Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.

The presence of anti-HLA donor-specific antibodies in lung allograft recipients does not correlate with C4d immunofluorescence in transbronchial biopsy specimens.

CONTEXT: C4d immunofluorescence (IF) is a surrogate for development of donor-specific antibodies (DSAs) against human leukocyte antigen (HLA) class I and II antigens in kidney and heart biopsy specimens for monitoring of antibody-mediated (humoral) allograft rejection (AMR). Use of C4d IF in monitoring of lung allografts has shown conflicting results. OBJECTIVE: To determine if C4d IF can be used as a reliable marker for AMR and if it correlates with the presence of DSAs and histologic findings on biopsy. DESIGN: All transbronchial biopsies in lung allograft recipients, performed at our institution in a 3-year period, were reviewed. A cohort of 92 patients with 110 corresponding biopsies met the inclusion criteria of (1) having a resulted DSA within 2 weeks of biopsy and (2) having C4d immunofluorescence studies performed and confirmed. RESULTS: Twenty-nine patients (31.5%) were positive for DSAs and 63 patients (68.5%) did not develop DSAs. Positive C4d capillary IF was seen in 18 of 110 total biopsy specimens (16.4%). Eight of these biopsy samples were from patients positive for DSAs and 10 were from patients negative for DSAs. The correlation coefficient between the presence of DSAs and C4d IF was 0.1628 (P = .09). CONCLUSIONS: A significant proportion of DSA-positive patients had negative C4d IF results and frequently have no histologic changes on biopsy specimens. DSA-negative patients can be positive for C4d and may show the same histologic changes as reported for DSA-positive patients. Diagnosis of AMR in lung may require a collaborative approach combining clinical data, DSA status, and histology.

Bortezomib in kidney transplant desensitization: a case report.

Although current therapies for pretransplant desensitization and antibody mediated rejection (AMR) have had some success, they do not specifically deplete plasma cells that produce antibodies to HLA. Bortezomib, a proteasome inhibitor, has been shown to induce plasma cell apoptosis and has been used in the treatment of AMR; however, there are no reported experiences in using this agent in pretransplant desensitization. We report using bortezomib in conjunction with Rituximab to desensitize a kidney transplant recipient on the waiting list. The patient's anti-HLA antibody titers (calculated PRA- (cPRA)) decreased from 57% to 31% prior to transplantation and the overall strength of his anti-HLA antibodies also decreased. He received a deceased-donor kidney to which he had a single low-level donor specific antibody that was undetectable post transplant. This case report demonstrates the potential safety of bortezomib therapy in treatment of highly sensitized patients awaiting kidney transplant and its association with decreased anti-HLA antibody levels.

The CD3epsilon proline-rich sequence, and its interaction with Nck, is not required for T cell development and function.

The CD3epsilon proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3epsilon PRS, we have generated mice that lack this motif (CD3epsilon.PRS(M)). Pull-down experiments demonstrated the inability of Nck to bind to the CD3epsilon PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3epsilon.PRS(M) T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3epsilon PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Function of CD80 and CD86 on monocyte- and stem cell-derived dendritic cells.

Dendritic cells (DCs) consist of a heterogeneous population of hematopoietic cells characterized by their unique dendritic morphology, their efficient antigen-presenting capability to activate naïve CD4+ and CD8+ T cells, as well as their lack of lineage-specific markers. Functional properties comparing umbilical cord blood monocyte-derived and umbilical cord blood stem cell-derived DCs have not yet been investigated. Human umbilical cord blood CD14+ monocytes and CD34+ stem cells were induced to differentiate into dendritic cells using 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 25 ng/mL interleukin (II)-4, 2.5 ng/mL tumor necrosis factor alpha (TNF-alpha) and 100 ng/mL GM-CSF, 25 ng/mL stem cell factor, and 2.5 ng/mL TNF-alpha, respectively. Differentiated dendritic cells were CD80+, CD86+, CD83+, CD54+, CD1a+, CD11b+, CD11c+, HLA-DR+, CD34-, CD3-, CD19-, CD14-, and CD16-. Reverse transcription polymerase chain reaction revealed that differentiating monocytes initially expressed CD86 mRNA while CD80 mRNA appeared on Day 2. Differentiating stem cells expressed both CD80 and CD86 mRNA on Day 2 of culture. Mixed lymphocyte reaction was employed to evaluate the two types of lineage-derived DCs. Monoclonal antibodies (mabs) to CD80 and CD86 were employed to assess their costimulatory roles. CD14 and CD34 derived DCs prior to the functional assay were stimulated for 18 h with 0.1 and 1.0 mg/mL Escherichia coli lipopolyssacharide, respectively. A decrease in stimulation as depicted by decreased T-cell activation was significant with mabs to both CD80 and CD86 on monocyte-derived DCs while only mabs to CD86 induced decreased T-cell activation by stem cell-derived DCs. The varied functional role of CD80 and CD86 costimulatory molecules is associated with DC differentiation from distinct cord blood-isolated hematopoietic lineages. These studies demonstrate that DC association with distinct hematopoietic lineages is of relevance in transplantation and vaccine therapies.

