduced in GCV-treated allografts compared with MCMV-infected grafts without GCV treatment (P<0.05). However, GCV failed to reduce these cell types to levels found in MCMV-uninfected allografts. By day 7 after cessation of GCV prophylaxis, dendritic cells, macrophages, and natural killer cells increased in number and became statistically indistinguishable from numbers of cells found in MCMV-infected allografts without GCV. GCV treatment did not affect the numbers of CD4+, CD8+, or CD19+/B220+ lymphocytes infiltrating the allografts. Infiltrates were confirmed histologically by immunofluorescent staining for CD3+ and CD11b+ cells. CONCLUSIONS: In this model, MCMV-infected allografts developed significantly greater innate and adaptive leukocytic infiltrates compared with uninfected grafts. GCV attenuated the MCMV-associated innate leukocyte infiltrates in infected allografts but not the lymphocytic infiltrates. The attenuated innate response was limited to the period of GCV prophylaxis.