A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

Human peripheral blood CD4 T cell-engrafted non-obese diabetic-scid IL2rγ(null) H2-Ab1 (tm1Gru) Tg (human leucocyte antigen D-related 4) mice: a mouse model of human allogeneic graft-versus-host disease.

Graft-versus-host disease (GVHD) is a life-threatening complication of human allogeneic haematopoietic stem cell transplantation. Non-obese diabetic (NOD)-scid IL2rγ(null) (NSG) mice injected with human peripheral blood mononuclear cells (PBMC) engraft at high levels and develop a robust xenogeneic (xeno)-GVHD, which reproduces many aspects of the clinical disease. Here we show that enriched and purified human CD4 T cells engraft readily in NSG mice and mediate xeno-GVHD, although with slower kinetics compared to injection of whole PBMC. Moreover, purified human CD4 T cells engraft but do not induce a GVHD in NSG mice that lack murine MHC class II (NSG-H2-Ab1(tm1Gru), NSG-Ab°), demonstrating the importance of murine major histocompatibility complex (MHC) class II in the CD4-mediated xeno-response. Injection of purified human CD4 T cells from a DR4-negative donor into a newly developed NSG mouse strain that expresses human leucocyte antigen D-related 4 (HLA-DR4) but not murine class II (NSG-Ab° DR4) induces an allogeneic GVHD characterized by weight loss, fur loss, infiltration of human cells in skin, lung and liver and a high level of mortality. The ability of human CD4 T cells to mediate an allo-GVHD in NSG-Ab° DR4 mice suggests that this model will be useful to investigate acute allo-GVHD pathogenesis and to evaluate human specific therapies.

Role of membrane immunoglobulin (Ig) crosslinking in membrane Ig-mediated, major histocompatibility-restricted T cell-B cell cooperation.

Resting murine B lymphocytes can present rabbit anti-Ig to T cell lines specific for normal rabbit globulin. The T cell-B cell interaction is major histocompatibility complex (MHC)-restricted, and leads to activation, proliferation, and differentiation of the resting B cell into an antibody-secreting cell. Efficient antigen presentation and B cell activation depends upon binding of rabbit globulin to (membrane) mIg. To investigate the role of mIg in this polyclonal model for a T cell-dependent primary antibody response, we determined whether crosslinking of mIg is required either for efficient antigen presentation, as measured by helper T cell activation, or for the B cell response to T cell help, since all the direct effects of anti-Ig on B cells require crosslinking of mIg. We found that monovalent Fab' fragments of anti-IgM or anti-IgD work as efficiently as their divalent counterparts. Therefore, a signal transduced through the antigen receptor seems not to be required when T cell help is provided by an MHC-restricted T helper cell recognizing antigen on the B cell surface. Moreover, rabbit globulin bound to class I MHC molecules in the form of anti-H-2K also results in efficient antigen presentation and T cell-dependent B cell activation. However, mIg still appears to be specialized for antigen presentation, since anti-Ig is presented about three- to fivefold more efficiently than anti-H-2K.

Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.

Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD154 monoclonal antibody.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and a brief course of anti-CD154 (anti-CD40 ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 days without further intervention. We now report adaptation of this protocol to the transplantation of islet and skin xenografts. We observed prolonged survival of rat islet xenografts in mice treated with donor-specific spleen cell transfusion and anti-CD154 monoclonal antibody (mAb). Challenge islet xenografts placed on these animals indicated that graft acceptance was species-specific but not strain specific. Spleen cells from recipients bearing intact grafts led to rejection of rat islet xenografts in scid mice, suggesting that graft acceptance was not due to complete clonal deletion of xenoreactive cells. We also observed prolonged survival of rat skin xenografts in mice treated with donor-specific transfusion and anti-CD154 mAb. Prolonged survival of skin xenografts was also species specific. We conclude that treatment with appropriately timed donor-specific transfusion and anti-CD154 mAb induces durable survival of both islet and skin xenografts in mice. Because this procedure is targeted directly at CD154, a co-activation molecule expressed predominantly by activated CD4+ T-cells, the results suggest that CD4+ cells have a major role in the cellular immune response to xenografts.

NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.