Effects of tumor-enhancing IgG2 on macrophage function.

Macrophages (M phi) play a significant role in allograft rejection. We investigated whether tumor-enhancing (te) IgG(2) acting as a cytophilic opsonin affects allograft destruction. Our results demonstrate that immune Tennessee Swiss (TS) (H-2(s)) mouse M phi destroyed greater numbers of target C3H(f)/He (H-2(k)) tumor cells than did nonimmune M phi. The percentage of (51)Cr release from labeled tumor cells induced by immune M phi was 39.10 + 3.24% compared to nonimmune M phi 28.0 + 3.87%, while te IgG suppressed cytotoxicity toward C3H(f)/He tumor cells of normal TS M phi as manifested by less isotope release (19.60 + 3.13%) than that produced by normal TS M phi with non-enhancing IgG(2) (39.90 + 5.8%). Rather than facilitating survival of allogeneic fibrosarcoma cells, te IgG(2) alloantibody potentiated their destruction by immune TS M phi (isotope release of 52.90 + 3.46%) compared with that produced by immune M phi alone (39.10 + 3.24%). Cytophilic te IgG(2) on TS M phi was demonstrated with either C3H(f)/He red cells or tumor cells. Electron micrographs of M phi revealed tumor cell ingestion and attachment of ferritin-labeled IgG(2) alloantibody on M phi and target tumor cells. In contrast to protection, te IgG(2) alloantibody facilitates macrophage-mediated allograft destruction.

Costimulatory function of umbilical cord blood CD14+ and CD34+ derived dendritic cells.

Dendritic cells (DCs) consist of a heterogeneous population of hematopoietic cells characterized by their unique dendritic morphology, their efficient antigen-presenting capability to activate naive CD4(+) and CD8(+) T cells, and their lack of lineage specific markers. Functional properties comparing umbilical cord blood monocyte-derived and umbilical cord blood stem cell-derived DCs have not yet been investigated. CD14(+) monocytes and CD34(+) stem cells were isolated from human umbilical cord blood and were induced to differentiate into dendritic cells using 100 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF), 25 ng/mL IL-4, 2.5 ng/mL tumor necrosis factor-alpha (TNF-alpha), 100 ng/mL GM-CSF, 25 ng/mL stem cell factor, and 2.5 ng/mL TNF-alpha, respectively. Flow cytometric analysis revealed that the 14-day-old dendritic cells were CD80(+), CD86(+), CD83(+), CD54(+), CD1a(+), CD11b(+), CD11c(+), HLA-DR(+), CD34(-), CD3(-), CD19(-), CD14(-), and CD16(-). Reverse transcription polymerase chain reaction was employed to detect expression of mRNA for CD80 and CD86. Differentiating monocytes initially expressed CD86 while CD80 appeared on day 2. Differentiating stem cells expressed CD80 and CD86 on day 2 of culture. The surface expression of CD80 and CD86 was studied over the course of differentiation. Mixed lymphocyte reaction was employed to evaluate the two types of lineage-derived DCs. Prior to the functional assay, CD14(+) and CD34(+) derived DCs were stimulated for 18 h with 0.1 mg/mL and 1.0 mg/mL E. coli lipopolyssacharide, respectively. Monoclonal antibodies (mabs) to CD80 and CD86 were employed to assess their costimulatory roles. A decrease of stimulation as depicted by decreased T cell activation was significant with mabs to both CD80 and CD86 on monocyte-derived DCs while only mabs to CD86 induced decreased T cell activation by stem cell-derived DCs. The varied functional role of CD80 and CD86 costimulatory molecules is associated with DC differentiation from distinct cord blood isolated hematopoietic lineages. These studies demonstrate that DC association with distinct hematopoietic lineages is of relevance in transplantation and vaccine therapies.

Architectural changes in the TCR:CD3 complex induced by MHC:peptide ligation.

A hallmark of T cell activation is the ligation-induced down-modulation of the TCR:CD3 complex. However, little is known about the molecular events that drive this process. The CD3 zeta-chain has been shown to play a unique role in regulating the assembly, transport, and cell surface expression of the TCR:CD3 complex. In this study we have investigated the relationship between CD3zeta and the TCRalphabetaCD3epsilondeltagamma complex after ligation by MHC:peptide complexes. Our results show that there is a significant increase in free surface CD3zeta, which is not associated with the TCR:CD3 complex, after T cell stimulation. This may reflect dissociation of CD3zeta from the TCRalphabetaCD3epsilondeltagamma complex or transport of intracellular CD3zeta directly to the cell surface. We also show that MHC:peptide ligation also results in exposure of the TCR-associated CD3zeta NH2 terminus, which is ordinarily buried in the complex. These observations appears to be dependent on Src family protein tyrosine kinases, which are known to be critical for efficient T cell activation. These data suggest a mechanism by which ligated TCR may be differentiated from unligated TCR and selectively down-modulated.