Fc gamma receptor effects on induction of c-myc mRNA expression in mouse B lymphocytes by anti-immunoglobulin.

Levels of c-myc mRNA have been assayed in mouse B cells cultured for 8 hr with Fab'2 anti-Ig or IgG anti-Ig. Fab'2 anti-Ig induces DNA synthesis in B cells, whereas the whole molecule inhibits anti-Ig-induced DNA synthesis by crosslinking the B cell Fc gamma R to mIg. Both the Fab'2 fragment and the IgG anti-Ig induce an increase in c-myc mRNA by 1 hr. Thereafter, levels in cells stimulated with submitogenic doses of Fab'2 anti-Ig or any dose of IgG anti-Ig returned to background, while levels in cultures containing a mitogenic dose of Fab'2 anti-Ig remained elevated.

Effects of IL-4 and Fc gamma receptor II engagement on Egr-1 expression during stimulation of B lymphocytes by membrane immunoglobulin crosslinking.

Egr-1 is an immediate early gene that is rapidly upregulated in response to mitogenic signals induced by antigen receptor crosslinking on murine B lymphocytes. It has been shown that levels of Egr-1 expression are closely correlated with B cell proliferation in several models of B cell activation and tolerance. We compared the expression of Egr-1 during B cell stimulation with Fab'2 and IgG anti-immunoglobulin (anti-Ig), since it is known that Fab'2 anti-Ig is mitogenic while IgG anti-Ig is not, owing to a dominant inhibitory effect of crosslinking the B cell Fc gamma RII to membrane Ig. While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in Egr-1 expression, IgG anti-Ig results in smaller increases in Egr-1 mRNA, comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig. However, the correlation between Egr-1 expression and B cell proliferation breaks down when IL-4 is added as a co-mitogen to induce B cell proliferation with IgG anti-Ig or submitogenic concentrations of Fab'2 anti-Ig. No corresponding increases in Egr-1 mRNA levels are observed when IL-4 is added. Therefore, IL-4 overcomes Fc receptor-mediated inhibition of B cell proliferation without affecting inhibition of Egr-1 mRNA induction, as demonstrated earlier for c-myc mRNA in this system.

Magnetic resonance imaging of the posterior fossa in autism.

The brainstem-cerebellar circuitry has been implicated in the pathophysiology of autism for several decades. Recent magnetic resonance imaging (MRI) studies of the posterior fossa have reported various abnormalities, the most noteworthy of which has been selective hypoplasia of the neocerebellar vermis. However, these initial MRI studies are limited by problems in both subject and control selection. The present study was undertaken to further investigate these MRI findings and the role of the cerebellum in autism, taking into consideration these methodologic issues. Eighteen high-functioning autistic subjects were recruited and matched with 18 normal controls on the basis of age, gender, IQ, race and socioeconomic status (SES). The midsagittal areas of the cerebellar vermis, vermal lobes, and the fourth ventricle were measured on 3 mm contiguous magnetic resonance images. Mean areas and standard deviations were comparable for all regions of interest and no statistically significant between-group differences were found. These negative findings argue against theories of autism based on gross structural abnormalities of the cerebellum. Previous reports of posterior fossa abnormalities may be related to technical and methodological factors, based on comparison of extant literature and recently available normative data.

Prolonged survival of mouse skin allografts in recipients treated with donor splenocytes and antibody to CD40 ligand.

Combined treatment with antibody against CD40 ligand and one transfusion of donor splenocytes prolonged survival of fully mismatched BALB/c skin allografts on C57BL/6 recipients, with approximately 20% of grafts surviving > 100 days. In vitro alloresponsiveness in treated animals was reduced in the immediate post-transplantation period, but by day 100 was increased despite the presence of a successful allograft. The presence of alloreactivity on day 100 was confirmed in vivo by adoptive transfer, which suggests that our protocol had induced either a state of "split tolerance" or "graft accommodation." Mice with skin grafts that had survived for > or = 100 days revealed no evidence of lymphoid chimerism. Treatment with donor splenocytes and antibody against CD40 ligand permits long-term survival of highly antigenic donor skin allografts despite the presence of functionally intact alloreactive lymphocytes.

Induction of immunological tolerance to islet allografts.

T-cell dependent activation of resting B cells involves the interaction of gp39 on T cells with its receptor, CD40, on B cells. We administered either a combination of T-cell-depleted splenic lymphocytes and anti-gp39 monoclonal antibody or antibody alone to establish islet allografts in mice without continuous immunosuppression. Fully allogeneic H-2q FVB islets were permanently accepted by chemically diabetic H-2b C57BL/6 mice provided that the recipients were pretreated with both T-cell-depleted donor spleen cells and anti-gp39 antibody. Antibody alone was less effective in prolonging allograft survival, but we did observe that anti-gp39 mAb alone can exert an independent, primary effect on islet allograft survival that was dose dependent. Targeting gp39, in combination with lymphocyte transfusion, might prove suitable for tolerance induction and allotransplantation without immunosuppression.

Purification and sequencing of neuropeptides from identified neurons in the marine mollusc, Tritonia.

Neuropeptides were characterized in two similar identified neurons termed pedal 5 and 6 (Pd5 and Pd6). Both neurons appear white, a characteristic of peptidergic neurons, and send peripheral axons down several nerves that innervate the foot and control locomotion. Gel electrophoresis of neurons incubated with labeled amino acids indicated that individually dissected Pd5 and Pd6 neurons synthesized peptide precursors of the same size and processed them in parallel. Using HPLC, several absorbance peaks that had retention times typical of peptides were identified that were specific to extracts of Pd5 and Pd6. Three peptides were purified from extracts of many pooled Pd5 and Pd6 neurons. The complete sequences of two 15-amino acid peptides were obtained and the sequence of a third 15-amino acid peptide was inferred from the partial sequence of an apparent processing intermediate. Each of the three peptides show sequence homology to Aplysia pedal peptide (Pep). HPLC of neurons incubated with labeled amino acids demonstrated that Pd5 and Pd6 each synthesized all three sequenced peptides.

Fc-dependent inhibition of mouse B cell activation by whole anti-mu antibodies.

We have been using whole rabbit anti-mouse mu antibodies to study Fc-dependent inhibition of mouse B cell activation by F(ab')2 anti-mu antibodies and antigen-nonspecific helper factors (SN). We show here that this inhibition does not appear to require adherent cells, appears to occur independently of cellular interactions, is reversible, and is not maintained solely by suppressive factors. In addition, occupancy of Fc receptors by rabbit antibody-antigen complexes is not sufficient to inhibit activation by F(ab')2 anti-mu and SN. These observations, in conjunction with the observation that blocking the Fc receptor-binding capacity of rabbit anti-mu antibodies by protein A prevents inhibition, suggest that cross-linking mlg and Fcgamma receptors on B cells prevents activation. However, the F(ab')2 anti-mu and SN-activated B cells become refractory to this inhibition by 48 hr in culture.

Subclass specificity of Fc gamma receptor-mediated inhibition of mouse B cell activation.

We have shown previously that cross-linking receptors for the Fc of IgG (Fc gamma R) to membrane Ig will inhibit membrane Ig-dependent mouse B cell activation. In this report we have determined which mouse IgG subclasses will mediate this inhibition by using monoclonal mouse anti-p-azophenylarsonate (anti-ars) antibodies to inhibit B cell activation by arsanilated rabbit Fab'2 anti-mouse IgM (arsFab'2 anti-mu). Cell recovery and Ig secretion after 4 days of culture with soluble helper factors and arsFab'2 anti-mu was inhibited by IgG1, IgG2b, and IgG2a anti-ars, but not IgM or Fab'2 anti-ars. In addition, we determined which of the inhibitory IgG subclasses are blocked by the monoclonal anti-mouse Fc gamma R antibody 2.4G2, which we have previously shown prevents inhibition by rabbit IgG anti-mu. These experiments demonstrated that 2.4G2 Fab could block inhibition mediated by IgG2a as well as that mediated by IgG1 and IgG2b. Because it has been reported that 2.4G2 does not bind the IgG2a receptor on macrophages, these data support our previous experiments, which indicated that this inhibition is not macrophage dependent.

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis.

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.